WO2023122805A1 - Sorbitol driven selection pressure method - Google Patents

Abstract

Recombinant yeast cells and methods of producing the same are provided. The present disclosure provides methods of making and culturing recombinant cells, and methods of preventing copy loss of genes of interest, such as cysteine rich polypeptides (CRPs). The present disclosure also provides recombinant yeast cells comprising, inter alia, a heterologous polynucleotide that prevents copy number loss when the recombinant yeast cells are grown in sorbitol.

Description

SORBITOL DRIVEN SELECTION PRESSURE METHOD

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of, and priority to, United States Provisional Application Serial No. 63/291,710, filed on December 20, 2021. The entire contents of the aforementioned application are incorporated herein.

SEQUENCE LISTING

[0002] This application incorporates by reference in its entirety the Sequence Listing entitled “225312-520930” (461 KB), which was created on December 27, 2022, at 5:33 PM, and filed electronically herewith.

TECHNICAL FIELD

[0003] The present disclosure provides recombinant yeast cells, methods of culturing the cells, and methods of preventing copy loss of genes of interest.

BACKGROUND

[0004] A goal in the production economically relevant, recombinantly-expressed proteins of interest, is to obtain these proteins in large quantities. Obtaining large quantities of these proteins can be accomplished through the use recombinant cells comprising multiple copies of heterologous polynucleotides operable to express the proteins of interest.

[0005] However, multiple integrations of heterologous polynucleotides carries risks that may compromise protein production yields. Namely, the ultimate number of copies integrated into the host cell’s genome can become unstable due to homologous out- recombination.

[0006] Furthermore, the production of recombinant cells with multiple copies of heterologous polynucleotides encoding proteins of interest is inefficient as, after a few generations, the integrated heterologous genes are likewise out-recombined via intrinsic DNA repair mechanisms — a problem that exacerbates as time progresses.

[0007] Recombinant cells that have lost one or more of the multiple copies of the heterologous polynucleotide are less encumbered by heterologous protein production; accordingly, cells that have lost one or more of the multiple copies are able to outcompete cells maintaining the multiple copies, because the latter are energetically burdened with heterologous protein production. [0008] Therefore, recombinant organisms, and methods of using the same, that are able to produce high yields of proteins of interest, and/or are able to maintain high copy numbers of multiple copies of heterologous encoding the proteins of interest, are needed.

SUMMARY

[0009] This invention describes a recombinant yeast cell having a heterologous polynucleotide that comprises (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[0010] In addition, the present disclosure describes how to increase expression of a heterologous polypeptide in a recombinant yeast cell, the method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, the heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[0011] In addition, the present disclosure describes a vector comprising: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein the 5’-homology arm and the 3’-homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein the vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide. [0012] In addition, the present disclosure describes a polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof.

[0013] In addition, the present disclosure describes how to increase expression of a Cysteine Rich Peptide (CRP) in a recombinant Kluyveromyces lactis cell, the method comprising: (a) inactivating or at least partially inactivating an endogenous sorbitol dehydrogenase (SDH) nucleotide sequence; (b) providing a vector comprising one or more copies of a heterologous polynucleotide, the heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) a nucleotide sequence operable to encode a heterologous SDH; and (ii) a nucleotide sequence operable to encode a CRP; (c) creating a recombinant Kluyveromyces lactis cell by transforming the vector into a Kluyveromyces lactis host cell; and (d) growing the recombinant Kluyveromyces lactis cell in a medium comprising a sole carbon source that is sorbitol; wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3; wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); aPhoneutria toxin; or an Atracotoxin (ACTX); and wherein growing the recombinant Kluyveromyces lactis cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the CRP, relative to a level of expression of the CRP when growing the recombinant Kluyveromyces lactis cell in a medium comprising a sole carbon source that is not sorbitol.

[0014] In addition, the present disclosure describes how to increase expression of a Cysteine Rich Peptide (CRP) in a recombinant Pichia pastoris cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, the heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) a nucleotide sequence operable to encode a heterologous SDH; and (ii) a nucleotide sequence operable to encode a CRP; (b) creating a recombinant Pichia pastoris cell by transforming the vector into a Pichia pastoris host cell; and (c) growing the recombinant Pichia pastoris cell in a medium comprising a sole carbon source that is sorbitol; wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3; wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX); and wherein growing the recombinant Pichia pastoris cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the CRP, relative to a level of expression of the CRP when growing the recombinant Pichia pastoris cell in a medium comprising a sole carbon source that is not sorbitol.

[0015] In addition, the present disclosure describes a recombinant Kluyveromyces lactis cell comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) two or more nucleotide sequences operable to encode a Cysteine Rich Peptide (CRP); wherein the ratio of (i) to (ii) is at least 1 :2; wherein the recombinant Kluyveromyces lactis has an endogenous SDH nucleotide sequence that has been inactivated or at least partially inactivated; and wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a Phoneutria toxin; or an Atracotoxin (ACTX).

[0016] In addition, the present disclosure describes a recombinant Pichia pastoris cell comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) two or more nucleotide sequences operable to encode a Cysteine Rich Peptide (CRP); wherein the ratio of (i) to (ii) is at least 1:2; and wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX).

BRIEF DESCRPTION OF THE DRAWINGS

[0017] FIG. 1 depicts a schematic representation of a vector diagram of the plasmid vector, pJUSor, having sorbitol dehydrogenase (lid), used to transform a wild type P. pastoris cell.

[0018] FIG. 2 shows a graph depicting growth of “BG10,” a wild type P. pastoris strain with no known sorbitol dehydrogenase (lid) gene. “JUSor2” shows growth of the same cell (BG10) transformed with the plasmid vector, pJUSor, which contains lid.

[0019] FIG. 3 depicts a plasmid vector, PKlDlid, containing an acetamidase selection marker gene (amdS), used in the knock-out of endogenous sorbitol dehydrogenase of K. lactis by replacing the endogenous lid with amdS.

[0020] FIG. 4 shows a photo of an agar plate comprising defined media with 4% sorbitol as a sole carbon source, and without com steep liquor (CSL). The section labeled “YCT306” corresponds to a wild-type K. lactis cell having one copy of endogenous sorbitol dehydrogenase or “lid.” The sections labeled il - i4 were confirmed lid knockouts in the wild-type YCT-306 background.

[0021] FIG. 5 depicts a graph showing the lid and amdS copy number estimates for K. Lactis lid knockout cells. “YCT-306” shows a wild-type K. lactis cell having one copy of endogenous lid. “VSTLBlOil” shows successful integration of the pKlDlid vector into a K. lactis cell by showing no amplification of sorbitol dehydrogenase (SDH) and one copy of amdS. “VSTLB10” shows successful out-recombination of the amdS marker by showing no amplification of both the endogenous sorbitol dehydrogenase and the amdS marker.

[0022] FIG. 6 depicts a plasmid vector, pLB10V5DS containing a heterologous polynucleotide comprising a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and a nucleotide sequence operable to encode a heterologous polypeptide, wherein the heterologous polypeptide is a cysteine-rich protein (CRP). Here, the CRP is hybrid+2-ACTX-Hvla. The plasmid vector pLB10V5DS is used for expression in T. Lactis of a heterologous polypeptide and a heterologous sorbitol dehydrogenase. The segments labeled “Seq-10V5DS-Sel-B” and “Seq-10V5DS-Sel,” correspond to the locations on the vector where sequencing primers bind, and are used to confirm that the correct selection marker was inserted into the plasmid.

[0023] FIG. 7 shows a graph depicting the estimated number of copies of lid in various transformants of pLB10V5DS. “YCT-306” shows a wild-type K. lactis cell having one copy of endogenous lid. “VSTLB10” shows a lid- and amdS-knocked out K. lactis cell. “lid-Hl, lid-H2, lid-H3, lid-Ml, lid-M2, lid-Ll, and lid-L2” are different transformants of pLB10V5DS into strain VSTLB 10 grown in media where sorbitol is the sole carbon source. Successful transformation was confirmed, and lid copy numbers measured, via quantitative polymerase chain reaction (qPCR) by the presence of lid.

[0024] FIG. 8 shows a photo of an agar plate comprising 4% sorbitol as the sole carbon source, and inoculated with transformants.. Here, larger colonies indicated significant improvement of sorbitol utilization suggesting successful integration of lid into VSTLB 10. [0025] FIG. 9 shows the HPLC results demonstrating expression of the heterologous polypeptide, Hybrid+2-ACTX-Hvla, in 44 VSTLB10 cells transformed with the pLB10V5DS vector. All 44 cells showed a 6300 peak 1 at 3.787 mins, suggesting that the sorbitol dehydrogenase selection marker can be used to identify and select colonies with successful integration of the heterologous polynucleotide and for the expression of a heterologous polypeptide. [0026] FIG. 10 shows a graph depicting copy loss in pLB10V5DS modified yeast cells cultivated in different media at generations 0, 13, 24, 34, 44, 54, and 65. “lidlOaSOR” shows a pLB10V5DS modified yeast cell grown in media containing sorbitol as the sole carbon source. “lidlObSOR” is a biological replicate of lidlOaSOR and shows a pLB10V5DS modified yeast cell grown in media containing sorbitol as the sole carbon source.

“lidlOaGLU” shows a pLB10V5DS modified yeast cell grown in media containing glucose as the sole carbon source. “lidlObGLU” is a biological replicate of lidlOaGLU and shows pLB10V5DS modified yeast cell grown in media containing glucose as the sole carbon source. Copy numbers were measured via qPCR.

[0027] FIG. 11 shows the relative quantification (RQ) of sorbitol dehydrogenase or “lid” based on a given strain. Here, “YCT306” corresponds to a wild-type K. lactis cell having one copy of endogenous sorbitol dehydrogenase or “lid.” Other cells evaluated were 68p, lid-Hl (Hl), VSTLB10 (LB10), and lid-M2 (M2).

[0028] FIGs. 12A-12D depict graphs showing growth rates in sorbitol and glucose. FIG. 12A shows the instant growth rate of transformed strains containing 0, 1, 3, or 5 SDH copies in media with sorbitol as the sole carbon source. FIG. 12B shows the instant growth rate of transformed strains containing 0, 1, 3, or 5 SDH copies in media with glucose as the sole carbon source. FIG. 12C shows the maximum instantaneous growth rate of transformed cells, over 98 hours of growth, containing 0, 1, 3, or 5 SDH copies in media with sorbitol as the sole carbon source (“SOR growth) or in media with glucose as the sole carbon source (“GLU growth). FIG. 12D shows the average instantaneous growth rate of transformed cells containing 0, 1, 3, or 5 SDH copies in media with sorbitol as the sole carbon source (“SOR growth) or in media with glucose as the sole carbon source (“GLU growth).

[0029] FIG. 13 depicts a plasmid vector map of pLB103bM165BD. This vector comprises a 5 ’-homology arm and a 3 ’-homology arm corresponding to loci in the LAC4 promoter (pLAC4) (allowing integration of the dual expression cassettes into the endogenous pLAC4 loci), and a heterologous polynucleotide comprising a dual expression cassette, wherein each expression cassette has a nucleotide sequence operable to encode the heterologous polypeptide, Av3165, having the amino acid sequence: “KSCCPCYWGGCPWGQNCYPEGCSGPKVG” (SEQ ID NO: 485). Here, cassette no. 1 (top) comprises an intact pLAC4 promoter; and cassette no. 2 comprises a pLAC12 promoter. Each cassette also comprises a nucleotide sequence operable to encode a heterologous polypeptide (i.e., Av3165), and an alpha-MF signal sequence. In addition, the vector comprises a single copy of an amdS transgene; and each cassette comprises: a Kex2 cleavage site; a multiple cloning site; a LAC4 terminator or a LAC 12 terminator; an ADH1 promoter; a P-lactamase (bla) gene; and an origin of replication site. Here, the two cassettes run in opposite directions, reflecting a bi-direction dual cassette strategy.

[0030] FIG. 14 shows a graph depicting the yield per copy number for two Av3 mutants, Av3 mutant 103b, as characterized by SEQ ID NO: 484, and Av3 mutant 165, characterized by SEQ ID NO: 485.

DETAILED DESCRPTION

[0031] DEFINITIONS

[0032] “5’-end” and “3’-end” refers to the directionality, i.e., the end-to-end orientation of a nucleotide polymer (e.g., DNA). The 5’-end of a polynucleotide is the end of the polynucleotide that has the fifth carbon.

[0033] “5’- and 3 ’-homology arms” or “5’ and 3’ arms” or “left and right arms” refer to the polynucleotide sequences in a vector and/or targeting vector that homologously recombine with the target genome sequence and/or endogenous gene of interest in the host organism in order to achieve successful genetic modification of the host organism’s chromosomal locus.

[0034] ‘T-CNTX-Pnla” or “y-CNTX-Pnla” or “gamma-CNTX-Pnla” or “gamma” refers to an insecticidal neurotoxin derived from the Brazilian armed spider, Phoneutria nigriventer. F-CNTX-Pn I a targets the N-methyl-D-aspartate (NMDA)-subtype of ionotropic glutamate receptor (GRIN), and sodium channels.

[0035] “Alpha mating factor (alpha-MF) peptide” or “alpha-MF signal” or “alpha- MF” or “alpha mating factor secretion signal” or “aMF secretion signal” (all used interchangeably) refers to a signal peptide that allows for secreted expression in a recombinant expression system, when the alpha-MF peptide is operably linked to a heterologous polypeptide of interest (e.g., a CRP). The Alpha-MF peptide directs nascent recombinant polypeptides to the secretory pathway of the recombinant expression system (e.g., a recombinant yeast cell).

[0036] “ACTX” or “ACTX peptide” or “atracotoxin” refers to a family of insecticidal ICK peptides that have been isolated from spiders belonging to the Atracinae family, and variants thereof. One such spider is known as the Australian Blue Mountains Funnel-web Spider, which has the scientific name Hadronyche versuta. Examples of ACTX peptides include the naturally occurring peptides: U-ACTX-Hvla, K-ACTX-Hvla, co-ACTX-Hvla; and the non-natural variants: U+2-ACTX-Hvla, K+2-ACTX-Hvla, and ®+2-ACTX-Hvla. [0037] “Alignment” refers to a method of comparing two or more sequences (e.g., nucleotide, polynucleotide, amino acid, peptide, polypeptide, or protein sequences) for the purpose of determining their relationship to each other. Alignments are typically performed by computer programs that apply various algorithms, however, it is also possible to perform an alignment by hand. Alignment programs typically iterate through potential alignments of sequences and score the alignments using substitution tables, employing a variety of strategies to reach a potential optimal alignment score. Commonly-used alignment algorithms include, but are not limited to, CLUSTALW (see Thompson J. D., Higgins D. G., Gibson T. J., CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research 22: 4673-4680, 1994); CLUSTALV (see Larkin M. A., et al., CLUSTALW2, ClustalW and ClustalX version 2, Bioinformatics 23(21): 2947-2948, 2007); Mafft; Kalign; ProbCons; and T-Coffee (see Notredame et al., T-Coffee: A novel method for multiple sequence alignments, Journal of Molecular Biology 302: 205-217, 2000). Exemplary programs that implement one or more of the foregoing algorithms include, but are not limited to, MegAlign from DNAStar (DNAStar, Inc. 3801 Regent St. Madison, Wis. 53705), MUSCLE, T-Coffee, CLUSTALX, CLUSTALV, JalView, Phylip, and Discovery Studio from Accelrys (Accelrys, Inc., 10188 Telesis Ct, Suite 100, San Diego, Calif 92121). In some embodiments, an alignment will introduce “phase shifts” and/or “gaps” into one or both of the sequences being compared in order to maximize the similarity between the two sequences, and scoring refers to the process of quantitatively expressing the relatedness of the aligned sequences.

[0038] “Arachnid” refers to a class of arthropods. For example in some embodiments, arachnid can mean spiders, scorpions, ticks, mites, harvestmen, or solifuges.

[0039] ‘Av2” or “ATX-II” or “neurotoxin 2” or “Anemonia viridis toxin 2” or 6-

AITX-Avdlc” refers to a toxin isolated from the venom of Anemonia sulcata. One example of an Av2 polypeptide is a polypeptide having the amino acid sequence of SEQ ID NO: 457. [0040] “Av3” refers to a polypeptide isolated from the sea anemone, Anemonia viridis, which can target receptor site 3 on a-subunit III of voltage-gated sodium channels. One example of an Av3 polypeptide is an Av3 polypeptide having the amino acid sequence of SEQ ID NO: 453 (NCBI Accession No. P01535.1). [0041] “AVP” or “Av3 variant polypeptides” refers to an Av3 polypeptide sequence and/or a polypeptide encoded by a variant Av3 polynucleotide sequence that has been altered to produce a non-naturally occurring polypeptide and/or polynucleotide sequence.

[0042] “bp” or “base pair” refers to a molecule comprising two chemical bases bonded to one another. For example, a DNA molecule consists of two winding strands, wherein each strand has a backbone made of an alternating deoxyribose and phosphate groups. Attached to each deoxyribose is one of four bases, i.e., adenine (A), cytosine (C), guanine (G), or thymine (T), wherein adenine forms a base pair with thymine, and cytosine forms a base pair with guanine.

[0043] “C-terminal” refers to the free carboxyl group (i.e., -COOH) that is positioned on the terminal end of a polypeptide.

[0044] “Carbon source” refers to a source of energy required by organisms for growth and development. For example, in some embodiments, a carbon source is metabolized by a cell, turning that carbon source into energy. In some embodiments, a carbon source allows an organism to carry out catabolic activities. For example, in some embodiments, a carbon source is a compound which is converted by the primary metabolism of a cell for the generation of energy. The term “sole carbon source,” as used herein, refers to a single carbon source that can be utilized by an organism to achieve catabolism. In some embodiments, the term “sole carbon source” means that, with the exception of the sole carbon source, no other carbon source is available to be used by the organism to achieve catabolism. For example, in some embodiments, a “sole carbon source” can be added to cell culture medium, and, with the exception of this sole carbon source, no other carbon source is present in the medium.

[0045] In yet other embodiments, the term “sole carbon source” means that, with the exception of the sole carbon source, any other carbon source available to be used by the organism to achieve catabolism is present in trivial or insignificant amounts, such that the organism cannot rely on that other carbon source to achieve catabolism. For example, in some embodiments, a “sole carbon source” can be added to cell culture medium, and, with the exception of this sole carbon source, any other carbon source present in the medium does not contribute the organism’s catabolic activities. In some embodiments, the sole carbon source is sorbitol.

[0046] ‘cDNA” or “copy DNA” or “complementary DNA” refers to a molecule that is complementary to a molecule of RNA. In some embodiments, cDNA may be either singlestranded or double-stranded. In some embodiments, cDNA can be a double-stranded DNA synthesized from a single stranded RNA template in a reaction catalyzed by a reverse transcriptase. In yet other embodiments, “cDNA” refers to all nucleic acids that share the arrangement of sequence elements found in native mature mRNA species, where sequence elements are exons and 3’ and 5’ non-coding regions. Normally mRNA species have contiguous exons, with the intervening introns removed by nuclear RNA splicing, to create a continuous open reading frame encoding the protein. In some embodiments, “cDNA” refers to a DNA that is complementary to and derived from an mRNA template. [0047] ‘Cleavable Linker” see Linker.

[0048] “Cloning” refers to the process and/or methods concerning the insertion of a polynucleotide segment (e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide) from one source and recombining it with a polynucleotide segment from another source (e.g., usually a vector, for example, a plasmid) and directing the recombined polynucleotide, or “recombinant DNA” to replicate, usually by transforming the recombined DNA into a bacteria or yeast host.

[0049] “Coding sequence” or “CDS” refers to a polynucleotide or nucleic acid sequence that can be transcribed (e.g., in the case of DNA) or translated (e.g., in the case of mRNA) into a peptide, polypeptide, or protein, when placed under the control of appropriate regulatory sequences and in the presence of the necessary transcriptional and/or translational molecular factors. The boundaries of the coding sequence are determined by a translation start codon at the 5’ (amino) terminus and a translation stop codon at the 3’ (carboxy) terminus. A transcription termination sequence will usually be located 3’ to the coding sequence. In some embodiments, a coding sequence may be flanked on the 5’ and/or 3’ ends by untranslated regions. In some embodiments, a coding sequence can be used to produce a peptide, a polypeptide, or a protein product. In some embodiments, the coding sequence may or may not be fused to another coding sequence or localization signal, such as a nuclear localization signal. In some embodiments, the coding sequence may be cloned into a vector or expression construct, may be integrated into a genome, or may be present as a DNA fragment.

[0050] “Codon optimization” refers to the production of a gene in which one or more endogenous, native, and/or wild-type codons are replaced with codons that ultimately still code for the same amino acid, but that are of preference in the corresponding host.

[0051] “Complementary” refers to the topological compatibility or matching together of interacting surfaces of two polynucleotides as understood by those of skill in the art. Thus, two sequences are “complementary” to one another if they are capable of hybridizing to one another to form a stable anti-parallel, double-stranded nucleic acid structure. A first polynucleotide is complementary to a second polynucleotide if the nucleotide sequence of the first polynucleotide is substantially identical to the nucleotide sequence of the polynucleotide binding partner of the second polynucleotide, or if the first polynucleotide can hybridize to the second polynucleotide under stringent hybridization conditions. Thus, the polynucleotide whose sequence 5'-TATAC-3' is complementary to a polynucleotide whose sequence is 5'- GTATA-3'.

[0052] “Conditioned medium” means the cell culture medium which has been used by cells and is enriched with cell derived materials but does not contain cells.

[0053] “Cone shell” or “cone snails” or “cones” refers to organisms belonging to the Conus genus of predatory marine gastropods. For example, in some embodiments, a cone shell can be one of the following species: Conus amadis; Conus catus; Conus ermineus; Conus geographus; Conus gloriamaris; Conus kinoshitai; Conus magus; Conus marmoreus; Conus purpurascens; Conus stercusmuscarum; Conus striatus; Conus textile; or Conus tulipa.

[0054] “Conotoxin” refers to the toxins isolated from cone shells that act by interfering with neuronal communication. For example, in some embodiments, a conotoxin can be an a-, co-, p-, 6-, or K-conotoxins. Briefly, the a-conotoxins (and aA- &cp-conotoxins) target nicotinic ligand gated channels; co-conotoxins target voltage-gated calcium channels; p-conotoxins target the voltage-gated sodium channels; 6-conotoxins target the voltage-gated sodium channel; and K-conotoxins target the voltage-gated potassium channel.

[0055] “Copy number” refers to the number of copies of a vector, an expression cassette, an amplification unit, a gene, a nucleotide sequence, a polynucleotide, a heterologous polynucleotide, e.g., heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, or indeed any defined nucleic acid sequence, that are present in a recombinant cell at any time. In some embodiments, copy number can refer to the number of copies, wherein the copies are transient and/or stably integrated. For example, in some embodiments, a heterologous polynucleotide, or another defined nucleotide sequence, may be present in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies in a recombinant cell (e.g., stably integrated into the chromosome of the recombinant cell). In other embodiments, a vector containing a heterologous polynucleotide or a nucleotide sequence, may be present in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies in a recombinant cell.

[0056] “CRP” refers to Cysteine Rich Peptide or Cysteine Rich Polypeptide or Cysteine Rich Protein (all used interchangeably). CRPs are peptides rich in cysteine residues that, in some embodiments, are operable to form disulfide bonds between such cysteine residues. In some embodiments, CRPs contain at least four (4), sometimes six (6), and sometimes eight (8) cysteine amino acids among proteins or peptides having at least 10 amino acids where the cysteines form two (2), three (3) or four (4) disulfide bonds. In some embodiments, the disulfide bonds contribute to the folding, three-dimensional structure, stability, and/or activity of a peptide. In some embodiments, the activity can be insecticidal activity. Indeed, in some embodiments, the cysteine-cysteine disulfide bonds, and the three dimensional structure they form, play a significant role in the insecticidal nature of insecticidal peptides.

[0057] In some embodiments, a CRP may or may not comprise a cystine knot. For example, in some embodiments, a CRP can have an inhibitor cystine knot (ICK) motif, a growth factor cystine knot (GFCK) motif, or a cyclic cystine knot (CCK) motif. In some embodiments, a CRP can have an ICK motif. For example, in some embodiments, a CRP with an ICK motif can be an ACTX peptide from a spider; in other embodiments, a CRP without an ICK motif, i.e., a non-ICK CRP, can be a peptide like Av2 and Av3, peptides isolated from sea anemones. Non-ICK CRPS can have 4-8 cysteines which form 2-4 disulfide bonds. These cysteine-cysteine disulfide bonds stabilized toxic peptides (CRPs) can have remarkable stability when exposed to the environment. Many CRPs are isolated from venomous animals such as spiders, scorpions, snakes and sea snails and sea anemones and they are toxic to insects.

[0058] “CRP construct” refers to the three-dimensional arrangement/orientation of peptides, polypeptides, and/or motifs of operably linked polypeptide segments (e.g., a CRP- modified protein). For example, a CRP expression ORF can include one or more of the following components or motifs: a CRP; an endoplasmic reticulum signal peptide (ERSP); a linker peptide (L); a translational stabilizing protein (STA); or any combination thereof. And, as used herein, the term “CRP construct” is used to describe the designation and/or orientation of the structural motif. In other words, the CRP construct describes the arrangement and orientation of the components or motifs contained within a given CRP expression ORF. For example, in some embodiments, a CRP construct describes, without limitation, the orientation of one of the following CRP-modified proteins: ERSP-CRP; ERSP-(CRP)N; ERSP-CRP-L; ERSP-(CRP)N-L; ERSP-(CRP-L)_N; ERSP-L-CRP; ERSP-L- (CRP)N; ERSP-(L-CRP)N; ERSP-STA-CRP; ERSP-STA-(CRP)N; ERSP-CRP-STA; ERSP- (CRP)N-STA; ERSP-(STA-CRP)N; ERSP-(CRP-STA)_N; ERSP-L-CRP-STA; ERSP-L-STA- CRP; ERSP-L-(CRP-STA)_N; ERSP-L-(STA-CRP)N; ERSP-L-(CRP)N-STA; ERSP-(L- CRP)N-STA; ERSP-(L-STA-CRP)N; ERSP-(L-CRP-STA)_N; ERSP-(L-STA)_N-CRP; ERSP- (L-CRP)N-STA; ERSP-STA-L-CRP; ERSP-STA-CRP-L; ERSP-STA-L-(CRP)N; ERSP- (STA-L)N-CRP; ERSP-STA-(L-CRP)N; ERSP-(STA-L-CRP)N; ERSP-STA-(CRP)N-L; ERSP-STA-(CRP-L)_N; ERSP-(STA-CRP)N-L; ERSP-(STA-CRP-L)_N; ERSP-CRP-L-STA; ERSP-CRP-STA-L; ERSP-(CRP)N-STA-L ERSP-(CRP-L)_N-STA; ERSP-(CRP-STA)_N-L; ERSP-(CRP-L-STA)N; or ERSP-(CRP-STA-L)N; wherein N is an integer ranging from 1 to 200. See also “Structural motif.”

[0059] “CRP ORF diagram” refers to the composition of one or more CRP ORFs, as written out in diagram or equation form. For example, a “CRP ORF diagram” can be written out as using acronyms or short-hand references to the DNA segments contained within the ORF. Accordingly, in one example, a “CRP ORF diagram” may describe the polynucleotide segments encoding the ERSP, L, STA, and CRP, by diagramming in equation form the DNA segments as “ersp” (i.e. , the polynucleotide sequence that encodes the ERSP polypeptide); "linker" or “Z” (i.e., the polynucleotide sequence that encodes the LINKER polypeptide); “sta” (i.e., the polynucleotide sequence that encodes the STA polypeptide), and "crp" (i.e., the polynucleotide sequence encoding a CRP), respectively. An example of a CRP ORF diagram is "ersp-sta-(linker crpp _i." or “ersp-(crpj-linkeri)N-sta” and/or any combination of the DNA segments thereof.

[0060] “CRP polynucleotide” refers to a polynucleotide or group of polynucleotides operable to express and/or encode an insecticidal protein comprising one or more CRPs in addition to one or more non-CRP polypeptides or proteins.

[0061] “CRP -modified protein” refers to any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, consisting of: (1) at least one CRP, or two or more CRPs; and (2) additional peptides, polypeptides, or proteins, wherein said additional peptides, polypeptides, or proteins have the ability to do one or more of the following: (a) increase the activity of CRP-modified protein, relative to a CRP alone (e.g., increase the mortality and/or inhibit the growth of insects when the insects are exposed to a CRP-modified protein, relative to a CRP alone); (b) increase the expression of said CRP-modified protein, e.g., in a host cell or an expression system; and/or (c) affect the post-translational processing of the CRP-modified protein. [0062] In some embodiments, a protein can comprise a one or more CRPs as disclosed herein. In some embodiments, a CRP -modified protein can be a polymer comprising two or more CRPs. In some embodiments, the insecticidal protein can comprise a CRP homopolymer, e.g., two or more CRP monomers that are the same CRP. In some embodiments, the protein can comprise a CRP heteropolymer, e.g., two or more CRP monomers, wherein the CRP monomers are different.

[0063] In some embodiments, a CRP-modified protein can be a polymer of amino acids that when properly folded or in its most natural thermodynamic state exerts an insecticidal activity against one or more insects.

[0064] In some embodiments, a CRP-modified protein can be a polymer comprising two or more CRPs, wherein the CRPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker. In some embodiments, a CRP-modified protein can refer to a one or more CRPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and/or any other combination thereof. In some embodiments, a CRP-modified protein can be a non-naturally occurring protein comprising (1) a wild-type CRP protein; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.

[0065] “Culture” or “cell culture” refers to the maintenance of cells in an artificial, in vitro environment.

[0066] “Culturing” refers to the propagation of organisms on or in various kinds of media. For example, the term “culturing” can mean growing a population of cells under suitable conditions in a liquid or solid medium. In some embodiments, culturing refers to fermentative recombinant production of a heterologous polypeptide of interest and/or other desired end products (typically in a vessel or reactor). In some embodiments, culturing refers to fermentative recombinant production of a heterologous polypeptide in a medium containing a sole carbon source (e.g., sorbitol).

[0067] “Cystine” refers to an oxidized cysteine-dimer. Cystines are sulfur-containing amino acids obtained via the oxidation of two cysteine molecules, and are linked with a disulfide bond.

[0068] “Defined medium” or “DM” refers to a medium that is composed of known chemical components but does not contain crude proteinaceous extracts or by-products such as yeast extract or peptone. [0069] “Degeneracy” or “codon degeneracy” refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein or polypeptide can vary due to degeneracies. As a result of the degeneracy of the genetic code, many nucleic acid sequences can encode a given polypeptide with a particular activity; such functionally equivalent variants are contemplated herein.

[0070] “Derived” or “derived from” refers to obtaining a peptide, polypeptide, protein or polynucleotide from a known and/or originating peptide, polypeptide, protein or polynucleotide. Thus, as used herein, the term “derived from” encompasses, without limitation: a protein or polynucleotide that is isolated or obtained directly from an originating source (e.g. an organism, such as a one or more species belonging to the Atracidae family); a synthetic or recombinantly generated protein or polynucleotide that is identical, substantially related to, or modified from, a protein or polynucleotide from an known/originating source; or protein or polynucleotide that is made from a protein or polynucleotide of an known/originating source or a fragment thereof. The term “substantially related”, as used herein, means that the protein may have been modified by chemical, physical or other means (e.g. sequence modification).

[0071] Accordingly, “derived” can refer to either directly or indirectly obtaining a protein or polynucleotide from a known and/or originating protein or polynucleotide. For example, in some embodiments, “derived” can refer to obtaining a protein or polynucleotide from a known and/or originating protein or polynucleotide by looking at the sequence of a known/originating protein or polynucleotide and preparing a protein or polynucleotide having a sequence similar, at least in part, to the sequence of the known and/or originating protein or polynucleotide. In yet other embodiments, “derived” can refer to obtaining a protein or polynucleotide from a known and/or originating protein or polynucleotide by isolating a protein or polynucleotide from an organism that is related to a known protein or polynucleotide. Other methods of “deriving” a protein or polynucleotide from a known protein or polynucleotide are known to one of skill in the art.

[0072] In some embodiments, “derived” in the context of a protein (e.g., “a protein derived from an organism”) describes a condition wherein said protein was originally identified in an organism, and has been reproduced therefrom via isolation from the organism, or through synthetic or recombinant means.

[0073] “Disulfide bond” means a covalent bond between two cysteine amino acids derived by the coupling of two thiol groups on their side chains. [0074] “DNA” refers to deoxyribonucleic acid, comprising a polymer of one or more deoxyribonucleotides or nucleotides (i.e., adenine [A], guanine [G], thymine [T], or cytosine [C]), which can be arranged in single-stranded or double-stranded form. For example, one or more nucleotides creates a polynucleotide.

[0075] “dNTPs” refers to the nucleoside triphosphates that compose DNA and RNA.

[0076] “Double expression cassette” refers to two heterologous polypeptide expression cassettes contained on the same vector.

[0077] “Double transgene peptide expression vector” or “double transgene expression vector” means a yeast expression vector that contains two copies of the heterologous polypeptide expression cassette.

[0078] “Endogenous” refers to a polynucleotide, peptide, polypeptide, protein, or process that naturally occurs and/or exists in an organism, e.g., a molecule or activity that is already present in the host cell before a particular genetic manipulation.

[0079] “ER” or “Endoplasmic reticulum” is a subcellular organelle common to all eukaryotes where some post translation modification processes occur.

[0080] “ERSP” or “endoplasmic reticulum signal peptide” is an N-terminus sequence of amino acids that — during protein translation of the mRNA molecule encoding a CRP — is recognized and bound by a host cell signal-recognition particle, which moves the protein translation ribosome/mRNA complex to the ER in the cytoplasm. The result is the protein translation is paused until it docks with the ER where it continues and the resulting protein is injected into the ER.

[0081] “ersp” refers to a polynucleotide encoding the peptide, ERSP.

[0082] “ER trafficking” means transportation of a cell expressed protein into ER for post-translational modification, sorting and transportation.

[0083] “Expression cassette” refers to all the DNA elements necessary to complete transcription of a transgene — e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide — in a recombinant expression system. Thus, in some embodiments, an “expression cassette” refers to a (1) a DNA sequence of interest, e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and one or more of the following: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements. The combination (1) with at least one of (2)-(6) is called an “expression cassette.” In some embodiments,

[0084] For example, in some embodiments, an expression cassette can be (1) a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and further comprising one or more: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements.

[0085] In some embodiments, an expression cassette can be (1) a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and further comprising one or more: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements; wherein each of the (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, further comprises one or more of (2)-(6).

[0086] For example, in some embodiments, an expression cassette can refer to (l)(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); further comprising one or more: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements. In other embodiments, an expression cassette can refer to (1 )(ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and further comprising one or more: (2) promoters, terminators, and/or enhancer elements; (3) an appropriate mRNA stabilizing polyadenylation signal; (4) an internal ribosome entry site (IRES); (5) introns; and/or (6) post-transcriptional regulatory elements.

[0087] In some embodiments, a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can comprise one or more expression cassettes. [0088] In some embodiments, there can be numerous expression cassettes cloned into a vector. For example, in some embodiments, there can be a first expression cassette comprising a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[0089] In alternative embodiments, there are two expression cassettes, each comprising a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide (i.e., a double expression cassette).

[0090] In yet other embodiments, there are two expression cassettes, wherein the first expression cassette comprises (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and the second expression cassette comprises (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[0091] In other embodiments, there are three expression cassettes operable to encode a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide (i.e., a triple expression cassette).

[0092] In some embodiments, a double expression cassette can be generated by subcloning a second expression cassette into a vector containing a first expression cassette. In some embodiments, a triple expression cassette can be generated by subcloning a third expression cassette into a vector containing a first and a second expression cassette. Methods concerning expression cassettes and cloning techniques are well-known in the art and described herein.

[0093] “Expression ORF” means a nucleotide sequence encoding a protein complex and is defined as the nucleotides in the ORF.

[0094] “Fermentation beer” refers to spent fermentation medium, i.e., fermentation medium supernatant after removal of organisms, that has been inoculated with and consumed by a transformed host cell (e.g., a yeast cell operable to express a heterologous SDH and a heterologous polypeptide (e.g., a CRP). of the present disclosure). In some embodiments, fermentation beer refers to the solution that is recovered following the fermentation of the transformed host cell. The term “fermentation” refers broadly to the enzymatic and anaerobic or aerobic breakdown of organic substances (e.g., a carbon substrate) nutrient substances by microorganisms under controlled conditions (e.g., temperature, oxygen, pH, nutrients, and the like) to produce fermentation products (e.g., one or more peptides of the present disclosure). While fermentation typically describes processes that occur under anaerobic conditions, as used herein it is not intended that the term be solely limited to strict anaerobic conditions, as the term “fermentation” used herein may also occur processes that occur in the presence of oxygen.

[0095] “Fermentation solid(s)” refers to solids (including dissolved) that remain from fermentation beer during the yeast-based fermentation process, and consists essentially of salts, complex protein source, vitamins, and additional yeast byproducts having a molecular weight cutoff of from about 200 kDa to about 1 kDa.

[0096] “Generation,” as used in the context of cells, refers to a doubling of a cell or population of cells (e.g., through cell division). Thus, a cell that has divided one time has gone through one generation; likewise, a population of cells that have divided one time have gone through one generation. In some embodiments, the term “generation” can refer to a population of cells at a given time point after a specific numbering of doublings. For example, after cells in a population have divided 10 times, these cells can be described as the cells after 10 generations. Methods of measuring generations of cells are well known in the art. For example, in some embodiments, a generation can be measured using a doubling of optical density (OD). In other embodiments, a generation can be measured based on the doubling of cell biomass (e.g., yeast biomass). Exemplary methods of measuring yeast generations, exponential growth, and non-exponential growth of yeast cells, are provided in U.S. Patent Application Publication No. US20150252319A1, the disclosure of which is incorporated herein by reference in its entirety.

[0097] ‘GFP” means a green fluorescent protein from the jellyfish, Aequorea victoria.

[0098] “Growing” when used in the context of a cell or population of cells (e.g., recombinant yeast cell in culture), refers to the propagation of said cell or population of cells on or in various kinds of media. For example, the term “growing” can refer to the propagation of a population of cells under suitable conditions in a liquid or solid medium.

[0099] “Heterologous” refers to a polynucleotide, peptide, polypeptide, protein, or process that does not naturally occur and/or exist in an organism, e.g., a molecule or activity that is not present in the host cell before a particular genetic manipulation. For example, a heterologous polynucleotide may a nucleic acid sequence that is not endogenous to the cell or part of the native genome in which it is present (e.g., a heterologous sorbitol dehydrogenase), and has been added to the cell by infection, transfection, microinjection, electroporation, microprojection, or the like. In some embodiments, a heterologous polynucleotide includes two or more distinct nucleotide sequences encoding two or more polypeptides and/or proteins that are foreign or not present in a host cell before manipulation.

[00100] “HIS” or “His” refers to histidine. For example, in some embodiments, “HIS” or His” may refer to a histidine tag, e.g., a histidine tag having an amino acid sequence as set forth in SEQ ID NO: 470.

[00101] “Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared *100. Thus, in some embodiments, the term “homologous” refers to the sequence similarity between two polypeptide molecules, or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology.

[00102] The term “homology,” when used in relation to nucleic acids, refers to a degree of complementarity. There may be partial homology, or complete homology and thus identical. “Sequence identity” refers to a measure of relatedness between two or more nucleic acids, and is given as a percentage with reference to the total comparison length. The identity calculation takes into account those nucleotide residues that are identical and in the same relative positions in their respective larger sequences.

[00103] “Homologous recombination” refers to the event of substitution of a segment of DNA by another one that possesses identical regions (homologous) or nearly so. For example, in some embodiments, “homologous recombination” refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA. Briefly, homologous recombination is most widely used by cells to accurately repair harmful breaks that occur on both strands of DNA, known as double-strand breaks. Although homologous recombination varies widely among different organisms and cell types, most forms involve the same basic steps: after a double-strand break occurs, sections of DNA around the 5' ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the broken DNA molecule then “invades” a similar or identical DNA molecule that is not broken. After strand invasion, the further sequence of events may follow either of two main pathways, i.e., the doublestrand break repair pathway, or the synthesis-dependent strand annealing pathway. Homologous recombination is conserved across all three domains of life as well as viruses, suggesting that it is a nearly universal biological mechanism. For example, in some embodiments, homologous recombination can occur using a site-specific integration (SSI) sequence, whereby there is a strand exchange crossover event between nucleic acid sequences substantially similar in nucleotide composition. These crossover events can take place between sequences contained in the targeting construct of the invention (i.e., the SSI sequence) and endogenous genomic nucleic acid sequences (e.g., the polynucleotide encoding the peptide subunit). In addition, in some embodiments, it is possible that more than one site-specific homologous recombination event can occur, which would result in a replacement event in which nucleic acid sequences contained within the targeting construct have replaced specific sequences present within the endogenous genomic sequences.

[00104] “Host cell” refers to a cell that is suitable for receiving and/or producing a heterologous polynucleotide or protein,

[00105] “ICK motif’ or “ICK motif protein” or “inhibitor cystine knot motif’ or “ICK peptides” or “cystine knot motif’ or “cystine knot peptides” refers to a 16 to 60 amino acid peptide with at least 6 half-cystine core amino acids having three disulfide bridges, wherein the 3 disulfide bridges are covalent bonds and of the six half-cystine residues the covalent disulfide bonds are between the first and fourth, the second and fifth, and the third and sixth half-cystines, of the six core half-cystine amino acids starting from the N-terminal amino acid. In general this type of peptide comprises a beta-hairpin secondary structure, normally composed of residues situated between the fourth and sixth core half-cystines of the motif, the hairpin being stabilized by the structural crosslinking provided by the moti s three disulfide bonds. Note that additional cysteine/cy stine or half-cystine amino acids may be present within the inhibitor cystine knot motif.

[00106] “ic ’ means a nucleotide encoding an ICK motif protein.

[00107] “Identity” refers to a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, as determined by comparing said sequences. The term “identity” also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. “Identity” and “similarity” can be readily calculated by any one of the myriad methods known to those having ordinary skill in the art, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994:, Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), the disclosures of which are incorporated herein by reference in their entireties. Furthermore, methods to determine identity and similarity are codified in publicly available computer programs. For example in some embodiments, methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990), the disclosures of which are incorporated herein by reference in their entireties.

[00108] “IGER” means a name for a short peptide, based on its actual sequence of one letter codes. It is an example of an intervening linker.

[00109] “in vivo” refers to the natural environment (e.g., an animal or a cell) and to processes or reactions that occur within a natural environment.

[00110] “Inactive” or “inactivation” refers to a condition wherein something is not in a state of use, e.g., lying dormant and/or not working. For example, when used in the context of a gene or when referring to a gene, the term inactive means said gene is no longer actively synthesizing a gene product, having said gene product translated into a protein, or otherwise having the gene perform its normal function. For example, in some embodiments, the term inactive can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes. “Partial inactivation” or “partially inactivated” refers to a condition wherein something is less than its normal state of use. For example, when used in the context of a gene or when referring to a gene, the term partially inactive means said gene exhibits less than normal synthetic levels of a gene product or otherwise having the gene perform less than its normal function.

[00111] “Increasing” or “increase” or “increased” refers to making something more or greater in size, amount, intensity, degree, or combination thereof (e.g., an amount of proteins expressed or a level of protein expression). As used herein, the term “increasing” or “increased” when used in the context of gene or protein expression, e.g., “increased expression” or “increased level of expression” refers to an increased capacity for a recombinant yeast cell to express a heterologous polypeptide (e.g., a CRP), e.g., when grown in a medium having sorbitol as the sole carbon source, relative to a recombinant yeast cell grown in a medium having a sole carbon source that is not sorbitol.

[00112] In some embodiments, a recombinant yeast cell of the present disclosure expresses an increased amount of a heterologous polypeptide (e.g., a CRP) when grown in a medium having sorbitol as the sole carbon source, relative to a recombinant yeast cell of the present disclosure when grown in a medium having a sole carbon source that is not sorbitol, in the same amount of time.

[00113] In some embodiments, the capacity for a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide (wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated), to express the heterologous polypeptide (e.g., a CRP) when grown in a medium having sorbitol as the sole carbon source — results in the following effect: an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00114] For example, in some embodiments, the term “increasing” includes any measurable increase of a level of expression of a heterologous polypeptide, e.g., there may be an increase of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more, of a level of expression of a heterologous polypeptide in a recombinant yeast cell, when growing the recombinant yeast cell in a medium comprising the sole carbon source that is sorbitol, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. Thus, in some embodiments, the term “increasing” e.g., when used in the phrase “increasing expression of a heterologous polypeptide,” refers to an increase in a level of expression of a heterologous polypeptide that is expressed by a recombinant yeast cell when growing the recombinant yeast cell in a medium comprising the sole carbon source that is sorbitol, wherein the increase of the level of expression of the heterologous polypeptide is at least about 0. 1%, at least about 0.2%, at least about 0.3%, at least about 0.4%, at least about 0.5%, at least about 0.6%, at least about 0.7%, at least about 0.8%, at least about 0.9%, at least about 1%, at least about 1.25%, at least about 1.5%, at least about 1.75%, at least about 2%, at least about 2.25%, at least about 2.5%, at least about 2.75%, at least about 3%, at least about 3.25%, at least about 3.5%, at least about 3.75%, at least about 4%, at least about 4.25%, at least about 4.5%, at least about 4.75%, at least about 5%, at least about 5.25%, at least about 5.5%, at least about 5.75%, at least about 6%, at least about 6.25%, at least about 6.5%, at least about 6.75%, at least about 7%, at least about 7.25%, at least about 7.5%, at least about 7.75%, at least about 8%, at least about 8.25%, at least about 8.5%, at least about 8.75%, at least about 9%, at least about 9.25%, at least about 9.5%, at least about 9.75%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, or a greater than 100%, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00115] In other embodiments, the term “increased expression” describes a reduction in the variation of protein expression levels across multiple independent cultures of recombinant yeast cells of the present disclosure, grown in media having sorbitol as the sole carbon source, relative to multiple independent cultures of recombinant yeast cells of the present disclosure grown in media having a sole carbon source that is not sorbitol; resulting in an overall higher mean level of expression across the cultures of the recombinant yeast cells of the present disclosure grown in media having sorbitol as the sole carbon source. Thus, in some embodiments, stochastic variation may result in any one culture of the recombinant yeast cells of the present disclosure not exhibiting higher peptide titers, but on average, the level of protein expression of a heterologous polypeptide will be increased when multiple independent assessments are made.

[00116] In yet other embodiments, “increased expression” refers to an increased level of expression of the heterologous polypeptide of the present disclosure, which results from the maintenance of multiple numbers of copies (e.g., two or more) of the heterologous polynucleotides of the present disclosure in a recombinant yeast cell. Here, maintaining the multiple copies of the heterologous polynucleotides of the present disclosure reduces the occurrence of stochastic copy loss events (e.g., out-recombination), affecting one or more copies of the heterologous polynucleotides and/or one or more copies nucleotide sequences operable to encode the heterologous polypeptide, wherein such stochastic copy loss events have a deleterious effect on heterologous polypeptide expression and/or yield.

[00117] “Inoperable” refers to the condition of a thing not functioning, malfunctioning, or no longer able to function. For example, when used in the context of a gene or when referring to a gene, the term inoperable means said gene is no longer able to operate as it normally would, either permanently or transiently. For example, “inoperable,” in some embodiments, means that a gene is no longer able to synthesize a gene product, having said gene product translated into a protein, or is otherwise unable to gene perform its normal function. For example, in some embodiments, the term inoperable can refer the failure of a gene to transcribe RNA, a failure of RNA processing (e.g., pre-mRNA processing; RNA splicing; or other post-transcriptional modifications); interference with non-coding RNA maturation; interference with RNA export (e.g., from the nucleus to the cytoplasm); interference with translation; protein folding; translocation; protein transport; and/or inhibition and/or interference with any of the molecules polynucleotides, peptides, polypeptides, proteins, transcription factors, regulators, inhibitors, or other factors that take part in any of the aforementioned processes.

[00118] “Integrative expression vector” or “integrative vector” means a yeast expression vector which can insert itself into a specific locus of the yeast cell genome and stably becomes a part of the yeast genome.

[00119] “Intervening linker” refers to a short peptide sequence in the protein separating different parts of the protein, or a short DNA sequence that is placed in the reading frame in the ORF to separate the upstream and downstream DNA sequences. For example, in some embodiments, an intervening linker may be used allowing proteins to achieve their independent secondary and tertiary structure formation during translation.

[00120] “Isolated” refers to separating a thing and/or a component from its natural environment, e.g., a toxin isolated from a given genus or species means that toxin is separated from its natural environment, e.g., taken out of a WT organism.

[00121] “Kappa-ACTX peptide” refers to an excitatory toxin that inhibits insect calcium-activated potassium (KCa) channels (Slo-type). As used herein, “Kappa-ACTX peptide” can refer to peptides isolated from the Australian Blue Mountains Funnel-web Spider, Hadronyche versuta, or variants thereof.

[00122] “kb” refers to kilobase, i.e., 1000 bases. As used herein, the term “kb” means a length of nucleic acid molecules. For example, 1 kb refers to a nucleic acid molecule that is 1000 nucleotides long. A length of double-stranded DNA that is 1 kb long, contains two thousand nucleotides (i.e., one thousand on each strand). Alternatively, a length of singlestranded RNA that is 1 kb long, contains one thousand nucleotides.

[00123] “kDa” refers to kilodalton, a unit equaling 1,000 daltons; a “dalton” or “Da” is a unit of molecular weight (MW).

[00124] ‘Knock in” or “knock-in” or “knocks-in” or “knocking-in” refers to the replacement of an endogenous gene with an exogenous or heterologous gene, or part thereof. For example, in some embodiments, the term “knock-in” refers to the introduction of a nucleic acid sequence encoding a desired protein to a target gene locus by homologous recombination, thereby causing the expression of the desired protein. In some embodiments, a “knock-in” mutation can introduce an exogenous or heterologous sorbitol dehydrogenase (SDH) gene. In some embodiments, a “knock-in” mutation can modify a gene sequence to create a loss-of-function or gain-of-function mutation, e.g., incorporation of an exogenous or heterologous SDH gene. The term “knock-in” can refer to the procedure by which an exogenous or heterologous polynucleotide sequence or fragment thereof is introduced into the genome (e.g., “they performed a knock-in” or “they knocked-in the heterologous gene”), or the resulting cell and/or organism (e.g., “the cell is a “knock-in” or “the animal is a “knock-in”).

[00125] ‘Knock out” or “knockout” or “knock-out” or “knocks-out” or “knocking-out” refers to a partial or complete suppression of the expression gene product (e.g., mRNA) of a protein encoded by an endogenous DNA sequence in a cell. In some embodiments, the “knock-out” can be effectuated by targeted deletion of a whole gene, or part of a gene encoding a peptide, polypeptide, or protein. As a result, the deletion may render a gene inactive, partially inactive, inoperable, partly inoperable, or otherwise reduce the expression of the gene or its products in any cell in the whole organism and/or cell in which it is normally expressed. The term “knock-out” can refer to the procedure by which an endogenous gene is made completely or partially inactive or inoperable (e.g., “they performed a knock-out” or “they knocked-out the endogenous gene”), or the resulting cell and/or organism (e.g., “ the cell is a “knock-out” or “the animal is a “knock-out”). In some embodiments, the cells are partially inactivated via knock-out of an endogenous SDH gene. [00126] “/” or “linker” refers to a nucleotide encoding linker peptide.

[00127] “L” in the proper context refers to a linker peptide, which links a translational stabilizing protein (STA) with an additional polypeptide, e.g., a heterologous peptide, and/or multiple heterologous peptides. When referring to amino acids, “L” can also mean leucine. [00128] ‘LAC4 promoter” or “pLAC4” or “Lac4 promoter” refers to a DNA segment comprising the promoter sequence derived from the K. lactis [3-galactosidase gene. The LAC4 promoters is strong and inducible reporter that is used to drive expression of exogenous genes transformed into yeast.

[00129] ‘LAC4 terminator” or “Lac4 terminator” refers to a DNA segment comprising the transcriptional terminator sequence derived from the K. lactis [3-galactosidase gene.

[00130] “Lid” or “lid,” as used herein, is an abbreviation for L-iditol 2-dehydrogenase, otherwise known as sorbitol dehydrogenase (SDH). The terms “lid”; “sorbitol dehydrogenase”; “SorDH” and “SDH” are used interchangeably.

[00131] “Linker” or “LINKER” or “peptide linker” or “L” or “intervening linker” refers to a short peptide sequence operable to link two peptides together. Linker can also refer to a short DNA sequence that is placed in the reading frame of an ORF to separate an upstream and downstream DNA sequences. In some embodiments, a linker can be cleavable by an insect protease. In some embodiments, a linker may allow proteins to achieve their independent secondary and tertiary structure formation during translation. In some embodiments, the linker can be either resistant or susceptible to cleavage in plant cellular environments, in the insect and/or lepidopteran gut environment, and/or in the insect hemolymph and lepidopteran hemolymph environment. In some embodiments, a linker can be cleaved by a protease, e.g., in some embodiments, a linker can be cleaved by a plant protease (e.g., papain, bromelain, ficin, actinidin, zingibain, and/or cardosins), an insect protease, a fungal protease, a vertebrate protease, an invertebrate protease, a bacteria protease, a mammal protease, a reptile protease, or an avian protease. In some embodiments, a linker can be cleavable or non-cleavable. In some embodiments, a linker comprises a binary or tertiary region, wherein each region is cleavable by at least two types of proteases: one of which is an insect and/or nematode protease and the other one of which is a human protease. In some embodiments, a linker can have one of (at least) three roles: to cleave in the insect gut environment, to cleave in the plant cell, or to be designed not to intentionally cleave. [00132] “Medium” (plural “media”) refers to a nutritive solution for culturing cells in cell culture. In some embodiments, the medium contains a sole carbon source (e.g., sorbitol). [00133] ‘MO A” refers to mechanism of action.

[00134] “Molecular weight (MW)” refers to the mass or weight of a molecule, and is typically measured in “daltons (Da)” or kilodaltons (kDa). In some embodiments, MW can be calculated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), analytical ultracentrifugation, or light scattering. In some embodiments, the SDS-PAGE method is as follows: the sample of interest is separated on a gel with a set of molecular weight standards. The sample is run, and the gel is then processed with a desired stain, followed by destaining for about 2 to 14 hours. The next step is to determine the relative migration distance (Rf) of the standards and protein of interest. The migration distance can be determined using the following equation:

Migration distance of the protein

Migration distance of the dye front Formula (III)

[00135] Next, the logarithm of the MW can be determined based on the values obtained for the bands in the standard; e.g., in some embodiments, the logarithm of the molecular weight of an SDS-denatured polypeptide and its relative migration distance (Rf) is plotted into a graph. After plotting the graph, interpolating the value derived will provide the molecular weight of the unknown protein band. [00136] “Motif’ refers to a polynucleotide or polypeptide sequence that is implicated in having some biological significance and/or exerts some effect or is involved in some biological process.

[00137] “Multiple cloning site” or “MCS” refers to a segment of DNA found on a vector that contains numerous restriction sites in which a DNA sequence of interest can be inserted.

[00138] “Mutant” refers to an organism, DNA sequence, amino acid sequence, peptide, polypeptide, or protein, that has an alteration or variation (for example, in the nucleotide sequence or the amino acid sequence), which causes said organism and/or sequence to be different from the naturally occurring or wild-type organism, wild-type sequence, and/or reference sequence with which the mutant is being compared. In some embodiments, this alteration or variation can be one or more nucleotide and/or amino acid substitutions or modifications (e.g., deletion or addition). In some embodiments, the one or more amino acid substitutions or modifications can be conservative; here, such a conservative amino acid substitution and/or modification in a “mutant” does not substantially diminish the activity of the mutant in relation to its non-mutant form. For example, in some embodiments, a “mutant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and/or claimed sequence, as indicated by a SEQ ID NO.

[00139] “N-terminal” refers to the free amine group (i.e. , -NH2) that is positioned on beginning or start of a polypeptide.

[00140] “NCBI” refers to the National Center for Biotechnology Information. [00141] ‘nm” refers to nanometers.

[00142] “Non-ICK CRPS” refers to peptides having 4-8 cysteines which form 2-4 disulfide bonds. Non-ICK peptides include cystine knot peptides that are not ICK peptides. Non-ICK peptides may have different disulfide bond connectivity patterns than ICKs. Examples of a Non-ICK CRP are peptides like Av2 and Av3, isolated from sea anemones; these anemone peptides are examples of a class of compounds that modulate sodium channels in the insect peripheral nervous system (PNS).

[00143] “Non-Polar amino acid” is an amino acid that is weakly hydrophobic and includes glycine, alanine, proline, valine, leucine, isoleucine, phenylalanine and methionine. Glycine or gly is the most preferred non-polar amino acid for the dipeptides of this invention. [00144] “Normalized peptide yield” means the peptide yield in the conditioned medium divided by the corresponding cell density at the point the peptide yield is measured. The peptide yield can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg/L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec. The cell density can be represented by visible light absorbance of the culture at wavelength of 600 nm (OD600). [00145] “OD” refers to optical density. Typically, OD is measured using a spectrophotometer. When measuring growth over time of a cell population, OD600 is preferable to UV spectroscopy; this is because at a 600 nm wavelength, the cells will not be harmed as they would under too much UV light.

[00146] “OD660nm” or “ODeeonm” refers to optical densities at 660 nanometers (nm).

[00147] “Omega peptide” or “omega toxin,” or “omega-ACTX-Hvla,” or “native omegaACTX-Hvla” all refer to an ACTX peptide which was first isolated from a spider known as the Australian Blue Mountains Funnel-web Spider, Hadronyche versuta. Omega peptide is a positive allosteric modulators of the nicotinic acetylcholine receptor, and may also be a dual antagonist to insect voltage-gated Ca²⁺ channels and voltage-gated K⁺ channels. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett. 2019 Jun; 593(12): 1336-1350; and Windley et al., Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors. Neuropharmacology. 2017 Dec; 127:224-242, the disclosures of which are incorporated herein by reference in their entireties.

[00148] “One letter code” means the peptide sequence which is listed in its one letter code to distinguish the various amino acids in the primary structure of a protein: alanine=A, arginine=R, asparagine=N, aspartic acid=D, asparagine or aspartic acid=B, cysteine=C, glutamic acid=E, glutamine=Q, glutamine or glutamic acid=Z, glycine=G, histidine=H, isoleucine=I, leucine=L, lysine=K, methionine=M, phenylalanine=F, proline=P, serine=S, threonine=T, tryptophan=W, tyrosine=Y, and valine=V.

[00149] “Operable” refers to the ability to be used, the ability to do something, and/or the ability to accomplish some function or result. For example, in some embodiments, “operable” refers to the ability of a polynucleotide, DNA sequence, RNA sequence, or other nucleotide sequence or gene to encode a peptide, polypeptide, and/or protein. For example, in some embodiments, a polynucleotide may be operable to encode a protein, which means that the polynucleotide contains information that imbues it with the ability to create a protein (e.g., by transcribing mRNA, which is in turn translated to protein).

[00150] “Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For example, in some embodiments, operably linked can refer to two or more DNA, peptide, or polypeptide sequences. In other embodiments, operably linked can mean that the two adjacent DNA sequences are placed together such that the transcriptional activation of one DNA sequence can act on the other DNA sequence. In yet other embodiments, the term “operably linked” can refer to two or more peptides and/or polypeptides, wherein said two or more peptides and/or polypeptides are connected in such a way as to yield a single polypeptide chain; alternatively, the term operably linked can refer to two or more peptides that are connected in such a way that one peptide exerts some effect on the other. In yet other embodiments, operably linked can refer to two adjacent DNA sequences are placed together such that the transcriptional activation of one can act on the other.

[00151] ‘ORF” or “open reading frame” refers to a length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG, respectively) and any one or more of the known termination codons, which encodes one or more polypeptide sequences. Put another way, the ORF describes the frame of reference as seen from the point of view of a ribosome translating the RNA code, insofar that the ribosome is able to keep reading (i.e., adding amino acids to the nascent protein) because it has not encountered a stop codon. Thus, “open reading frame” or “ORF” refers to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. Here, the terms “initiation codon” and “termination codon” refer to a unit of three adjacent nucleotides (i.e., a codon) in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[00152] In some embodiments, an ORF is a continuous stretch of codons that begins with a start codon (usually ATG for DNA, and AUG for RNA) and ends at a stop codon (usually UAA, UAG or UGA). In other embodiments, an ORF can be length of RNA or DNA sequence, between a translation start signal (e.g., AUG or ATG) and any one or more of the known termination codons, wherein said length of RNA or DNA sequence encodes one or more polypeptide sequences. In some other embodiments, an ORF can be a DNA sequence encoding a protein which begins with an ATG start codon and ends with a TGA, TAA or TAG stop codon. ORF can also mean the translated protein that the DNA encodes. Generally, those having ordinary skill in the art distinguish the terms “open reading frame” and “ORF,” from the term “coding sequence,” based upon the fact that the broadest definition of “open reading frame” simply contemplates a series of codons that does not contain a stop codon. Accordingly, while an ORF may contain introns, the coding sequence is distinguished by referring to those nucleotides (e.g., concatenated exons) that can be divided into codons that are actually translated into amino acids by the ribosomal translation machinery (i.e., a coding sequence does not contain introns); however, as used herein, the terms “coding sequence”; “CDS”; “open reading frame”; and “ORF,” are used interchangeably.

[00153] “Out-recombined” or “out-recombination” refers to the removal of a gene and/or polynucleotide sequence (e.g., an endogenous gene, a transgene, a heterologous polynucleotide, etc.) that is flanked by two site-specific recombination sites (e.g., the 5’- and 3’ - nucleotide sequence of a target gene that is homologous to the homology arms of a target vector) during in vivo homologous recombination. In some embodiments, the term “out- recombined” refers to the process wherein an endogenous gene is removed, e.g., during homologous recombination. In other embodiments, the term “out-recombined” refers to the process wherein a heterologous polynucleotide is removed via molecular mechanisms intrinsic to the host cell.

[00154] “Peptide yield” or “protein yield” refers to the peptide concentration (e.g., the concentration of a heterologous sorbitol dehydrogenase (SDH) and/or a heterologous polypeptide (e.g., a CRP) in the conditioned medium, which is produced from the recombinant cells of the present disclosure. In some embodiments, peptide yield can be represented by the mass of the produced peptide in a unit of volume, for example, mg per liter or mg/L, or by the UV absorbance peak area of the produced peptide in the HPLC chromatograph, for example, mAu.sec.

[00155] “Plasmid” refers to a DNA segment that acts as a carrier for a gene of interest, and, when transformed or transfected into an organism, can replicate and express the DNA sequence contained within the plasmid independently of the host organism. Plasmids are a type of vector, and can be “cloning vectors” (i.e., simple plasmids used to clone a DNA fragment and/or select a host population carrying the plasmid via some selection indicator) or “expression plasmids” (i.e., plasmids used to produce large amounts of polynucleotides and/or polypeptides).

[00156] “Pichia pastoris ” or “ . pastoris" refers to a species of yeast. Those having ordinary skill in the art will recognize that phylogenetic analysis and genome sequencing of P. pastoris has resulted in its being reassigned to the genus Komagataella, with the species split into the three species: Komagataella phaffli, Komagataella pastoris, and Komagataella pseudopastoris. As used herein, the term “Pichia pastoris, ” when describing a yeast species, encompasses and includes the species: Komagataella phaffli, Komagataella pastoris, and Komagataella pseudopastoris. See De Schutter et al., Genome sequence of the recombinant protein production host Pichia pastoris. Nat Biotechnol. 2009 Jun;27(6):561-6; Heistinger et al., Microbe Profile: Komagataella phaffii: a methanol devouring biotech yeast formerly known as Pichia pastoris. Microbiology (Reading). 2020 Jul;166(7):614-616; Strumberger et al., Refined Pichia pastoris reference genome sequence. J Biotechnol. 2016 Oct 10; 235: 121-131.

[00157] “Polar amino acid” is an amino acid that is polar and includes serine, threonine, cysteine, asparagine, glutamine, histidine, tryptophan and tyrosine; preferred polar amino acids are serine, threonine, cysteine, asparagine and glutamine; with serine being most highly preferred.

[00158] “Polynucleotide” refers to a polymeric-form of nucleotides (e.g., ribonucleotides, deoxyribonucleotides, or analogs thereof) of any length; e.g., a sequence of two or more ribonucleotides or deoxyribonucleotides. As used herein, the term “polynucleotide” includes double- and single-stranded DNA, as well as double- and singlestranded RNA; it also includes modified and unmodified forms of a polynucleotide (modifications to and of a polynucleotide, for example, can include methylation, phosphorylation, and/or capping). In some embodiments, a polynucleotide can be one of the following: a gene or gene fragment (for example, a probe, primer, EST, or SAGE tag); genomic DNA; genomic DNA fragment; exon; intron; messenger RNA (mRNA); transfer RNA; ribosomal RNA; ribozyme; cDNA; recombinant polynucleotide; branched polynucleotide; plasmid; vector; isolated DNA of any sequence; isolated RNA of any sequence; nucleic acid probe; primer or amplified copy of any of the foregoing.

[00159] In yet other embodiments, a polynucleotide can refer to a polymeric-form of nucleotides operable to encode the open reading frame of a gene.

[00160] In some embodiments, a polynucleotide can refer to cDNA.

[00161] In some embodiments, polynucleotides can have any three-dimensional structure and may perform any function, known or unknown. The structure of a polynucleotide can also be referenced to by its 5’- or 3’- end or terminus, which indicates the directionality of the polynucleotide. Adjacent nucleotides in a single-strand of polynucleotides are typically joined by a phosphodiester bond between their 3’ and 5’ carbons. However, different intemucleotide linkages could also be used, such as linkages that include a methylene, phosphoramidate linkages, etc. This means that the respective 5’ and 3’ carbons can be exposed at either end of the polynucleotide, which may be called the 5’ and 3’ ends or termini. The 5’ and 3’ ends can also be called the phosphoryl (PO4) and hydroxyl (OH) ends, respectively, because of the chemical groups attached to those ends. The term polynucleotide also refers to both double- and single-stranded molecules. Unless otherwise specified or required, any embodiment that makes or uses a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.

[00162] In some embodiments, a polynucleotide can include modified nucleotides, such as methylated nucleotides and nucleotide analogs (including nucleotides with nonnatural bases, nucleotides with modified natural bases such as aza- or deaza-purines, etc.). If present, modifications to the nucleotide structure can be imparted before or after assembly of the polynucleotide.

[00163] In some embodiments, a polynucleotide can also be further modified after polymerization, such as by conjugation with a labeling component. Additionally, the sequence of nucleotides in a polynucleotide can be interrupted by non-nucleotide components. One or more ends of the polynucleotide can be protected or otherwise modified to prevent that end from interacting in a particular way (e.g. forming a covalent bond) with other polynucleotides.

[00164] In some embodiments, a polynucleotide can be composed of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T). Uracil (U) can also be present, for example, as a natural replacement for thymine when the polynucleotide is RNA. Uracil can also be used in DNA. Thus, the term “sequence” refers to the alphabetical representation of a polynucleotide or any nucleic acid molecule, including natural and non-natural bases.

[00165] The term “RNA molecule” or ribonucleic acid molecule refers to a polynucleotide having a ribose sugar rather than deoxyribose sugar and typically uracil rather than thymine as one of the pyrimidine bases. An RNA molecule of the invention is generally single-stranded, but can also be double-stranded. In the context of an RNA molecule from an RNA sample, the RNA molecule can include the single-stranded molecules transcribed from DNA in the cell nucleus, mitochondrion or chloroplast, which have a linear sequence of nucleotide bases that is complementary to the DNA strand from which it is transcribed.

[00166] In some embodiments, a polynucleotide can further comprise one or more heterologous regulatory elements. For example, in some embodiments, the regulatory element is one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; or combinations thereof. [00167] In some embodiments, a polynucleotide can be a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide. In other embodiments, a polynucleotide can nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH). In yet other embodiments, a polynucleotide can be nucleotide sequence operable to encode a heterologous polypeptide, e g., a CRP.

[00168] “Post-transcriptional gene silencing”, or “PTGS”, means a cellular process within living cells that suppress the expression of a gene.

[00169] “Post-transcriptional regulatory elements” are DNA segments and/or mechanisms that affect mRNA after it has been transcribed. Post-transcriptional mechanisms include splicing events, capping, addition of a Poly (A) tail, and other mechanisms known to those having ordinary skill in the art.

[00170] ‘Promoter” refers to a region of DNA to which RNA polymerase binds and initiates the transcription of a gene.

[00171] “Protein” has the same meaning as “peptide” and/or “polypeptide” in this document.

[00172] “Providing” refers to supplying or making a material available. For example, in some embodiments, providing (when used in the context of a vector) refers to suppling said vector and/or making said vector available to a yeast cell.

[00173] “Ratio” refers to the quantitative relation between two amounts or between two objects, which shows the relationship (in amount or quantity) between the two or more amounts, or between the two or more objects. Accordingly, in some embodiments, a ratio shows the number of times a first value contains, or is contained, within a second value. In some embodiments, there can be a ratio between the number of copies of (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), relative to the number of copies of (ii) a nucleotide sequence operable to encode a heterologous polypeptide: thus, the ratio of (i): (ii) describes the relationship in quantity between the copy number of (i) relative to the copy number of (ii). For example, a ratio of (i): (ii) shows the number of times a first value (i.e., (i) a nucleotide sequence operable to encode a heterologous SDH) contains, or is contained, within a second value (i.e., (ii) a nucleotide sequence operable to encode a heterologous polypeptide).

[00174] “Reading frame” refers to one of the six possible reading frames, three in each direction, of the double stranded DNA molecule. The reading frame that is used determines which codons are used to encode amino acids within the coding sequence of a DNA molecule. In some embodiments, a reading frame is a way of dividing the sequence of nucleotides in a polynucleotide and/or nucleic acid (e.g., DNA or RNA) into a set of consecutive, non-overlapping triplets.

[00175] ‘Recombinant cell” means a cell into which foreign DNA has been inserted (e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide).

[00176] ‘Recombinant DNA” or “rDNA” refers to DNA that comprises two or more different DNA segments.

[00177] ‘Recombinant vector” means a DNA plasmid into which foreign DNA has been inserted.

[00178] “Recombinant yeast cell” or “transformant” refers to host cell that has been transformed, e.g., with a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide), including the progeny of such cells. Recombinant yeast cells include “transformants” and “transformed cells,” which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Thus, in some embodiments, a recombinant yeast cell is a yeast cell into which foreign DNA has been inserted (e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide).

[00179] “Regulatory elements” refers to a genetic element that controls some aspect of the expression and/or processing of nucleic acid sequences. For example, in some embodiments, a regulatory element can be found at the transcriptional and post- transcriptional level. Regulatory elements can be cis -regulatory elements (CREs), or trans- regulatory elements (TREs). In some embodiments, a regulatory element can be one or more promoters; enhancers; silencers; operators; splicing signals; polyadenylation signals; termination signals; RNA export elements, internal ribosomal entry sites (IRES); poly-U sequences; and/or other elements that influence gene expression, for example, in a tissuespecific manner; temporal-dependent manner; to increase or decrease expression; and/or to cause constitutive expression. [00180] “Restriction enzyme” or “restriction endonuclease” refers to an enzyme that cleaves DNA at a specified restriction site. For example, a restriction enzyme can cleave a plasmid at an EcoRI, SacII or BstXI restriction site allowing the plasmid to be linearized, and the DNA of interest to be ligated.

[00181] “Restriction site” refers to a location on DNA comprising a sequence of 4 to 8 nucleotides, and whose sequence is recognized by a particular restriction enzyme.

[00182] “SDH” or “SorDH” or “lid” are used interchangeably, and refers to sorbitol dehydrogenase.

[00183] ‘Sea anemone” refers to a group of marine animals of the order Actiniaria. Sea anemones are named after the anemone, which is a terrestrial flowering plant, due to colorful appearance many sea anemones possess. For example, in some embodiments, a sea anemone is one of the following species: Actinia equine; Anemonia erythraea; Anemonia sulcata; Anemonia viridis; Anthopleura elegantissima; Anthopleura fuscoviridis;

Anthopleura xanthogrammica; Bunodosoma caissarum; Bunodosoma cangicum; Bunodosoma granulifera; Heteractis crispa; Parasicyonis actinostoloides; Radianthus paumotensis; or Stoichactis helianthus.

[00184] “Selection marker” means a gene or an exogenous or heterologous polynucleotide sequence which confers an advantage for a genetically modified organism to grow under the selective pressure.

[00185] “Sorbitol” refers to (2S,3R,4R,5R)-Hexane-l,2,3,4,5,6-hexol, a sugar alcohol that is generally obtained by the reduction of glucose. In some embodiments, sorbitol serves as a carbon source. See “carbon source.”

[00186] “Sorbitol dehydrogenase” or “SDH” is an enzyme that catalyzes the reversible NAD⁺-dependent oxidation of various sugar alcohols. SDH is mostly active with D-sorbitol (D-glucitol), L-threitol, xylitol and ribitol as substrates, leading to the C2-oxidized products D-fructose, L-erythrulose, D-xylulose, and D-ribulose, respectively. SDH is a key enzyme in the polyol pathway, and it interconverts glucose and fructose via sorbitol, which constitutes an important alternate route for glucose metabolism.

[00187] “s/?.” refers to species.

[00188] "ssp." or "subsp." refers to subspecies.

[00189] “Subcloning” or “subcloned” refers to the process of transferring DNA from one vector to another, usually advantageous vector. For example, polynucleotide encoding a heterologous peptide can be subcloned into a pKlacl plasmid subsequent to selection of yeast colonies transformed with pKLACl plasmids. [00190] “SSI” is an acronym that is context dependent. In some contexts, it can refer to “site-specific integration,” which is used to refer to a sequence that will permit in vivo homologous recombination to occur at a specific site within a host organism’s genome. Thus, in some embodiments, the term “site-specific integration” refers to the process directing a transgene to a target site in a host-organism’s genome, allowing the integration of genes of interest into pre-selected genome locations of a host-organism.

[00191] “STA” or “Translational stabilizing protein” or “stabilizing domain” or “stabilizing protein” (used interchangeably herein) means a peptide or protein with sufficient tertiary structure that it can accumulate in a cell without being targeted by the cellular process of protein degradation. The protein can be between 5 and 50 amino acids long. The translational stabilizing protein is coded by a DNA sequence for a protein that is operably linked with a sequence encoding a CRP in the ORF. The operably-linked STA can either be upstream or downstream of the CRP and can have any intervening sequence between the two sequences (STA and CRP) as long as the intervening sequence does not result in a frame shift of either DNA sequence. The translational stabilizing protein can also have an activity which increases delivery of the CRP across the gut wall and into the hemolymph of the insect. [00192] “sta” means a nucleotide encoding a translational stabilizing protein.

[00193] “Strain” refers to a genetic variant, an isolate, a subtype, a group thereof, or a culture thereof, exhibiting phenotypic and/or genotypic traits belonging to the same lineage, distinct from those of other members of the same species. For example, in some embodiments, the term “strain” can refer to one or more yeast cells having one or more characteristics that makes them differ in some way relative to other yeast cells of their species, wherein said other yeast cells do not possess the one or more characteristics. For example, one or more yeast cells comprising an endogenous sorbitol dehydrogenase that is partially inactivated, can be described as a strain.

[00194] “Structural motif’ refers to the three-dimensional arrangement of peptides and/or polypeptides, and/or the arrangement of operably linked polypeptide segments. For example, a polypeptide having an ERSP motif, an STA motif, a LINKER motif, and a CRP polypeptide motif, has an overall “structural motif’ of ERSP-STA-L-CRP. See also “CRP construct.”

[00195] “Talb” or “Ul-agatoxin-Talb” or “TalbWT” or “wild-type Ul-agatoxin- Talb” refers to a polypeptide isolated from the Hobo spider, Eratigena agrestis. One example of a Ul-agatoxin-Talb is a polypeptide having the amino acid sequence of SEQ ID NO: 195 (NCBI Accession No. 046167.1). [00196] “Talb variant polynucleotide” or “Ul-agatoxin-Talb variant polynucleotide” refers to a polynucleotide or group of polynucleotides operable to express and/or encode an insecticidal protein comprising one or more TVPs. The term “Ul-agatoxin-Talb variant polynucleotide” when used to describe the Ul-agatoxin-Talb variant polynucleotide sequence contained in a TVP expression ORF, its inclusion in a vector, and/or when describing the polynucleotides encoding an insecticidal protein, is described as “tvp” and/or “T /?.”

[00197] “Toxin” refers to a venom and/or a poison, especially a protein or conjugated protein produced by certain animals, higher plants, and pathogenic bacteria. Generally, the term “toxin” is reserved natural products, e.g., molecules and peptides found in scorpions, spiders, snakes, poisonous mushrooms, etc., whereas the term “toxicant” is reserved for manmade products and/or artificial products e.g., man-made chemical pesticides. However, as used herein, the terms “toxin” and “toxicant” are used synonymously

[00198] “Transfection” and “transformation” both refer to the process of introducing exogenous and/or heterologous DNA or RNA (e.g., a vector containing a polynucleotide that encodes a CRP) into a host organism (e.g., a prokaryote or a eukaryote). Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and/or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and/or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the term “transformation” and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and/or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

[00199] “Transgenic cell” means a cell which is transformed with a gene and has been selected for its transgenic status via an additional selection gene.

[00200] “Triple expression cassette” refers to three heterologous polypeptide (e.g., a CRP)/heterologous SDH expression cassettes contained on the same vector.

[00201] “TSP” or “total soluble protein” means the total amount of protein that can be extracted from a plant tissue sample and solubilized into the extraction buffer.

[00202] “TVP” or “Ul-agatoxin-Talb Variant Polypeptides (TVPs)” or “Talb Variant Polypeptides (TVPs)” refers to mutants or variants of the wild-type Ul-agatoxin-Talb polypeptide sequence and/or a polynucleotide sequence encoding a wild-type Ul-agatoxin- Talb polypeptide, that have been altered to produce a non-naturally occurring polypeptide and/or polynucleotide sequence. An exemplary wild-type Ul-agatoxin-Talb polypeptide sequence is provided herein, having the amino acid sequence of SEQ ID NO: 195. An exemplary wild-type Ul-agatoxin-Talb precursor polypeptide sequence is provided herein, having the amino acid sequence of SEQ ID NO: 194 (NCBI Accession No. 046167.1), which includes the signal sequence “MKLQLMICLVLLPCFFC” (SEQ ID NO:471). In some embodiments, a TVP can have an amino acid sequence according to any of the amino acid sequences listed in Table 1. Accordingly, the term “TVP” refers to peptides having one or more mutations relative to the amino acid sequence set forth in SEQ ID NO: 195. In some embodiments, a TVP can have an amino acid sequence according to Formula (I):

E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E- C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7

Formula (I)

[00203] wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent.

[00204] In some embodiments, a TVP can have an amino acid sequence according to Formula (II):

E-P-D-E-I-C-R-A-X1-M-T-N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E- C-F-R-N-D-V-Y-Zi-A-C-H-E-A-Q-K-G

Formula (II)

[00205] wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is R or Q; and Zi is T or A; or a pharmaceutically acceptable salt thereof.

[00206] “U-ACTX-Hvla” or “hybrid peptide” or “hybrid toxin” or “hybrid-ACTX- Hvla” or “native hybridACTX-Hvla” or “U peptide” or “U toxin” or “native U” or “native U-ACTX-Hvla,” all refer to an ACTX peptide, which was discovered from a spider known as the Australian Blue Mountains Funnel-web Spider, Hadronyche versuta. U-ACTX-Hvla is a positive allosteric modulators of the nicotinic acetylcholine receptor, and may also be a dual antagonist to insect voltage-gated Ca²⁺ channels and voltage-gated K⁺ channels. See Chambers et al., Insecticidal spider toxins are high affinity positive allosteric modulators of the nicotinic acetylcholine receptor. FEBS Lett. 2019 Jun; 593(12): 1336-1350; and Windley et al., Lethal effects of an insecticidal spider venom peptide involve positive allosteric modulation of insect nicotinic acetylcholine receptors. Neuropharmacology. 2017 Dec; 127:224-242, the disclosures of which are incorporated herein by reference in their entireties. An exemplary U-ACTX-Hvla peptide is provided in SEQ ID NO: 186.

[00207] “U+2 peptide” or “U+2 protein” or “U+2 toxin” or “U+2” or “U+2-ACTX-

Hvla” or “Spear” or “Hybrid+2-ACTX-Hvla” or “H+2-ACTX-Hvla” all refer to a U- ACTX-Hv la having an additional dipeptide operably linked to the native peptide. The additional dipeptide that is operably linked to the U peptide is indicated by the “+2” or “plus 2” can be selected from among several peptides, any of which may result in a “U+2 peptide” with unique properties as discussed herein. In some preferred embodiments, the dipeptide is “GS”; an exemplary U+2-ACTX-Hvla peptide is set forth in SEQ ID NO: 187.

[00208] “UBI” refers to ubiquitin. For example, in some embodiments, UBI can refer to a ubiquitin monomer isolated from Zea mays.

[00209] ~var." refers to varietas or variety. The term “var.” is used to indicate a taxonomic category that ranks below the species level and/or subspecies (where present). In some embodiments, the term “var.” represents members differing from others of the same subspecies or species in minor but permanent or heritable characteristics.

[00210] “Variant” or “variant sequence” or “variant peptide” refers to an amino acid sequence that possesses one or more conservative amino acid substitutions or conservative modifications. The conservative amino acid substitutions in a “variant” does not substantially diminish the activity of the variant in relation to its non-varied form. For example, in some embodiments, a “variant” possesses one or more conservative amino acid substitutions when compared to a peptide with a disclosed and/or claimed sequence, as indicated by a SEQ ID NO.

[00211] “Vector” refers to the DNA segment that accepts a foreign polynucleotide of interest (e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide). The polynucleotide of interest is known as an “insert” or “transgene.” In some embodiments, a vector can be a plasmid (e.g., a pJUSor, pKLD, or a pLB10V5 plasmid). [00212] “Wild type” or “WT” refers to the phenotype and/or genotype (i.e., the appearance or sequence) of an organism, polynucleotide sequence, and/or polypeptide sequence, as it is found and/or observed in its naturally occurring state or condition.

[00213] “Yeast cell” refers to a eukaryotic, single-celled microorganism that is a member of the fungus kingdom. “Yeast cells” can be identified by the genera to which they belong to (e.g., Saccharomyces, Pichia, Kluyveromyces, etc.) and/or the species that they derived from (Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc.).

[00214] “Yeast expression vector” or “expression vector” or “vector” means a plasmid which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated.

[00215] “Yield” refers to the production of a peptide, and increased yields can mean increased amounts of production, increased rates of production, and an increased average or median yield and increased frequency at higher yields.

[00216] Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e., one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

[00217] The present disclosure is performed without undue experimentation using, unless otherwise indicated, conventional techniques of molecular biology, microbiology, virology, recombinant DNA technology, solid phase and liquid nucleic acid synthesis, peptide synthesis in solution, solid phase peptide synthesis, immunology, cell culture, and formulation. Such procedures are described, for example, in Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Second Edition (1989), whole of Vols I, II, and III; DNA Cloning: A Practical Approach, Vols. I and II (D. N. Glover, ed., 1985), IRL Press, Oxford, whole of text; Oligonucleotide Synthesis: A Practical Approach (M. J. Gait, ed, 1984) IRL Press, Oxford, whole of text, and particularly the papers therein by Gait, ppl-22; Atkinson et al, pp35-81; Sproat et al, pp 83- 115; and Wu et al, pp 135-151; 4. Nucleic Acid Hybridization: A Practical Approach (B. D. Hames & S. J. Higgins, eds., 1985) IRL Press, Oxford, whole of text; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford, whole of text; Perbal, B., A

Practical Guide to Molecular Cloning (1984); Methods In Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.), whole of series; J. F. Ramalho Ortigao, “The Chemistry of Peptide Synthesis” In: Knowledge database of Access to Virtual Laboratory website (Interactiva, Germany); Sakakibara, D., Teichman, J., Lien, E. Land Fenichel, R. L. (1976). Biochem. Biophys. Res. Commun. 73 336-342; Merrifield, R. B. (1963). J. Am. Chem. Soc. 85, 2149-2154; Barany, G. and Merrifield, R. B. (1979) in “The Peptides” (Gross, E. and Meienhofer, 3. eds.), vol. 2, pp. 1-284, Academic Press, New York. 12. Wiinsch, E., ed. (1974) Synthese von Peptiden in Houben-Weyls Metoden der Organischen Chemie (Muler, E., ed.), vol. 15, 4th ed., Parts 1 and 2, Thieme, Stuttgart; Bodanszky, M. (1984) Principles of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. & Bodanszky, A. (1984) The Practice of Peptide Synthesis, Springer-Verlag, Heidelberg; Bodanszky, M. (1985) Int. J. Peptide Protein Res. 25, 449-474; Handbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C. Blackwell, eds., 1986, Blackwell Scientific Publications); and Animal Cell Culture: Practical Approach, Third Edition (John R. W. Masters, ed., 2000); each of these references are incorporated herein by reference in their entireties.

[00218] Throughout this specification, unless the context requires otherwise, the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of elements or integers. [00219] All patent applications, patents, and printed publications referred to herein are incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. And, all patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers, or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.

[00220] RECOMBINANT YEAST CELLS

[00221] The present disclosure provides recombinant yeast cells, and methods for making the same, wherein the recombinant yeast cell comprises a heterologous polynucleotide, wherein said heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and wherein (a) the recombinant yeast cell is selected from a yeast species that does not possess an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene); or (b) the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene), that is at least partially inactivated.

[00222] When the recombinant yeast cells of the present disclosure are grown in media comprising a sole carbon source that is sorbitol, the recombinant yeast cells can advantageously maintain copy numbers and/or prevent copy loss of the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00223] The heterologous polynucleotide comprises at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH). Sorbitol dehydrogenase (Enzyme Commission No. EC 1.1.1.14; SDH) is an enzyme capable of converting sorbitol into fructose. Sorbitol dehydrogenase has been found primarily in rosaceous species (i.e., apples and peaches) in plants and also exists in bacteria. The nucleic acid and protein sequences for sorbitol dehydrogenase from a variety of species are known in the art and can be used with the disclosed recombinant yeast cells.

[00224] Additionally, the heterologous polynucleotide comprises at least one nucleotide sequence operable to encode a heterologous polypeptide. In some embodiments, the heterologous polypeptide is a desirable polypeptide, e.g., without limitation, a Cysteine Rich Peptide (CRP).

[00225] CRPs are peptides rich in cysteine residues that, in some embodiments, are operable to form disulfide bonds between such cysteine residues. In some embodiments, CRPs contain at least four (4), sometimes six (6), and sometimes eight (8) cysteine amino acids among proteins or peptides having at least 10 amino acids where the cysteines form two (2), three (3) or four (4) disulfide bonds. In some embodiments, the disulfide bonds contribute to the folding, three-dimensional structure, stability, and/or activity of a peptide. In some embodiments, the activity can be insecticidal activity. Indeed, in some embodiments, the cysteine-cysteine disulfide bonds, and the three dimensional structure they form, play a significant role in the insecticidal nature of insecticidal peptides. In some embodiments, a CRP may or may not comprise a cystine knot. For example, in some embodiments, a CRP can have an inhibitor cystine knot (ICK) motif, a growth factor cystine knot (GFCK) motif, or a cyclic cystine knot (CCK) motif. In some embodiments, a CRP can have an ICK motif. For example, in some embodiments, a CRP with an ICK motif can be an ACTX peptide from a spider; in other embodiments, a CRP without an ICK motif, i.e., a non-ICK CRP, can be a peptide like Av2 and Av3, peptides isolated from sea anemones. Non-ICK CRPS can have 4- 8 cysteines which form 2-4 disulfide bonds. These cysteine-cysteine disulfide-bond-stabilized peptides can have remarkable stability when exposed to the environment. Many CRPs are isolated from venomous animals such as spiders, scorpions, snakes and sea snails and sea anemones and they are toxic to insects.

[00226] The recombinant yeast cell of the present disclosure can be transformed with the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, using a variety of techniques known in the art. Exemplary methods of transforming the recombinant yeast cell with the heterologous polynucleotide are described below. Likewise, yeast cells (or host cells) operable to be transformed with the heterologous polynucleotide of the present disclosure are also described herein.

[00227] Multi-copy integrations of polynucleotides operable to encode heterologous peptides exhibit copy number loss as a result of the repeated regions of homology in close proximity. And, copy number can correlate positively with peptide expression — accordingly, the loss of copies can result in a decrease in yield of peptides. Furthermore, if a copy loss event (e.g., “out-recombination”) is accompanied by a fitness benefit in growth, selection can promote the increase in frequency of the lower copy number cells in a mixed culture over time, thus reducing yield further.

[00228] “Copy number loss,” or “copy loss,” as used herein, refers to a decrease in copy number(s) of heterologous polynucleotides that have integrated into a host cell’s genome. Copy loss occurs when the ultimate number of copies integrated into the host cell’s genome are reduced due to homologous out-recombination. For example, after a few generations, a recombinant cell comprising multiple copies of an integrated heterologous polynucleotide, can experience copy loss, wherein one or more copies of the integrated heterologous polynucleotides are out-recombined via intrinsic DNA repair mechanisms — a problem that exacerbates as time progresses. And, recombinant cells that have lost one or more of the multiple copies of the heterologous polynucleotide are less encumbered by heterologous protein production; accordingly, recombinant cells that have lost one or more of the multiple copies are able to outcompete cells maintaining the multiple copies, because the latter are energetically burdened with heterologous protein production. In some embodiments, the decrease in copy number can be a decrease in the number of copies of (1) a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (2) a heterologous nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); (3) a heterologous nucleotide sequence operable to encode a heterologous polypeptide; or (4) any combination thereof.

[00229] The copy number, or number of copies, is determined as the number of copies relative to an initial number of copy numbers, after an amount of time and/or other metric. For example, in some embodiments, the initial number of copies can be assessed relative to the number of copies after the cell has undergone replication for a certain number of generations. In yet other embodiments, the initial number of copies can be assessed relative to the number of copies after the cell has undergone treatment with a modality and/or been exposed to experimental conditions.

[00230] For example, in some embodiments, a cell that losses copy number(s) can be a cell that has an initial number of copies (e.g., 10 copy numbers of a vector; or 10 copies of a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide) and, after said cell has replicated for a certain number of generations, the cell can contain a lower amount of number of copies of the vector or heterologous polynucleotide (e.g., 9 or less copy numbers of a vector). Here, the reduction of 10 copies to 9 copies represents the “copy loss” or the “copy number loss.” [00231] In some embodiments, the recombinant yeast cells of the present disclosure are able to maintain copies of the heterologous polynucleotide. The terms “maintenance” or “copy number maintenance,” as used herein, refers to the prevention, minimization, or retardation of copy number loss in a cell (e.g., a yeast cell) that has been recombinantly modified with one or more heterologous polypeptide.

[00232] Thus, in some embodiments, a recombinant yeast cell of the present disclosure comprises a heterologous polynucleotide, wherein said heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and wherein (a) the recombinant yeast cell is selected from a yeast species that does not possess an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene); or (b) the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene), that is at least partially inactivated; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in a prevention of copy number loss and/or copy number maintenance of the heterologous polynucleotide, relative to a copy number when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00233] As discussed above, copy number (or the maintenance thereof) can correlate positively with levels of peptide expression; and, as the loss of copy number of a heterologous polynucleotide encoding a peptide of interest can result in a decrease in yield of said peptides, a maintenance of copy number can alternatively prevent such a decrease. [00234] Accordingly, in some embodiments, a recombinant yeast cell of the present disclosure comprises a heterologous polynucleotide, wherein said heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and wherein (a) the recombinant yeast cell is selected from a yeast species that does not possess an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene); or (b) the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene), that is at least partially inactivated; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00235] Methods of making the recombinant yeast cell; aspects of the heterologous polynucleotide, including SDH and heterologous polypeptides (e.g., a Cysteine Rich Peptide (CRP)), are discussed in detail in the sections below.

[00236] HOST CELLS

[00237] Host cells are cells that are suitable for receiving and/or producing a heterologous polynucleotide or protein; thus, host cells are cells that are operable to be transformed, e.g., with a heterologous polynucleotide, or, into which a heterologous polynucleotide may be introduced — wherein the cells are likewise operable to express the heterologous polynucleotide (transcribe) and/or the protein encoded by the same (translate). For example, in some embodiments, a recombinant yeast cell of the present disclosure is created by transforming the vector comprising a heterologous polynucleotide comprises (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, into a yeast host cell. [00238] A wide variety of yeast cells can be utilized as the yeast host cell of the present disclosure. Exemplary cells that may be utilized as host cells in accordance with the present disclosure include those of eukaryotes.

[00239] In some embodiments, the yeast host cell can be a cell belonging to the kingdom: fungi.

[00240] In some embodiments, the yeast host cell may be a cell from a fungi belonging to one of the following genera: Aspergillus, Cladosporium, Magnaporthe, Morchella, Neurospora, Penicillium, Saccharomyces, Cryptococcus, or Ustilago.

[00241] In some embodiments, yeast host cell may be a member of the Saccharomycetaceae family. For example, in some embodiments, the yeast cell may be one of the following genera within the Saccharomycetaceae family: Brettanomyces, Candida, Citer omyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.

[00242] In some embodiments, the yeast host cell may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[00243] In some embodiments, the yeast host cell may be a species within the Candida genus. For example, the yeast cell may be one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[00244] In some embodiments, the yeast host cell may be a species within the Kluyveromyces genus. For example, the yeast host cell may be one of the following: Kluyveromyces aestuariv, Kluyveromyces aestuarii ATCC 18862,' Kluyveromyces dobzhanskii Kluyveromyces dobzhanskii CBS 2104,' Kluyveromyces hubeiensis,' Kluyveromyces lactis; Kluyveromyces lactis NRRL Y-1140; Kluyveromyces lactis x Kluyveromyces marxianus,' Kluyveromyces lactis x Kluyveromyces wickerhamii,' Kluyveromyces marxianus,' Kluyveromyces marxianus CBS 712; Kluyveromyces marxianus DMKU3-1042; Kluyveromyces marxianus var. bulgaricus; Kluyveromyces marxianus var. marxianus Kluyveromyces marxianus var. marxianus KCTC 17555; Kluyveromyces cf. marxianus TO77.2; Kluyveromyces nonfermentans; Kluyveromyces siamensis;

Kluyveromyces starmeri; Kluyveromyces wickerhamii; Kluyveromyces wickerhamii UCD 54- 210; unclassified Kluyveromyces; Kluyveromyces sp. ; Kluyveromyces sp. 1S-1;

Kluyveromyces sp. 5s-l; Kluyveromyces sp. CCTCC M2011385; Kluyveromyces sp. CW3.1; Kluyveromyces sp. CW3. 7; Kluyveromyces sp. CW3.8; Kluyveromyces sp. CW4.3;

Kluyveromyces sp. EY12114; Kluyveromyces sp. GX8-5A; Kluyveromyces sp. HF12172; Kluyveromyces sp. HN10-4-2; Kluyveromyces sp. HNC-3; Kluyveromyces sp. IFO 11072; Kluyveromyces sp. IFO 1884; Kluyveromyces sp. JI; Kluyveromyces sp. JSK2016;

Kluyveromyces sp. KJS-2016; Kluyveromyces sp. MMSTP-xj_4; Kluyveromyces sp. NCIM 3565; Kluyveromyces sp. NI 18; Kluyveromyces sp. PCH397; Kluyveromyces sp. RWT25; Kluyveromyces sp. ST-343; Kluyveromyces sp. SY-20/3 Kluyveromyces sp. WM04.172; Kluyveromyces sp. Y3; Kluyveromyces sp. YS 9F; Kluyveromyces sp. YSNB1; Kluyveromyces sp. YSF9; Kluyveromyces sp. ZJ15-G; Kluyveromyces sp. ZMS1; or Kluyveromyces sp. ZMS3. [00245] In some embodiments, the yeast host cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans, or Kluyveromyces wickerhamii.

[00246] In some embodiments, the yeast host cell may be a species within the Komagataella genus. For example, in some embodiments, the yeast host cell may be one of the following: Komagataella kurtzmanii; Komagataella mondaviorum; Komagataella pastoris (e.g., Komagataella pastoris DSMZ 70382); Komagataella phaffli (e.g., Komagataella phaffli CBS 7435; Komagataella phaffli GS115; Komagataella phaffli JC308); Komagataella populi; Komagataella pseudopastoris; Komagataella ulmi; or unclassified Komagataella (e.g., Komagataella sp., Komagataella sp. 11-1192, Komagataella sp. EN- 2017b).

[00247] In some embodiments, the yeast host cell may be a species within the Komagataella genus. For example, in some embodiments, the yeast host cell may be one of the following: Komagataella phaffli, Komagataella pastoris, or Komagataella pseudopastoris. [00248] In some embodiments, the yeast host cell may be a species within the Pichia genus. For example, the yeast host cell may be one of the following: Pichia aff. alni PL5WP, Pichia aff. alni PLE2W3; Pichia barker! Pichia bruneiensis; Pichia cactophila; Pichia cecembensis; Pichia cephalocereana.’ Pichia chihodasensis.’ Pichia deserticola; Pichia dushanensis.' Pichia eremophila; Pichia exigua; Pichia fermentans; Pichia aff. fermentans; Pichia aff. fermentans B-WHX-12-15; Pichia aff. fermentans IMUFRJ 51964; Pichia aff. fermentans Y0 143; Pichia aff. fermentans Y0 149; Pichia aff. fermentans Y0 150; Pichia aff. fermentans Y153.’ Pichia aff. fermentans Y3 166; Pichia aff. fermentans Y3 167; Pichia aff. fermentans YM26086; Pichia garciniae.’ Pichia gijzeniarum; Pichia heedff Pichia insulana; Pichia cf. insulana TO4.2.P, Pichia kluyveri; Pichia kudriavzevii Pichia kudriavzevii CAB39-6420; Pichia kudriavzevii M12; Pichia manshurica.' Pichia cf. manshurica MY 1A; Pichia cf. manshurica MY IB; Pichia cf. manshurica MY 1C; Pichia membranifaciens; Pichia membranifaciens NRRL Y-2026; Pichia nakasei; Pichia nanzhaoensis; Pichia nongkratonensis; Pichia norvegensis; Pichia occidentalis; Pichia paraexigua; Pichia porticicola; Pichia pseudocactophila; Pichia punctispora; Pichia rarassimilans; Pichia scaptomyzae; Pichia scutulata; Pichia sporocuriosa; Pichia terricola; Pichia aff. terricola; Pichia cf. trehaloabstinens UWO(PS)99-304.4; Pichia aff. trypodendroni MKL6DP 3; Pichia/Candida clade; [Candida] awuae; [Candida] cabralensis; [Candida] californica; [Candida] ethanolica; [Candida] cf. ethanolica; [Candida] inconspicua; [Candida] phayaonensis; [Candida] pseudolambica; [Candida] aff. pseudolambica EXF-7983; [Candida] rugopelliculosa; [Candida] thaimueangensis;

[Candida] sp. JCM 28228; [Candida] sp. KJS-2016; Pichia membranifaciens x Pichia sp.; unclassified Pichia; Pichia sp. ; Pichia sp. 'Issatchenkia siamensis '; Pichia sp. j ' aroonii '; Pichia sp. 'namnaoensis'; Pichia sp. 1 CS-2017; Pichia sp. 1 TMS-2011; Pichia sp. 11-1203; Pichia sp. 11-4867; Pichia sp. 15c-l; Pichia sp. 2 TMS-2011; Pichia sp. 2c; Pichia sp. 2d; Pichia sp. 3p-2; Pichia sp. 3p-B; Pichia sp. 5W-2; Pichia sp. 6D1; Pichia sp. 6F21; Pichia sp. 7W-1; Pichia sp. ANT03-414; Pichia sp. AQGWD 7; Pichia sp. AS2.3480; Pichia sp. ATNov07DSX-Y7; Pichia sp. AUMC 7755; Pichia sp. AUMC 7766; Pichia sp. AWRI 1272; Pichia sp. B1 1 3 SC14; Pichia sp. B3 2 3 SC12; Pichia sp. BCCSM1; Pichia sp. BS51; Pichia sp. BYB-3; Pichia sp. BZ159; Pichia sp. CanS-40; Pichia sp. CBS 241; Pichia sp. CBS 8578; Pichia sp. CC-88204; Pichia sp. Cfrav47; Pichia sp. CH3II; Pichia sp. CH3IV; Pichia sp. ChihlrhP305; Pichia sp. ChihpsP314; Pichia sp. GJ; Pichia sp. CLIB 1633; Pichia sp. ColRe-ITSl; Pichia sp. control78; Pichia sp. CS9; Pichia sp. D50 3; Pichia sp. Dp-Y5; Pichia sp. EF17; Pichia sp. EJ7S01; Pichia sp. EN17S12; Pichia sp. EN27S10; Pichia sp. EN29S03; Pichia sp. EN9S05; Pichia sp. ESI 79; Pichia sp. EU11S06; Pichia sp. EXF-5650; Pichia sp. F4; Pichia sp. FE-7-11-7; Pichia sp. feni 101; Pichia sp. feni 106 Pichia sp. feni 107; Pichia sp. feni 108; Pichia sp. FI09; Pichia sp. FLS-2010; Pichia sp. FS26; Pichia sp. FTJZZJ 10; Pichia sp. GATQ-1; Pichia sp. GATQ-2; Pichia sp. GAXQ-1; Pichia sp. GAXQ-2; Pichia sp. GE1S02; Pichia sp. GE20S12; Pichia sp. GJ5M13; Pichia sp. GoEmiLpsP 380; Pichia sp. GoRoLruMA344; Pichia sp. GoToruMP329; Pichia sp. GV1 05; Pichia sp. GY16S05; Pichia sp. GY18L01; Pichia sp. GY5S07; Pichia sp. H9; Pichia sp. H9Y8; Pichia sp. HA1559; Pichia sp. HK-2008a; Pichia sp. HSD08; Pichia sp. IFO 10088; Pichia sp. IFO 1788; Pichia sp. IMBG193; Pichia sp. IR08; Pichia sp. JJP-2009a; Pichia sp. JP-2008; Pichia sp. JP39; Pichia sp. KCY-5; Pichia sp. KKK 24; Pichia sp. KQ27; Pichia sp. KS36-1; Pichia sp. KS36-2; Pichia sp. KY-328; Pichia sp. KY-490; Pichia sp. KY-491; Pichia sp. L2- 4; Pichia sp. L3-5x; Pichia sp. LCF-18; Pichia sp. LCF-28; Pichia sp. LHE; Pichia sp. LHY1; Pichia sp. ELI 1 118; Pichia sp. LM023; Pichia sp. LN0004; Pichia sp. LY19; Pichia sp.

Mil; Pichia sp. M25; Pichia sp. M3; Pichia sp. M31; Pichia sp. M42; Pichia sp. M45; Pichia sp. M46; Pichia sp. M50; Pichia sp. M51; Pichia sp. M56; Pichia sp. M60; Pichia sp. M8; Pichia sp. MAR; Pichia sp. MBIC4209; Pichia sp. MD6; Pichia sp. MKS10W1; Pichia sp. MR-3; Pichia sp. MT-LUC0001; Pichia sp. MT-LUC0002; Pichia sp. MT-LUC0003; Pichia sp. MT-LUC0004; Pichia sp. MT-LUC0005; Pichia sp. MT-LUC0006; Pichia sp. MT- LUC0007; Pichia sp. MT-LUC0008; Pichia sp. MT-LUC0009; Pichia sp. MT-LUC0012; Pichia sp. MT-LUC0016; Pichia sp. MY/130; Pichia sp. MYf84; Pichia sp. mYJddl4; Pichia sp. N006; Pichia sp. N02-2.3; Pichia sp. N1 XX-2012; Pichia sp. Nl-1; Pichia sp. NBRC 105024; Pichia sp. NBRC 105025; Pichia sp. NBRC 106867; Pichia sp. NBRC 106874; Pichia sp. NCIM 3421; Pichia sp. NCIM 3621; Pichia sp. NCIM3251; Pichia sp. NCYC 4037; Pichia sp. NCYC 4038; Pichia sp. NCYC 4044; Pichia sp. NI 03; Pichia sp. NI 12; Pichia sp. NI 14; Pichia sp. NI 15; Pichia sp. NRRL Y-11513; Pichia sp. NRRL Y-11569; Pichia sp. NRRL Y-12824; Pichia sp. NRRL Y-12827; Pichia sp. NRRL Y-12830; Pichia sp. NRRL Y-17803; Pichia sp. NRRL Y -27259; Pichia sp. NRRL Y -27261; Pichia sp. NRRL Y- 27293; Pichia sp. NRRL Y-7615; Pichia sp. NRRL YB-1305; Pichia sp. NRRL YB-1982;

Pichia sp. NRRL YB-2437; Pichia sp. NRRL YB-4149; Pichia sp. NYNU 13710; Pichia sp. NYNU 14770; Pichia sp. NYNU 161119; Pichia sp. P 34709; Pichia sp. PiUnl; Pichia sp. PN-2013; Pichia sp. PR1; Pichia sp. PR2; Pichia sp. PS-4; Pichia sp. QAUPK01; Pichia sp. QAUPK02; Pichia sp. QAUPK03; Pichia sp. QAUPK04; Pichia sp. QAUPK05; Pichia sp. RG; Pichia sp. RND14; Pichia sp. RT; Pichia sp. S4; Pichia sp. S413Y4; Pichia sp. S432Y3; Pichia sp. S432Y30; Pichia sp. S432Y31; Pichia sp. S432Y42; Pichia sp. S433Y13; Pichia sp. S433Y17; Pichia sp. S5; Pichia sp. S513Y6; Pichia sp. S513Y7; Pichia sp. S612Y5; Pichia sp. SA18S01; Pichia sp. SA18S04; Pichia sp. SA8S03; Pichia sp. SC10S07; Pichia sp. SC2S03; Pichia sp. SC5L12; Pichia sp. SCC-2006a; Pichia sp. SCN-2006a; Pichia sp. SF5S11; Pichia sp. SG2S06.’ Pichia sp. SG3L01; Pichia sp. SG4S01; Pichia sp. SG6I.04.' Pichia sp. S12;

Pichia sp. SJ7S11; Pichia sp. SN-2013.’ Pichia sp. SN3S09.' Pichia sp. SO; Pichia sp. ST-236,' Pichia sp. ST-3,' Pichia sp. ST-30,' Pichia sp. ST-314,' Pichia sp. ST-334,' Pichia sp. ST-335,' Pichia sp. ST-339,' Pichia sp. ST-4,' Pichia sp. ST-433,' Pichia sp. ST-445,' Pichia sp. SW106; Pichia sp. TCJ 24; Pichia sp. TCJ105; Pichia sp. TCJ133; Pichia sp. TCJ3; Pichia sp. TCL; Pichia sp. TLD; Pichia sp. UFMG-C156; Pichia sp. UFMG-CM-Y3335; Pichia sp. UFMG- CM-Y531; Pichia sp. UFMG-IA11.1 ; Pichia sp. UFMG-IO 16. 7; Pichia sp. UFMGCB L43-2; Pichia sp. UWO(PS)85-301.3; Pichia sp. UWO(PS)99-305.1; Pichia sp. UWO(PS)99-530. 3;

Pichia sp. UWO(PS)99-666. 3; Pichia sp. W1311 Pichia sp. wl314; Pichia sp. wl315; Pichia sp. W8Y21; Pichia sp. XM03C; Pichia sp. XMO5E; Pichia sp. Yl; Pichia sp. Yl(2015);

Pichia sp. Y4; Pichia sp. Y7H7b7epi,' Pichia sp. YC-01; Pichia sp. Yeast 2; Pichia sp. YF04a,' Pichia sp. yHKS152; Pichia sp. yHQL2294; Pichia sp. yHQL2295; Pichia sp. yHQL2296;

Pichia sp. yHQL2297; Pichia sp. yHQL2298; Pichia sp. YM24322,' Pichia sp. YS 10F; Pichia sp. YS 110,' Pichia sp. YS 73,' Pichia sp. YS 78; Pichia sp. YSDN22; Pichia sp. YS104,' Pichia sp. YS 15; Pichia sp. YS16A; Pichia sp. YS16B; Pichia sp. YS22; Pichia sp. YS31; Pichia sp. YS5; Pichia sp. YS80; Pichia sp. YSF 10; Pichia sp. YSF11; Pichia sp. YSF116; Pichia sp. YSF 14; Pichia sp. YSF5; Pichia sp. YSF8; Pichia sp. YW; Pichia sp. Z22; Pichia sp. Z4Y31; Pichia sp. Z54; Pichia sp. Z58; Pichia sp. Z6(l 70); ox Pichia sp. Z8Y15.

[00249] In some embodiments, the yeast host cell may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.

[00250] In some embodiments, the yeast host cell may be a species within the Saccharomyces genus. For example, the yeast cell may be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[00251] In some embodiments, the yeast host cell may be one of the following: Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, or Pichia pastoris.

[00252] In some embodiments, the yeast host cell can be from the species Saccharomyces including any species of Saccharomyces, for example Saccharomyces cerevisiae species selected from following strains: INVScl, YNN27, S150-2B, W303-1B, CG25, W3124, JRY188, BJ5464, AH22, GRF18, W303-1A and BJ3505.

[00253] In some embodiments, the yeast cell can be any member of the Pichia species including any species of Pichia, for example the Pichia species, Pichia pastoris, for example, the Pichia pastoris is selected from following strains: Bg08, BglO, Y-11430, X-33, GS115, GS190, JC220, JC254, GS200, JC227, JC300, JC301, JC302, JC303, JC304, JC305, JC306, JC307, JC308, YJN165, KM71, MC100-3, SMD1163, SMD1165, SMD1168, GS241, MS105, any pep4 knock-out strain and any prbl knock-out strain, as well as Pichia pastoris selected from following strains: Bg08, BglO, X-33, SMD1168 and KM71.

[00254] In some embodiments, any Kluyveromyces species can be used as the yeast host cell, including any species of Kluyveromyces, for example, Kluyveromyces lactis, and we teach that the stain of Kluyveromyces lactis can be but is not required to be selected from following strains: GG799, YCT306, YCT284, YCT389, YCT390, YCT569, YCT598, NRRL Y-1140, MW98-8C, MSI, CBS293.91, Y721, MD2/1, PM6-7A, WM37, K6, K7, 22AR1, 22A295-1, SD11, MG1/2, MSK110, JA6, CMK5, HP101, HP108 and PM6-3C, in addition to Kluyveromyces lactis species is selected from GG799, YCT306 and NRRL Y-1140.

[00255] In some embodiments, the yeast host cell can be an Aspergillus oryzae.

[00256] In some embodiments, the yeast host cell can be an Aspergillus japonicas.

[00257] In some embodiments, the yeast host cell can be an Aspergillus niger.

[00258] In some embodiments, the yeast host cell can be a Trichoderma reesei.

[00259] In some embodiments, the yeast host cell can include any species of Hansenula species including any species of Hansenula and preferably Hansenula polymorpha.

[00260] In some embodiments, the yeast host cell can be any species of Yarrowia species for example, Yarrowia lipolytica. [00261] In some embodiments, the yeast host cell can be any species of Schizosaccharomyces species including any species of Schizosaccharomyces and preferably Schizosaccharomyces pombe.

[00262] In some embodiments, the yeast host cell can be a yeast cell that has an endogenous nucleotide sequence operable to encode the enzyme sorbitol dehydrogenase (SDH) (e.g., an endogenous SDH gene).

[00263] In some embodiments, the yeast host cell can be a yeast cell that does not have an endogenous nucleotide sequence operable to encode the enzyme sorbitol dehydrogenase (SDH) (e.g., an endogenous SDH gene).

[00264] In some embodiments, the yeast host cell having an endogenous SDH nucleotide sequence may be from a species within the Candida genus. For example, the yeast cell may be one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[00265] In some embodiments, the yeast cell having an endogenous SDH nucleotide sequence may be from a species within the Kluyveromyces genus. For example, the yeast cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans , or Kluyveromyces wickerhamii.

[00266] In some embodiments, the yeast cell having an endogenous SDH nucleotide sequence may be from a species within the Saccharomyces genus. For example, the yeast cell may be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[00267] In some embodiments, the yeast cell can be selected from a yeast species that does not have an endogenous SDH nucleotide sequence.

[00268] In some embodiments, the yeast cell lacking an endogenous SDH nucleotide sequence may be from a species within the Pichia genus. For example, the yeast cell may be one of the following: Pichia farinose, Pichia anomala, Pichia heedii, Pichia guilliermondii, Pichia kluyveri, Pichia membranifaciens, Pichia norvegensis, Pichia ohmeri, Pichia pastoris, Pichia methanolica, or Pichia subpelliculosa.

[00269] SORBITOL DEHYDROGENASE (SDH)

[00270] The present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide where the heterologous polynucleotide comprises (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00271] Sorbitol dehydrogenase (Enzyme Commission No. EC 1.1.1.14; SDH) is an enzyme capable of, inter alia, converting sorbitol into fructose. Sorbitol dehydrogenase has been found primarily in rosaceous species (i.e., apples and peaches) in plants and also exists in bacteria. The nucleic acid and protein sequences for sorbitol dehydrogenase from a variety of species are known in the art and can be used with the disclosed recombinant yeast cells.

[00272] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is derived from a cell that is a member of the Saccharomycetaceae family. For example, in some embodiments, the nucleotide sequence operable to encode a heterologous SDH may be derived from a species belonging to one of the following genera within the Saccharomycetaceae family: Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.

[00273] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH may be derived from one of the following species: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[00274] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH may be derived from a cell that may be a species within the Candida genus. For example, the nucleotide sequence operable to encode a heterologous SDH may be derived from one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[00275] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can be derived from a species within the Kluyveromyces genus. For example, the nucleotide sequence operable to encode a heterologous SDH may be derived from one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans , or Kluyveromyces wickerhamii.

[00276] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH may be derived from a species within the Saccharomyces genus. For example, the nucleotide sequence operable to encode a heterologous SDH may be derived from one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[00277] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is derived from a yeast cell. For example, a Kluyveromyces lactis, Kluyveromyces marxianus or, Saccharomyces cerevisiae. [00278] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is derived from Kluyveromyces lactis.

[00279] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can have a nucleic acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least

75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least

83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least

87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least

91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least

95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least

99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the nucleic acid sequence set forth in SEQ ID NO: 1.

[00280] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can have a nucleic acid sequence according to the nucleic acid sequence set forth in SEQ ID NO: 1.

[00281] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH is operable to encode an SDH having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 2.

[00282] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is operable to encode an SDH having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 2.

[00283] In some embodiments, the SDH amino acid sequence can be SEQ ID NO: 3 (Xyl2; NCBI Accession No. QEU60545), SEQ ID NO: 4 (KLLA0B00451p; NCBI Accession No. CAHO1943), or SEQ ID NO: 5 (KLLA0D19929p; NCBI Accession No. CAR64382). [00284] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH is operable to encode an SDH having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 3.

[00285] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH is operable to encode an SDH having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 4.

[00286] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH is operable to encode an SDH having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 5. [00287] In some embodiments, the SDH nucleic acid or amino acid sequence is derived from Kluyveromyces marxianus. In some embodiments, the SDH amino acid sequence can be SEQ ID NO: 6 (SORD1; NCBI Accession No. QGN18033.1; XP_022677663.1; BAO41892.1).

[00288] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH is operable to encode an SDH having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 6.

[00289] CYSTEINE RICH PEPTIDES (CRPs)

[00290] The heterologous polynucleotide of the present disclosure comprises at least one nucleotide sequence operable to encode a heterologous polypeptide. In some embodiments, the heterologous polypeptide is a desirable polypeptide, e.g., without limitation, a Cysteine Rich Peptide (CRP).

[00291] CRPs are peptides rich in cysteine residues that, in some embodiments, are operable to form disulfide bonds between such cysteine residues. In some embodiments, CRPs contain at least four (4), sometimes six (6), and sometimes eight (8) cysteine amino acids among proteins or peptides having at least 10 amino acids where the cysteines form two (2), three (3) or four (4) disulfide bonds.

[00292] In some embodiments, the disulfide bonds contribute to the folding, three- dimensional structure, and activity of the peptide. The cysteine-cysteine disulfide bonds, disulfide bond topology, and the resulting three dimensional structure they form, play a significant role in the nature of these peptides (e.g., in some embodiments, the structure plays a role in stability and/or insecticidal nature).

[00293] In some embodiments, a CRP may have a cysteine knot, e.g., an inhibitor cysteine knot (ICK), a growth factor cysteine knot (GFCK), or a cyclic cysteine knot (CCK). In other embodiments, the CRP may not comprise a cysteine knot, e.g., inhibitor cystine knot (ICK) motif. For example, in some embodiments, a CRP with an ICK motif can be an ACTX peptide from a spider; in other embodiments, a CRP without an ICK motif, i.e., a non-ICK CRP, can be a peptide like Av2 and Av3, peptides isolated from sea anemones. Non-ICK CRPS can have 4-8 cysteines which form 2-4 disulfide bonds. These cysteine-cysteine disulfide-bond-stabilized toxic peptides (CRPs) can have remarkable stability when exposed to the environment.

[00294] Several types of CRPs are contemplated and taught herein. Any of the CRPs described herein can be implemented in the practice of the present disclosure. For example, any of the following CRPs can be encoded by a nucleotide sequence operable to encode a heterologous polypeptide of the present disclosure.

[00295] Spider peptides and toxins

[00296] In some embodiments, the present disclosure provides a nucleotide sequence operable to encode a heterologous polypeptide, wherein the heterologous polypeptide is a spider peptide or toxin.

[00297] In some embodiments, the present disclosure provides a nucleotide sequence operable to encode a CRP, wherein the CRP can be a spider toxin peptide or protein derived from one of the following: Phoneutria nigriventer; Allagelena opulenta; Cupiennius salei; Plectreurys tristis; Coremiocnemis valida; Haplopelma huwenum; Agelena orientalis; Allagelena opulenta; Segestria florentina, Apomastus schlingeri; Phoneutria keyserlingi; Macrothele gigas; Macrothele raveni; Missulena bradleyi; Pireneitega luctuosa; Phoneutria reidyi; Illawara wisharti; Eucratoscelus constrictus; Agelenopsis aperta; Hololena curta; Oxyopes lineatus; Brachypelma albiceps; or Brachypelma smithi.

[00298] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP derived from Hadronyche versuta, or the Blue Mountain funnel web spider, Hadronyche venenata, Atrax robustus, Atrax formidabilis, or Atrax infensus.

[00299] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode any of the following spider peptides, polypeptides, and/or toxins: U+2-ACTX-Hvla; T-CNTX-Pnla ; U13-ctenitoxin-Pnla, U13-ctenitoxin- Pnlb,U13-ctenitoxin-Pnlc, Ul-agatoxin-Aopla, Ul-ctenitoxin-Csla, Ul-nemetoxin-Cspla, Ul-nemetoxin-Csplb, Ul-nemetoxin-Csplc, Ul-plectoxin-Ptla, Ul-plectoxin-Ptlb, Ul- plectoxin-Ptlc, Ul-plectoxin-Ptld, Ul-plectoxin-Ptlf, Ul-theraphotoxin-Cvla, Ul- theraphotoxin-Hhla l, Ul-theraphotoxin-Hhla_2, Ul-theraphotoxin-Hhla_3, Ul- theraphotoxin-Hhlb, Ul-theraphotoxin-Hhlc_l, Ul-theraphotoxin-Hhlc_2, Ul- theraphotoxin-Hhld, Ul-theraphotoxin-Hhle, Ul-theraphotoxin-Hhlf_l, Ul-theraphotoxin- Hhlf_2, Ul-theraphotoxin-Hhlf_3, Ul-theraphotoxin-Hhlf_4, Ul-theraphotoxin-Hhlg, U2- agatoxin-Aola, U2-agatoxin-Aopla, U2-ctenitoxin-Csla, U2-ctenitoxin-Pnla, U2- cyrtautoxin-Asla, U2-segestritoxin-Sfl a, U2-segestritoxin-Sflb, U2-segestritoxin-Sfl c, U2- segestritoxin-Sfld, U2-segestritoxin-Sfle, U2-segestritoxin-Sflf, U2-segestritoxin-Sflg, U2- segestritoxin-Sflh, U2-theraphotoxin-Hhla, U3-cyrtautoxin-Asla, U3 -pl ectoxin-Pt la, U5- ctenitoxin-Pnla, U7-ctenitoxin-Pkla, P-hexatoxin-Mgl a, P-hexatoxin-Mrl a, E-ctenitoxin- Pnla, 6-actinopoditoxin-Mbla, 6-Amaurobitoxin-Plla, 6-Amaurobitoxin-Pllb, 6- Amaurobitoxin-Pllc, 6-Amaurobitoxin-Plld, 6-ctenitoxin-Asp2e, 6-ctenitoxin-Pnla_l, 6- ctenitoxin-Pnla_2, 6-ctenitoxin-Pnlb, 6-ctenitoxin-Pn2a, 6-ctenitoxin-Pn2b, 6-ctenitoxin- Pn2c6-ctenitoxin-Pr2d, 6-hexatoxin-Arl a, 6-hexatoxin-Hvla, 6-hexatoxin-Hvlb, 6- hexatoxin-Iwla, 6-hexatoxin-Mgla, 6-hexatoxin-Mglb, K-hexatoxin-Hfla, K-hexatoxin- Hvla, K-hexatoxin-Hvlb, K-hexatoxin-Hvlc_l, K-hexatoxin-Hvlc_2, K-hexatoxin-Hvlc_3, K-hexatoxin-Hvlc_4, K-hexatoxin-Hvld, K-hexatoxin-Hvle, K-theraphotoxin-Ec2a, K- theraphotoxin-Ec2b, p-agatoxin-Aala, p-agatoxin-Aalb, p-agatoxin-Aalc, p-agatoxin-Aald, p-agatoxin-Aale, p-agatoxin-Aalf, p-agatoxin-Hcla, p-agatoxin-Hcl b. p-agatoxin-Hclc, p- hexatoxin-Mgla, p-hexatoxin-Mglb, p-hexatoxin-Mglc, p-hexatoxin-Mg2a, p- theraphotoxin-Hhla, co-actinopoditoxin-Mbla, co-agatoxin-Aa4a, co-agatoxin-Aa4b, co- agatoxin-Aa4c, co-hexatoxin- Ari a l, co-hexatoxin-Arla_3, co-hexatoxin- Ari b l, co- hexatoxin-Arl d_l , co -hexatoxin- Ar ld_4, , co-hexatoxin- Ari e_l co-hexatoxin- Arif, co- hexatoxin-Arlg_l, co -hexatoxin- Ar Ih, co-hexatoxin- Ar2a, co-hexatoxin- Ar2b, co-hexatoxin- Ar2c, co-hexatoxin-Ar2d, co-hexatoxin-Ar2e_l, co-hexatoxin- Ar2e_2, co-atracotoxin-Asp2a, co-hexatoxin- Asp2b, co-hexatoxin-Hf 1 a, co-hexatoxin-Hila_l, co-hexatoxin-Hila_2, co- hexatoxin-Hila_3, co-hexatoxin-Hilb_l, co-hexatoxin-Hilb_10, co-hexatoxin-Hilb_2, co- hexatoxin-Hilb_5, co-hexatoxin-Hilb_8, co-hexatoxin-Hilc_l, co-hexatoxin-Hilc_2, co- hexatoxin-Hvla, co-hexatoxin-Hvlb, co-hexatoxin-Hvlc, co-hexatoxin-Hvld, co-hexatoxin- Hvle, co-hexatoxin-Hvlf, co-hexatoxin-Hvlg_l, co-hexatoxin-Hvlg_5co-hexatoxin-Hvlg_6co- hexatoxin-Hv2a, co-hexatoxin-Hv2b_l, co-hexatoxin-Hv2b_2, co-hexatoxin-Hv2b_3, co- hexatoxin-Hv2b_4, co-hexatoxin-Hv2b_5, co-hexatoxin-Hv2b_6, co-hexatoxin-Hv2b_7, co- hexatoxin-Hv2c, co-hexatoxin-Hv2d_l, co-hexatoxin-Hv2d_2, co-hexatoxin-Hv2d_3, co- hexatoxin-Hv2e, co-hexatoxin-Hv2f, co-hexatoxin-Hv2g, co-hexatoxin-Hv2h_l, co-hexatoxin- Hv2h_2, co-hexatoxin-Hv2i, co-hexatoxin-Hv2j_l, co-hexatoxin-Hv2j_2, co-hexatoxin-Hv2k, co-hexatoxin-Hv21, co-hexatoxin-Hv2m_l, co-hexatoxin-Hv2m_2, co-hexatoxin-Hv2m_3, co- hexatoxin-Hv2n, co-hexatoxin-Hv2o, co-hexatoxin-Hvnla, co-hexatoxin-Hvnlb_l, co- hexatoxin-Hvnlb_2, co-hexatoxin-Hvnlb_3, co-hexatoxin-Hvnlb_4, co-hexatoxin-Hvnlb_6, co-hexatoxin-Iw2a, co-oxotoxin-011b, co-plectoxin-Ptla, co-theraphotoxin-Aspla, co- theraphotoxin-Asplf, co-theraphotoxin-Asplg, co-theraphotoxin-Bala, co-theraphotoxin-Balb, co-theraphotoxin-Bsla, co-theraphotoxin-Bs2a, or co-theraphotoxin-Hh2a.

[00300] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a spider toxin peptide or protein having an amino acid sequence as set forth in any one of SEQ ID NOs: 7-185.

[00301] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least

70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least

82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least

86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least

90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least

94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least

98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 7-185.

[00302] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 7-185. [00303] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 7-185.

[00304] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 7-185.

[00305] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 7-185.

[00306] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 7-185.

[00307] ACTX Peptides

[00308] In some embodiments, a recombinant yeast cell comprises, consists essentially of, or consists of: a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide is a CRP. In some embodiments, the CRP can be an ACTX peptide.

[00309] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode one or more of the following ACTX peptides: U- ACTX-Hvla, U+2-ACTX-Hvla, rU-ACTX-Hvla, rU-ACTX-Hvlb, nc-ACTX-Hvlc, ®- ACTX-Hvla, and/or ®-ACTX-Hvla+2.

[00310] Exemplary ACTX peptides include: U-ACTX-Hvla, having the amino acid sequence “QYCVPVDQPCSLNTQPCCDDATCTQERNENGHTVYYCRA” (SEQ ID NO: 186); U+2-ACTX-Hvla, having the amino acid sequence

Omega-ACTX-Hvla, having the amino acid sequence “SPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 188); “®+2- ACTX-Hvla+2” (or Omega+2-ACTX-Hvla) having the amino acid sequence “GSSPTCIPSGQPCPYNENCCSQSCTFKENENGNTVKRCD” (SEQ ID NO: 189); and Kappa+2-ACTX-Hvla (or K+2-ACTX-Hvla), having the amino acid sequence “GSAICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP” (SEQ ID NO: 190). [00311] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a “Kappa-ACTX-Hvla” (or K+2-ACTX-Hvla) having the amino acid sequence “AICTGADRPCAACCPCCPGTSCKAESNGVSYCRKDEP” (SEQ ID NO: 191).

[00312] In some embodiments, nucleotide sequence operable to encode a heterologous polypeptide is operable to encode an ACTX peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: SEQ ID NOs: 186-191.

[00313] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 186-191.

[00314] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00315] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least

99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00316] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00317] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00318] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00319] r-CNTX-Pnla peptides [00320] In some embodiments, a recombinant yeast cell comprises, consists essentially of, or consists of: a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide is a CRP. In some embodiments, the CRP can be a -CNTX-Pnla or y-CNTX-Pnla toxin. [00321] The T-CNTX-Pnla peptide is an insecticidal neurotoxin derived from the Brazilian armed spider, Phoneutria nigriventer . T-CNTX-Pnla targets the N-methyl-D- aspartate (NMDA)-subtype of ionotropic glutamate receptor (GRIN), and sodium channels. An exemplary wild-type full length T-CNTX-Pnla peptide has an amino acid sequence of: MKVAIVFLSLLVLAFASESIEENREEFPVEESARCADINGACKSDCDCCGDSVTCDCY WSDSCKCRESNFKIGMAIRKKFC (SEQ ID NO: 192) (NCBI Accession No. P59367). A recombinant mature -CNTX-Pnla peptide is provided, having an amino acid sequence of “GSCADINGACKSDCDCCGDSVTCDCYWSDSCKCRESNFKIGMAIRKKFC” (SEQ ID NO: 193).

[00322] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a -CNTX-Pnla having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00323] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193. [00324] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00325] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00326] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00327] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00328] Wild-Type Ul-agatoxins and TVPs

[00329] ‘Hobo spiders” (Eratigena agrestis, formerly Tegenaria agrestis) are venomous spiders that are members of the Agelenidae family of spiders, or funnel web weavers. See Ingale A, Antigenic epitopes prediction and MHC binder of a paralytic insecticidal toxin (ITX-1) of Tegenaria agrestis (hobo spider). 4 August 2010 Volume 2010:2 pp 97-103. The venom of Hobo spiders has been implicated as possessing insecticidal activity. See Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch Insect Biochem Physiol. 1998; 38(1): 19-31 ; Klint et al., Production of Recombinant Disulfide-Rich Venom Peptides for Structural and Functional Analysis via Expression in the Periplasm of E. coli. PLoS One. 2013; 8(5): e63865.

[00330] The Hobo spider — along with several other spiders in the Agelenidae family, produce venom containing agatoxins — which exhibit insecticidal activity. Agatoxins are a chemically diverse group of toxins that can induce various insecticidal effects depending on the target species; .e.g., agatoxins cause slow-onset spastic paralysis in coleopterans, lepidopterans, and dipterans; increase the rate of neuron firing in the central nervous system (CNS) of houseflies (Musca domestica , and are lethal to other insects (e.g., the blowfly, Lucilia cuprina). Accordingly, agatoxins are implicated in targeting the CNS. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators. Structure 23: 1283-1292, and Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19-31(1998).

[00331] Two types of agatoxins include Ul-agatoxin-Tala and Ul-agatoxin-Talb, which are both members of the helical arthropod-neuropeptide-derived (HAND) toxins family. In addition to spiders, these toxins can also be found in the venom of centipedes. The agatoxins are evolutionary offshoots of an ancient ecdysozoan hormone family, i.e., the ion transport peptide/ crustacean hyperglycemic hormone (ITP/CHH) family. See Undheim et al., Weaponization of a hormone: convergent recruitment of hyperglycemic hormone into the venom of arthropod predators. Structure 23: 1283-1292, and Johnson et al., Novel insecticidal peptides from Tegenaria agrestis spider venom may have a direct effect on the insect central nervous system. Arch. Insect Biochem. Physiol. 38:19-31(1998).

[00332] The Hobo-spider-derived Ul-agatoxin-Talb toxin has a full amino acid sequence of “MKLQLMICLVLLPCFFCEPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRN DVYTACHEAQKG (SEQ ID NO: 194)” which includes a signal peptide from amino acid positions 1-17, and the mature toxin from positions 18-68. Id. The protein comprises four tightly packed a-helices, with no [3-strands present, and the molecular mass of the mature toxin is 5700.39 Daltons (Da). Id.

[00333] An exemplary mature wild-type Ul-agatoxin-Talb polypeptide from Eratigena agrestis is provided having the amino acid sequence: “EPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQKG” (SEQ ID NO: 195).

[00334] During protein processing, the mature wild-type Ul-agatoxin-Talb toxin undergoes an excision event of the C-terminal glycine, yielding the following amino acid sequence: EPDEICRARMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYTACHEAQK (SEQ ID NO: 486). A subsequent post-translational event result in the mature wild-type Ul-agatoxin- Talb toxin having a C-terminal amidation.

[00335] In some embodiments, the present disclosure provides a nucleotide sequence operable to encode a heterologous polypeptide, wherein the heterologous polypeptide is a CRP, wherein the CRP is a wild-type Ul-agatoxin-Talb having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 194-195, or 486.

[00336] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 194-195, or 486.

[00337] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 194-195, or 486.

[00338] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 194-195, or 486.

[00339] Ul-agatoxin-Talb Variant Polypeptides (TVPs)

[00340] Ul-agatoxin-Talb Variant Polypeptides (TVPs) are mutants or variants that differ from the wild-type Ul-agatoxin-Talb (SEQ ID NO: 195) in some way, e.g., in some embodiments, this variance can be an amino acid substitution, deletion, or addition; or a change to the polynucleotide encoding the wild-type Ul-agatoxin-Talb resulting in an amino acid substitution, deletion, or addition. The result of this variation is a non-naturally occurring polypeptide and/or polynucleotide sequence encoding the same that possesses enhanced insecticidal activity against one or more insect species relative to the wild-type Ul-agatoxin- Talb.

[00341] In some embodiments, a recombinant yeast cell comprises, consists essentially of, or consists of: a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide is a CRP. In some embodiments, the CRP can be a TVP.

[00342] In some embodiments, the a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least

90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least

94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least

98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least

99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-248, as shown in Table 1. [00343] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-248.

[00344] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and

(c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-248.

[00345] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-248.

[00346] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-248.

[00347] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-248.

[00348] Table 1. TVPs of the present disclosure.

[00349] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP having an amino acid set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

[00350] In some embodiments, a nucleotide sequence operable to encode a TVP has a polynucleotide sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to any polynucleotide sequence set forth in Table 2.

[00351] Table 2. Nucleotide sequences operable to encode a TVP of the present disclosure.

[00352] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP that comprises one or more mutations relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195. For example, in some embodiments, a TVP can have a first, second, or third mutation relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195.

[00353] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP that can have a first mutation relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195 wherein the first mutation is an amino acid substitution of R9Q; K18A; R38A; A8N; A8S; R9N; T1 IP; or T43A.

[00354] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP that can have a first and second mutation relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, e.g., R9QAG; K18AAG; R38AAG; A8NAG; A8SAG; R9NAG; T11PAG; or T43AAG; wherein the first mutation is an amino acid substitution of R9Q; K18A; R38A; A8N; A8S; R9N; or TUP; and wherein the second mutation is a deletion of the C-terminal Glycine.

[00355] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP that can have a first and second mutation relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, e.g., R9QT43A; K18AT43A; R38AT43A; A8NT43A; A8ST43A; R9NT43A; or THPT43A; wherein the first mutation is an amino acid substitution of R9Q; K18A; R38A; A8N; A8S; R9N; or T1 IP; and wherein the second mutation is a T43A amino acid substitution that results in a TVP that is not glycosylated.

[00356] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP that can have a first, second, and third mutation relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, e.g., R9QT43AAG; K18AT43AAG; R38AT43AAG; A8NT43AAG; A8ST43AAG; R9NT43AAG; or T11PT43AAG; wherein the first mutation is an amino acid substitution of R9Q; K18A; R38A; A8N; A8S; R9N; or TUP; and wherein the second mutation is a T43A amino acid substitution that results in a TVP that is not glycosylated; and wherein the third mutation is a deletion of the C-terminal Glycine.

[00357] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a TVP that can have an amino acid sequence according to SEQ ID NOs: 472-477 as shown in Table 3.

[00358] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 472-477.

[00359] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 472-477.

[00360] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 472-477.

[00361] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 472-477. [00362] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 472-477.

[00363] Table 3. Additional TVPs of the present disclosure.

[00364] Exemplary TVPs

[00365] An exemplary description of TVPs, and polynucleotides operable to encode TVP, is provided in International Application No. PCT/US21/28254, the disclosure of which is incorporated herein by reference in its entirety.

[00366] In some embodiments, a TVP comprises one or more mutations relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195. For example, in some embodiments, a TVP can have a first, second, or third mutation relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195.

[00367] In some embodiments, the heterologous polypeptide of the present disclosure can be an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), wherein the TVP can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3-N-K-E-F-T-Y- X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E-A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wildtype sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO:195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X₄ is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent, or a pharmaceutically acceptable salt thereof.

[00368] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP has one amino acid substitution at Xi, X₂, X₃, X4, or X5, or a pharmaceutically acceptable salt thereof.

[00369] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP has one amino acid substitution at Xi, X₂, X₃, X4, or X5; and wherein X7 is Glycine, or a pharmaceutically acceptable salt thereof. [00370] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-XI-X₂-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X₄ is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP has one amino acid substitution at Xi, X₂, X3, X4, or X5; and wherein X7 is absent, or a pharmaceutically acceptable salt thereof.

[00371] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3-

N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP has one amino acid substitution at Xi, X₂, X₃, X4, or X5; and wherein Xe and X7 are absent, or a pharmaceutically acceptable salt thereof.

[00372] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-XI-X₂-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X₄ is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP comprises an amino sequence as set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248, or a pharmaceutically acceptable salt thereof.

[00373] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X₄ is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP is encoded by a polynucleotide sequence as set forth in any one of SEQ ID NOs: 250-302, or a complementary nucleotide sequence thereof.

[00374] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP further comprises a homopolymer or heteropolymer of two or more TVPs, wherein the amino acid sequence of each TVP is the same or different.

[00375] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP is a fused protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each TVP may be the same or different.

[00376] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X4 is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein the TVP is a fused protein comprising two or more TVPs separated by a cleavable or non-cleavable linker, and wherein the amino acid sequence of each TVP may be the same or different, and wherein the linker is cleavable inside the gut or hemolymph of an insect.

[00377] In some embodiments, the linker has an amino acid sequence as set forth in any one of SEQ ID NOs: 187-190, 193, 303-307.

[00378] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (I): E-P-D-E-I-C-R-X1-X2-M-X3- N-K-E-F-T-Y-X4-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-X5-N-D-V-Y-Z1-A-C-H-E- A-Q-X6-X7, wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is A, S, or N; X₂ is R, Q, N, A, G, N, L, D, V, M, I, C, E, T, or S; X₃ is T or P; X₄ is K or A; X₅ is R or A; Zi is T, S, A, F, P, Y, K, W, H, A, G, N, L, V, M, I, Q, C, E, or R; X₆ is K or absent; and X7 is G or absent; and wherein if Zi is T or S, then the TVP is glycosylated, or a pharmaceutically acceptable salt thereof.

[00379] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence “EPDEICRAQMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYAACHEAQKG” (SEQ ID NO: 212).

[00380] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (II): E-P-D-E-I-C-R-A-Xi-M-T- N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-R-N-D-V-Y-Zi-A-C-H-E- A-Q-K-G; wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is R or Q; and Zi is T or A; or a pharmaceutically acceptable salt thereof.

[00381] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (II): E-P-D-E-I-C-R-A-Xi-M-T- N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-R-N-D-V-Y-Zi-A-C-H-E- A-Q-K-G; wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is R or Q; and Zi is T or A; or a pharmaceutically acceptable salt thereof; wherein if Zi is T then the TVP is glycosylated. [00382] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence according to Formula (II): E-P-D-E-I-C-R-A-Xi-M-T- N-K-E-F-T-Y-K-S-N-V-C-N-N-C-G-D-Q-V-A-A-C-E-A-E-C-F-R-N-D-V-Y-Zi-A-C-H-E- A-Q-K-G; wherein the polypeptide comprises at least one amino acid substitution relative to the wild-type sequence of Ul-agatoxin-Talb as set forth in SEQ ID NO: 195, and wherein Xi is R or Q; and Zi is T or A; or a pharmaceutically acceptable salt thereof, wherein Xi is Q; and Zi is A.

[00383] In some embodiments, an insecticidal Ui-agatoxin-Talb variant polypeptide (TVP), can be a TVP comprising an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence as set forth in any one of SEQ ID NOs: 196, 210, or 212, or a pharmaceutically acceptable salt thereof.

[00384] In some embodiments, the TVP may comprise an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence: “EPDEICRAQMTNKEFTYKSNVCNNCGDQVAACEAECFRNDVYAACHEAQKG” (SEQ ID NO: 212).

[00385] In some preferred embodiments, a TVP can be a TVP-R9Q/T43A (SEQ ID NO: 212).

[00386] Scorpion peptides and toxins

[00387] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode any of the following scorpion peptides, polypeptides, and/or toxins: Imperatoxin-A (IpTxa), Potassium channel toxin alpha-KTx 10.2 (Cobatoxin- 2), Potassium channel toxin alpha-KTx 11.1 (Parabutoxin-1), Potassium channel toxin alpha- KTx 11.2 (Parabutoxin-2), Potassium channel toxin alpha-KTx 11.3 (Parabutoxin-10), Potassium channel toxin alpha-KTx 12.1 (Butantoxin), Potassium channel toxin alpha-KTx

12.2 (Butantoxin), Potassium channel toxin alpha-KTx 12.3 (Butantoxin-like peptide), Potassium channel toxin alpha-KTx 15.1 (Peptide Aal), Potassium channel toxin alpha-KTx

15.3 (Toxin AmmTX3), Potassium channel toxin alpha-KTx 15.6 (Discrepin), Potassium channel toxin alpha-KTx 16.1 (Tamulotoxin), Potassium channel toxin alpha-KTx 19.1 (Neurotoxin BmBKTxl), Potassium channel toxin alpha-KTx 1.3 (Iberiotoxin), Potassium channel toxin alpha-KTx 1.4 (Limbatotoxin), Potassium channel toxin alpha-KTx 1.7 (Lqh 15-1), Potassium channel toxin alpha-KTx 1.9 (Hongotoxin-2), Potassium channel toxin alpha-KTx 1.10 (Parabutoxin-3), Potassium channel toxin alpha-KTx 1.11 (Slotoxin), Potassium channel toxin alpha-KTx 1.13 (Charybdotoxin c), Potassium channel toxin alpha- KTx 2.1 (Noxiustoxin), Potassium channel toxin alpha-KTx 2.2 (Margatoxin), Potassium channel toxin alpha-KTx 2.3 (CllTxl), Potassium channel toxin alpha-KTx 2.4 (Noxiustoxin- 2), Potassium channel toxin alpha-KTx 2.5 (Hongotoxin-1), Potassium channel toxin alpha- KTx 2.6 (Hongotoxin-3), Potassium channel toxin alpha-KTx 2.7 (CllTx2), Potassium channel toxin alpha-KTx 2.8 (Toxin Cel), Potassium channel toxin alpha-KTx 2.9 (Toxin Ce2), Potassium channel toxin alpha-KTx 2.10 (Toxin Ce3), Potassium channel toxin alpha- KTx 2.11 (Toxin Ce4), Potassium channel toxin alpha-KTx 2.12 (Toxin Ce5), Potassium channel toxin alpha-KTx 3.1 (Kaliotoxin-1), Potassium channel toxin alpha-KTx 3.2 (Agitoxin-2), Potassium channel toxin alpha-KTx 3.3 (Agitoxin-3), Potassium channel toxin alpha-KTx 3.4 (Agitoxin-1), Potassium channel toxin alpha-KTx 3.7 (OsK-1), Potassium channel toxin alpha-KTx 3.8 (Charybdotoxin-like peptide Bs 6), Potassium channel toxin alpha-KTx 3.9 (Kaliotoxin-3), Potassium channel toxin alpha-KTx 4.1 (Tityustoxin K-alpha), Potassium channel toxin alpha-KTx 4.3 (Toxin TdKl), Potassium channel toxin alpha-KTx 4.4 (Toxin Tc30), Potassium channel toxin alpha-KTx 5.1 (Leiurotoxin-1), Potassium channel toxin alpha-KTx 5.2 (Lei urotoxin I-like toxin P05), Potassium channel toxin alpha- KTx 5.4 (Tamapin), Potassium channel toxin alpha-KTx 5.5 (Tamapin-2), Potassium channel toxin alpha-KTx 6.1 (Potassium channel-blocking toxin 1), Potassium channel toxin alpha- KTx 6.2 (Maurotoxin), Potassium channel toxin alpha-KTx 6.3 (Neurotoxin HsTXl), Potassium channel toxin alpha-KTx 6.12 (Anuroctoxin), Potassium channel toxin alpha-KTx 6.13 (Spinoxin), Potassium channel toxin alpha-KTx 6.14 (HgeTxl), Potassium channel toxin alpha-KTx 7.2 (Toxin PiTX-K-beta), Potassium channel toxin gamma-KTx 1.2 (Ergtoxin- like protein 1), Potassium channel toxin gamma-KTx 1.3 (Ergtoxin-like protein 1), Potassium channel toxin gamma-KTx 1.4 (Ergtoxin-like protein 1), Potassium channel toxin gamma- KTx 1.5 (Ergtoxin-like protein 1), Potassium channel toxin gamma-KTx 1.6 (Ergtoxin-like protein 1), Potassium channel toxin gamma-KTx 4.2 (Ergtoxin-like protein 5), Insectotoxin- II. Small toxin (Peptide I), Insectotoxin-I3 (BeI3), Insectotoxin-I4 (BeI4), Insectotoxin-I5A, Neurotoxin 8 (Neurotoxin VIII), Probable toxin Lqh 8/6, Neurotoxin 9 (Neurotoxin IX), Maurocalcin (MCa), Chlorotoxin-like peptide Bs 14 (Bsl4), Chlorotoxin (CTX), Neurotoxin P2, Insectotoxin-I5 (Beto), Potassium channel toxin alpha-KTx 6.15 (Hemitoxin), Toxin GaTxl, AahlTl, Phaiodotoxin, BaIT2, BotITl, BotIT2, BmK Ml, BmK-M2, BmK-M4, BmK-M7, BmK IT-AP, Bom3, Bom4, BjalT, Bj-xtrlT, BjIT2, LqhalT, Lqhbl, LqhIT2, LqhdprIT3a, Lgh-xtrlT, Lqh3, Lqh6, Lqh7, LqqlTl, LqqIT2, Lqq3, OD1, Tsl, or Tzl. [00388] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a scorpion peptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 308-411.

[00389] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 308-411.

[00390] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 308-411. [00391] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 308-411.

[00392] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 308-411.

[00393] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 308-411.

[00394] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode an imperatoxin. Imperatoxins are peptide toxins derived from the venom of the African scorpion (Pandinus imperato ).

[00395] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode an imperatoxin, wherein the imperatoxin is Imperatoxin A (IpTx-a), or a variant thereof.

[00396] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode an IpTx-a having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence: GDCLPHLKRCKADNDCCGKKCKRRGTNAEKRCR (SEQ ID NO: 303).

[00397] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode an AalTl toxin. The protein toxin, AalTl, is a sodium channel site 4 toxin from North African desert scorpion (Androctonus australis'). An exemplary AalTl toxin is a peptide having the amino acid sequence according to SEQ ID NO: 308 (NCBI accession No. P01497.2). AalTl is a site 4 toxin, which forces the insect sodium channel to open by lowering the activation reaction energy barrier.

[00398] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a scorpion peptide or toxin having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 303 or 308-411.

[00399] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 303 or 308-411.

[00400] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 303 or 308-411.

[00401] Sea anemone peptides and toxins

[00402] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a peptide derived from a sea anemone. For example, in some embodiments, the sea anemone can be Actinia equina,' Anemonia erythraea,' Anemonia sulcata, Anemonia viridis,' Anthopleura elegantissima,' Anthopleura fuscoviridis,' Anthopleura xanthogrammica,' Bunodosoma caissarum,' Bunodosoma cangicum; Bunodosoma granulifera, Heteractis crispa,' Parasicyonis actinostoloides,' Radianthus paumotensis,' or Stoichactis helianthus. In yet other embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode sea anemone toxin that can be Av2; an Av3; or a variant thereof.

[00403] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode one of the following sea anemone toxins: Toxin AETX- 1 (AETX I), Toxin APETxl, Toxin APETx2, Antihypertensive protein BDS-1 (Blood depressing substance I), Antihypertensive protein BDS-2 (Blood depressing substance II), Neurotoxin Bg-2 (Bg II), Neurotoxin Bg-3 (Bg III), Toxin APE 1-1, Toxin APE 1-2, Neurotoxin- 1 (Toxin ATX-I), Neurotoxin- 1 (Neurotoxin I), Neurotoxin 1 (Toxin RTX-I), Neurotoxin 1 (Toxin SHP-I), Toxin APE 2-1, Toxin APE 2-2, Neurotoxin-2 (Toxin ATX-II), (aka AV2)Neurotoxin-2 (Toxin AFT-II), Neurotoxin 2 (Toxin RTX-II), Neurotoxin 2 (Neurotoxin II), Neurotoxin 3 homolog (Neurotoxin III homolog), Neurotoxin 3 (Toxin RTX-III), Neurotoxin 3 (Neurotoxin-III), Neurotoxin 4 (Toxin RTX-IV), Neurotoxin-5 (Toxin ATX-V), Neurotoxin 5 (Toxin RTX-V), Anthopleurin-A (Toxin AP-A), Anthopleurin-B (Toxin AP-B), Anthopleurin-C (Toxin AP-C), Potassium channel toxin Aek, Potassium channel toxin Bgk, Major neurotoxin Belli, Neurotoxin BcIV, Cangitoxin (CGTX), Potassium channel toxin ShK, Toxin PCR1 (PCR1-2), Toxin PCR2 (PCR2-5), Toxin PCR3 (PCR2-1), Toxin PCR4 (PCR2-10), Toxin PCR6 (PCR3-7), Cangitoxin-2 (Cangitoxin II), or Cangitoxin-3 (Cangitoxin III).

[00404] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a sea anemone peptide having an amino acid sequence as set forth in SEQ ID NOs: 412-452.

[00405] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-452.

[00406] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-452.

[00407] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-452.

[00408] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-452.

[00409] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-452.

[00410] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode one or more polypeptides derived from the sea anemone, Anemonia viridis, which possesses a variety of toxins that it uses to defend itself. One of the toxins derived ixomAnemonia viridis is the neurotoxin “Av3.” Av3 is a type III sea anemone toxin that inhibits the inactivation of voltage-gated sodium (Na⁺) channels at receptor site 3, resulting in contractile paralysis. The binding of an Av3 toxin to site 3 results in the inactivated state of the sodium channel to become destabilized, which in turn causes the channel to remain in the open position (see Blumenthal et al., Voltage-gated sodium channel toxins: poisons, probes, and future promise. Cell Biochem Biophys. 2003; 38(2):215- 38). Av3 shows high selectivity for crustacean and insect sodium channels, and low selectivity for mammalian sodium channels (see Moran et al., Sea anemone toxins affecting voltage-gated sodium channels - molecular and evolutionary features, Toxicon. 2009 Dec 15; 54(8): 1089-1101). An exemplary Av3 polypeptide fxomAnemonia viridis is provided having the amino acid sequence of SEQ ID NO:453.

[00411] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode an Av3 variant polypeptide (AVP). In some embodiments, AVPs can have the following amino acid variations from SEQ ID NO:453: an N-terminal amino acid substitution of R1K relative to SEQ ID NO:453, changing the polypeptide sequence from the wild-type “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “KSCCPCYWGGCPWGQNCYPEGCSGPKV” (SEQ ID NO:454); C-terminal amino acid can be deleted relative to SEQ ID NO:453, changing the polypeptide sequence from the wildtype “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “RSCCPCYWGGCPWGQNCYPEGCSGPK” (SEQ ID NO:455); and/or an N-terminal mutation and a C-terminal mutation, wherein the N-terminal amino acid can have a substitution of R1K relative to SEQ ID NO:453, and the C-terminal amino acid can be deleted relative to SEQ ID NO:453, changing the polypeptide sequence from the wild-type “RSCCPCYWGGCPWGQNCYPEGCSGPKV” to “KSCCPCYWGGCPWGQNCYPEGCSGPK” (SEQ ID NO:456).

[00412] In some embodiments, an illustrative Av3 peptide or variant thereof is described in the Applicant’s PCT application (Application No. PCT/US19/51093) filed Sept. 13, 2019, entitled “Av3 Mutant Insecticidal Polypeptides and Methods for Producing and Using Same,” the disclosure of which, and the disclosure of Av3 peptides or variants thereof, are described and are incorporated by reference herein in its entirety.

[00413] In some embodiments, a polynucleotide encoding a sea anemone peptide can encode a sea anemone peptide having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NOs: 412-457.

[00414] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-457.

[00415] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-457.

[00416] In some embodiments, a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, comprises: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-457.

[00417] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide; and wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-457.

[00418] In some embodiments, a polynucleotide of the present disclosure comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof; wherein the heterologous polypeptide has an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 412-457.

[00419] Cone shell peptides and conotoxins

[00420] Conotoxins are toxins isolated from cone shells; these toxins act by interfering with neuronal communication. Examples of conotoxins include the a-, co-, p-, 6-, and K- conotoxins. Briefly, the a-conotoxins (and aA- &cp-conotoxins) target nicotinic ligand gated channels; co-conotoxins target voltage-gated calcium channels; p-conotoxins target the voltage-gated sodium channels; 6-conotoxins target the voltage-gated sodium channel; and K- conotoxins target the voltage-gated potassium channel.

[00421] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP derived from organisms belonging to the Conus genus, wherein the peptide isolated is a conotoxin.

[00422] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP derived from Conus amadis; Conus catus; Conus ermineus; Conus geographus; Conus gloriamaris; Conus kinoshitai,' Conus magus,' Conus marmoreus; Conus purpurascens,' Conus stercusmuscarum; Conus striatus,' Conus textile,' or Conus tulipa.

[00423] Other CRPs

[00424] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that can be a toxin, peptide, or protein (otherwise known as a venom- or poison- peptide or protein) that is produced and/or isolated from an arthropod, a spider, a scorpion, an insect, a bee, a wasp, a centipede, a crustacean, a reptile, a snake, a lizard, an amphibian, a frog, a salamander, a mollusk, a cone shell, a cnidarian, a sea anemone, a jellyfish, a hydrozoan, a cephalopod, an octopus, a squid, a cuttlefish, a fish, or a mammal.

[00425] In some embodiments, a nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a protein derived from a snake venom, or toxin therefrom.

[00426] CRP-modified Proteins [00427] CRP -modified proteins are any protein, peptide, polypeptide, amino acid sequence, configuration, or arrangement, consisting of: (1) at least one CRP, or two or more CRPs; and (2) additional non-CRP peptides, polypeptides, or proteins that, e.g., in some embodiments, have the ability to do the following: increase the mortality and/or inhibit the growth of insects when the insects are exposed to a CRP-modified protein, relative to a CRP alone; increase the expression of said CRP-modified protein, e.g., in a host cell or an expression system; and/or affect the post-translational processing of the CRP-modified protein.

[00428] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated; and wherein the heterologous polypeptide is a CRP-modified protein.

[00429] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the heterologous polypeptide is a CRP-modified protein.

[00430] In some embodiments, a CRP-modified protein can be a polymer comprising two or more CRPs. In some embodiments, a CRP-modified protein can be a polymer comprising two or more CRPs, wherein the CRPs are operably linked via a linker peptide, e.g., a cleavable and/or non-cleavable linker.

[00431] In some embodiments, a CRP-modified protein can refer to a one or more CRPs operably linked with one or more proteins such as a stabilizing domain (STA); an endoplasmic reticulum signaling protein (ERSP); an insect cleavable or insect non-cleavable linker (L); and/or any other combination thereof.

[00432] In some embodiments, a CRP-modified protein can be a non-naturally occurring protein comprising (1) a wild-type CRP; and (2) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.

[00433] In some embodiments, a CRP-modified protein can be a non-naturally occurring protein comprising (1) a wild-type CRP; and (2) a non-naturally occurring CRP. [00434] In some embodiments, a CRP-modified protein can be a non-naturally occurring protein comprising (1) a wild-type CRP; and (2) a non-naturally occurring CRP; and (3) additional peptides, polypeptides, or proteins, e.g., an ERSP; a linker; a STA; a UBI; or a histidine tag or similar marker.

[00435] In some embodiments, a CRP-modified protein can comprise any of the CRPs described herein.

[00436] In some embodiments, an insecticidal protein can comprise a one or more CRPs as disclosed herein. In some embodiments, the insecticidal protein can comprise a CRP homopolymer, e.g., two or more CRP monomers that are the same CRP. In some embodiments, the insecticidal protein can comprise a CRP heteropolymer, e.g., two or more CRP monomers, wherein the CRP monomers are different.

[00437] In some embodiments, an insecticidal protein can comprise a fused protein comprising two or more CRPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRP may be the same or different.

[00438] In some embodiments, an insecticidal protein can comprise a fused protein comprising two or more CRPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRP may be the same or different, wherein the linker is cleavable inside the gut or hemolymph of an insect.

[00439] In some embodiments, an insecticidal protein can comprise a fused protein comprising two or more CRPs separated by a cleavable or non-cleavable linker, wherein the amino acid sequence of each CRP may be the same or different, wherein the linker is cleavable inside the gut of a mammal. [00440] Exemplary methods for the generation of cleavable and non-cleavable linkers can be found in U.S. Patent Application No. 15/727,277; and PCT Application No. PCT/US2013/030042, the disclosure of which are incorporated by reference herein in their entirety.

[00441] Exemplary CRPs

[00442] Exemplary CRPs encoded by the nucleotide sequence operable to encode a heterologous polypeptide, include without limitation, a Ul-agatoxin-Talb peptide; a Ul- agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; and/or an Atracotoxin (ACTX).

[00443] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a Ul-agatoxin-Talb peptide having an amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 195. In some embodiments, the Ul-agatoxin-Talb peptide has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 195.

[00444] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a Ul-agatoxin-Talb peptide having an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 195. In some embodiments, the Ul-agatoxin-Talb peptide has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 195.

[00445] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a TVP having an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

[00446] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a TVP having an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

[00447] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a TVP having an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

[00448] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a sea anemone toxin, wherein the sea anemone toxin can be an Av2 toxin, or an Av3 toxin. In some embodiments, the Av2 toxin can have an amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in amino acid sequence set forth in SEQ ID NO: 457.

[00449] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a sea anemone toxin that can be an Av2 toxin, or an Av3 toxin. In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an Av2 toxin having amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 457.

[00450] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an Av2 toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 457. [00451] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an Av3 toxin having an amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in amino acid sequence set forth in SEQ ID NO: 453.

[00452] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an Av3 toxin having an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 453.

[00453] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an Av3 toxin having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 453.

[00454] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an AVP having an amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

[00455] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an AVP having an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

[00456] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an AVP having an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 454-456. [00457] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a ctenitoxin (CNTX).

[00458] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CNTX, wherein the CNTX can be T-CNTX-Pnla.

[00459] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a CRP that is a T-CNTX-Pnla having an amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00460] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a T-CNTX-Pnla having an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00461] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode a T-CNTX-Pnla having an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 193.

[00462] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX.

[00463] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX, wherein the ACTX can be a U-ACTX peptide, Omega- ACTX peptides, or Kappa- ACTX peptide.

[00464] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX that is a U-ACTX-Hvla, a U+2-ACTX- Hvla, a rU-ACTX-Hvla, a rU-ACTX-Hvlb, a K-ACTX-Hvla, a K+2-ACTX-Hvla, a ®- ACTX-Hvla, or a ®+2-ACTX-Hvla.

[00465] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX having amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00466] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX having an amino acid sequence that is that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to an amino acid sequence set forth in SEQ ID NO: 187.

[00467] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX having an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00468] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX having an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 187.

[00469] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX having an amino acid sequence consisting of an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00470] In some embodiments, the nucleotide sequence operable to encode a heterologous polypeptide can encode an ACTX having an amino acid sequence consisting of an amino acid sequence set forth in SEQ ID NO: 187.

[00471] METHODS OF THE PRESENT DISCLOSURE

[00472] This section describes methods for making the recombinant yeast cells of the present disclosure (described above); methods of making vectors; methods of making polynucleotides; along with methods for increasing the expression of a heterologous polypeptide. The present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00473] In some embodiments, a host cell (e.g., a yeast host cell) can be transformed using recombinant techniques known in the art, in order to produce a recombinant yeast cell of the present disclosure, and/or to produce a recombinant yeast cell as part of a method of increasing expression of a heterologous polypeptide.

[00474] In some embodiments, the yeast cell selected can be any yeast cell described herein (see “HOST CELL” section above). For example, in some embodiments, a yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as the yeast cell to be modified, to produce a recombinant yeast cell.

[00475] In some embodiments, to produce a recombinant yeast cell of the present disclosure, a yeast host cell (e.g., any yeast cell described herein) can be transformed with one or more vectors, wherein the one or more vectors comprises a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide. In some embodiments, the heterologous polynucleotide can comprise one or more nucleotide sequence operable to encode a heterologous SDH (e.g., 1 to 20 copies), and/or one or more nucleotide sequence operable to encode a heterologous polypeptide (e.g., 1 to 20 copies).

[00476] The terms “transformation” and “transfection” both describe the process of introducing exogenous and/or heterologous DNA or RNA to a host organism. Generally, those having ordinary skill in the art sometimes reserve the term “transformation” to describe processes where exogenous and/or heterologous DNA or RNA are introduced into a bacterial cell; and reserve the term “transfection” for processes that describe the introduction of exogenous and/or heterologous DNA or RNA into eukaryotic cells. However, as used herein, the term “transformation” and “transfection” are used synonymously, regardless of whether a process describes the introduction exogenous and/or heterologous DNA or RNA into a prokaryote (e.g., bacteria) or a eukaryote (e.g., yeast, plants, or animals).

[00477] Making the heterologous polynucleotide

[00478] The heterologous polynucleotide of the present disclosure, comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, can be created using a variety of techniques known in the art. The sections below describe how to make the heterologous nucleotide, and how to make each of its component parts, i.e., the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and nucleotide sequence operable to encode a heterologous polypeptide.

[00479] Making nucleotide sequences operable to encode a heterologous SDH [00480] Nucleotide sequences encoding a heterologous SDH, and the resulting amino acid sequences encoded by the same, are well known in the art. And the nucleotide and amino acid sequences of sorbitol dehydrogenase from a variety of species are likewise well known in the art, and can be used with the recombinant yeast cells, constructs, and/or methods disclosed herein.

[00481] In some embodiments, a recombinant yeast cell of the present disclosure comprises a heterologous polynucleotide, wherein said heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and wherein (a) the recombinant yeast cell is selected from a yeast species that does not possess an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene); or (b) the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene), that is at least partially inactivated; and wherein the at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH that is operable to convert sorbitol into fructose. [00482] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a eukaryote. [00483] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a prokaryote. [00484] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a plant. [00485] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a rosaceous species (e.g., apples and peaches).

[00486] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a bacteria. [00487] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a fungi. [00488] In some embodiments, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), encodes any SDH derived from a yeast cell. [00489] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH has a nucleic acid or amino acid sequence that is derived from an organism belonging to the Saccharomycetaceae family. For example, in some embodiments, the organism may be one of the following genera within the Saccharomycetaceae family: Brettanomyces, Candida, Citeromyces, Cyniclomyces, Debaryomyces, Issatchenkia, Kazachstania, Kluyveromyces, Komagataella, Kuraishia, Lachancea, Lodderomyces, Nakaseomyces, Pachysolen, Pichia, Saccharomyces, Spathaspora, Tetrapisispora, Vanderwaltozyma, Torulaspora, Williopsis, Zygosaccharomyces, or Zygotorulaspora.

[00490] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can be derived from a cell that may be one of the following: Aspergillus flavus, Aspergillus terreus, Aspergillus awamori, Cladosporium elatum, Cladosporium Herbarum, Cladosporium Sphaerospermum, Cladosporium cladosporioides, Magnaporthe grisea, Magnaporthe oryzae, Magnaporthe rhizophila, Morchella deliciosa, Morchella esculenta, Morchella conica, Neurospora crassa, Neurospora intermedia, Neurospora tetrasperma, Penicillium notatum, Penicillium chrysogenum, Penicillium roquefortii, or Penicillium simplicissimum.

[00491] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can be derived from a species within the Candida genus. For example, the nucleotide sequence operable to encode a heterologous SDH can be derived from one of the following: Candida albicans, Candida ascalaphidarum, Candida amphixiae, Candida antarctica, Candida argentea, Candida atlantica, Candida atmosphaerica, Candida auris, Candida blankii, Candida blattae, Candida bracarensis, Candida bromeliacearum, Candida carpophila, Candida carvajalis, Candida cerambycidarum, Candida chauliodes, Candida corydalis, Candida dosseyi, Candida dubliniensis, Candida ergatensis, Candida fructus, Candida glabrata, Candida fermentati, Candida guilliermondii, Candida haemulonii, Candida humilis, Candida insectamens, Candida insectorum, Candida intermedia, Candida jeffresii, or Candida kefyr.

[00492] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can be derived from a species within the Kluyveromyces genus. For example, the cell may be one of the following: Kluyveromyces aestuarii, Kluyveromyces dobzhanskii, Kluyveromyces lactis, Kluyveromyces marxianus, Kluyveromyces nonfermentans , or Kluyveromyces wickerhamii .

[00493] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is derived from a species within the Saccharomyces genus. For example, the cell may be one of the following: Saccharomyces arboricolus, Saccharomyces bayanus, Saccharomyces bulderi, Saccharomyces cariocanus, Saccharomyces cariocus, Saccharomyces cerevisiae, Saccharomyces cerevisiae var boulardii, Saccharomyces chevalieri, Saccharomyces dairenensis, Saccharomyces ellipsoideus, Saccharomyces eubayanus, Saccharomyces exiguous, Saccharomyces florentinus, Saccharomyces fragilis, Saccharomyces kudriavzevii, Saccharomyces martiniae, Saccharomyces mikatae, Saccharomyces monacensis, Saccharomyces norbensis, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces spencerorum, Saccharomyces turicensis, Saccharomyces unisporus, Saccharomyces uvarum, or Saccharomyces zonatus.

[00494] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is derived from a yeast cell. For example, a Kluyveromyces lactis, Kluyveromyces marxianus or, Saccharomyces cerevisiae.

[00495] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH is derived from Kluyveromyces lactis.

[00496] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can have a nucleotide sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least 83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least 87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least 91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least 95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least 99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least

99.8% identical, at least 99.9% identical, or 100% identical to the nucleotide set forth in SEQ ID NO: 1.

[00497] In some embodiments, the nucleotide sequence operable to encode a heterologous can have a nucleotide consisting of the nucleotide sequence as set forth in SEQ ID NO: 1.

[00498] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least

75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least

83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least

87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least

91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least

95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least

99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 2.

[00499] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence consisting of the amino acid sequence as set forth in SEQ ID NO: 2.

[00500] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least

75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least

83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least

87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least

91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least

95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least

99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 3. [00501] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence consisting of an amino acid sequence as set forth in SEQ ID NO: 3 (Xyl2; NCBI Accession No. QEU60545).

[00502] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least

75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least

83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least

87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least

91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least

95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least

99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 4.

[00503] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence consisting of an amino acid sequence as set forth in SEQ ID NO: 4 (KLLA0B00451p; NCBI Accession No. CAHO1943).

[00504] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least

75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least

83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least

87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least

91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least

95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least

99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 5.

[00505] In some embodiments, the nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence consisting of an amino acid sequence as set forth in SEQ ID NO: 5 (KLLA0D19929p; NCBI Accession No. CAR64382).

[00506] In some embodiments, a nucleotide sequence operable to encode a heterologous SDH can encode an amino acid sequence that is at least 50% identical, at least 55% identical, at least 60% identical, at least 65% identical, at least 70% identical, at least 75% identical, at least 80% identical, at least 81% identical, at least 82% identical, at least

83% identical, at least 84% identical, at least 85% identical, at least 86% identical, at least

87% identical, at least 88% identical, at least 89% identical, at least 90% identical, at least

91% identical, at least 92% identical, at least 93% identical, at least 94% identical, at least

95% identical, at least 96% identical, at least 97% identical, at least 98% identical, at least

99% identical, at least 99.5% identical, at least 99.6% identical, at least 99.7% identical, at least 99.8% identical, at least 99.9% identical, or 100% identical to the amino acid sequence to the amino acid sequence SEQ ID NO: 6.

[00507] In some embodiments, the SDH nucleic acid or amino acid sequence is derived from Kluyveromyces marxianus. In some embodiments, the SDH amino acid sequence can be SEQ ID NO: 6 (SORD1; NCBI Accession No. QGN18033. 1; XP_022677663.1; BAO41892.1).

[00508] Making nucleotide sequences encoding a heterologous polypeptide

[00509] In some embodiments, a recombinant yeast cell of the present disclosure comprises a heterologous polynucleotide, wherein said heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; and wherein (a) the recombinant yeast cell is selected from a yeast species that does not possess an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene); or (b) the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene), that is at least partially inactivated; and wherein the at least one nucleotide sequence operable to encode a heterologous polypeptide encodes a CRP.

[00510] The nucleotide sequence operable to encode a heterologous polypeptide, wherein the heterologous polypeptide is a CRP, can be derived from a wild-type source. For example, in some embodiments, a CRP can be obtained directly from the source (e.g., isolating said CRP from an organism). Subsequently, the nucleotide sequence can be determined using techniques known to those having ordinary skill in the art. Likewise, a mutant CRP can be generated by creating a mutation in the wild-type CRP polynucleotide sequence; inserting that CRP polynucleotide sequence into the appropriate vector; transforming a host organism in such a way that the nucleotide sequence encoding a CRP is expressed; culturing the host organism to generate the desired amount of CRP; and then purifying the CRP from in and/or around host organism. [00511] Producing a mutation in wild-type CRP polynucleotide sequence can be achieved by various means that are well known to those having ordinary skill in the art. Methods of mutagenesis include Kunkel’s method; cassette mutagenesis; PCR site-directed mutagenesis; the “perfect murder” technique (delitto perfetto),- direct gene deletion and sitespecific mutagenesis with PCR and one recyclable marker; direct gene deletion and sitespecific mutagenesis with PCR and one recyclable marker using long homologous regions; transplacement “pop-in pop-out” method; and CRISPR-Cas 9. Exemplary methods of site- directed mutagenesis can be found in Ruvkun & Ausubel, A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1; 289(5793):85-8; Wallace et al., Oligonucleotide directed mutagenesis of the human beta-globin gene: a general method for producing specific point mutations in cloned DNA. Nucleic Acids Res. 1981 Aug 11;

9(15):3647-56; Dalbadie-McFarland et al., Oligonucleotide-directed mutagenesis as a general and powerful method for studies of protein function. Proc Natl Acad Sci U S A. 1982 Nov; 79(21):6409-13; Bachman. Site-directed mutagenesis. Methods Enzymol. 2013; 529:241-8; Carey et al., PCR-mediated site-directed mutagenesis. Cold Spring Harb Protoc. 2013 Aug 1; 2013(8):738-42; and Cong et al., Multiplex genome engineering using CRISPR/Cas systems. Science. 2013 Feb 15; 339(6121):819-23, the disclosures of all of the aforementioned references are incorporated herein by reference in their entireties.

[00512] Wild-type CRPs, e.g., spider, scorpion, and/or other toxins can be isolated from the venom. For example, spider venom can be isolated from the venom glands of spiders (e.g., spiders such as Eratigena agresiis). using any of the techniques known to those having ordinary skill in the art. For example, in some embodiments, venom can be isolated from spiders according to the methods described in U.S. Patent No 5,688,764, the disclosure of which is incorporated herein by reference in its entirety.

[00513] A wild-type CRP polynucleotide sequence can be obtained by screening a genomic library using primer probes directed to the CRP polynucleotide sequence. Alternatively, wild-type CRP polynucleotide sequence and/or mutant CRP polynucleotide sequences can be chemically synthesized. For example, a CRP polynucleotide sequence and/or mutant CRP polynucleotide sequence can be generated using the oligonucleotide synthesis methods such as the phosphoramidite; triester, phosphite, or H-Phosphonate methods. See Engels, J. W. and Uhlmann, E. (1989), Gene Synthesis (New Synthetic Methods (77)). Angew. Chem. Int. Ed. Engl., 28: 716-734, the disclosure of which is incorporated herein by reference in its entirety.

[00514] Chemically synthesizing a heterologous polynucleotide [00515] In some embodiments, a heterologous polynucleotide and/or the nucleotide sequence encoding a heterologous polypeptide and/or a heterologous SDH can be chemically synthesized using commercially available polynucleotide synthesis services such as those offered by GENEWIZ® (e.g., TurboGENE™; PriorityGENE; and FragmentGENE), or SIGMA- ALDRICH® (e.g., Custom DNA and RNA Oligos Design and Order Custom DNA Oligos). Exemplary methods for generating DNA and or custom chemically synthesized polynucleotides are well known in the art, and are illustratively provided in U.S. Patent No. 5,736,135, Serial No. 08/389,615, filed on Feb. 13, 1995, the disclosure of which is incorporated herein by reference in its entirety. See also Agarwal, et al., Chemical synthesis of polynucleotides. Angew Chem Int Ed Engl. 1972 Jun; 11 (6):451-9; Ohtsuka et al., Recent developments in the chemical synthesis of polynucleotides. Nucleic Acids Res. 1982 Nov 11; 10(21): 6553-6570; Sondek & Shortle. A general strategy for random insertion and substitution mutagenesis: substoichiometric coupling of trinucleotide phosphorami dites. Proc Natl Acad Sci U S A. 1992 Apr 15; 89(8): 3581-3585; Beaucage S. L., et al., Advances in the Synthesis of Oligonucleotides by the Phosphorami dite Approach. Tetrahedron, Elsevier Science Publishers, Amsterdam, NL, vol. 48, No. 12, 1992, pp. 2223-2311; Agrawal (1993) Protocols for Oligonucleotides and Analogs: Synthesis and Properties; Methods in Molecular Biology Vol. 20, the disclosures of which are incorporated herein by reference in their entirety.

[00516] Chemically synthesizing polynucleotides allows for a DNA sequence to be generated that is tailored to produce a desired polypeptide based on the arrangement of nucleotides within said sequence (i.e., the arrangement of cytosine [C], guanine [G], adenine [A] or thymine [T] molecules); the mRNA sequence that is transcribed from the chemically synthesized DNA polynucleotide can be translated to a sequence of amino acids, each amino acid corresponding to a codon in the mRNA sequence. Accordingly, the amino acid composition of a polypeptide chain that is translated from an mRNA sequence can be altered by changing the underlying codon that determines which of the 20 amino acids will be added to the growing polypeptide; thus, mutations in the DNA such as insertions, substitutions, deletions, and frameshifts may cause amino acid insertions, substitutions, or deletions, depending on the underlying codon.

[00517] Obtaining a nucleotide sequence operable to encode a heterologous SDH and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP) from a chemically synthesized DNA polynucleotide sequence and/or a wild-type DNA polynucleotide sequence that has been altered via mutagenesis can be achieved by cloning the DNA sequence into an appropriate vector. There are a variety of expression vectors available, host organisms, and cloning strategies known to those having ordinary skill in the art. For example, the vector can be a plasmid, which can introduce a heterologous gene and/or expression cassette into yeast cells to be transcribed and translated. The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A vector may contain “vector elements” such as an origin of replication (ORI); a gene that confers antibiotic resistance to allow for selection; multiple cloning sites; a promoter region; a selection marker for non-bacterial transfection; and a primer binding site. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated herein by reference. In addition to encoding a CRP polynucleotide, a vector may encode a targeting molecule. A targeting molecule is one that directs the desired nucleic acid to a particular tissue, cell, or other location.

[00518] Copies of the heterologous polynucleotide and ratios of the nucleotide sequences

[00519] In some embodiments, the recombinant yeast cell can be transformed with one or more copies of a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide. For example, in some embodiments, the recombinant yeast cell may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies of the heterologous polynucleotide.

[00520] In some embodiments, a heterologous polynucleotide of the present disclosure can comprise multiple copies of: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof. For example, in some embodiments, the polynucleotide may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies of (i), (ii), or a combination thereof. [00521] In some embodiments, a vector of the present disclosure can comprise multiple copies of: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof. For example, in some embodiments, the polynucleotide may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies of (i), (ii), or a combination thereof.

[00522] In some embodiments, the recombinant yeast cell may have from about 1 to about 20 copies; from about 2 to about 20 copies; from about 3 to about 20 copies; from about 4 to about 20 copies; from about 5 to about 20 copies; from about 6 to about 20 copies; from about 7 to about 20 copies; from about 8 to about 20 copies; from about 9 to about 20 copies; from about 10 to about 20 copies; from about 11 to about 20 copies; from about 12 to about 20 copies; from about 13 to about 20 copies; from about 14 to about 20 copies; from about 15 to about 20 copies; from about 16 to about 20 copies; from about 17 to about 20 copies; from about 18 to about 20 copies; from about 19 to about 20 copies; of the heterologous polynucleotide.

[00523] In yet other embodiments, the recombinant yeast cell can have from about 1 to about 10 copies; from about 2 to about 10 copies; from about 3 to about 10 copies; from about 4 to about 10 copies; from about 5 to about 10 copies; from about 6 to about 10 copies; from about 7 to about 10 copies; from about 8 to about 10 copies; from about 9 to about 10 copies; from about 10 to about 10 copies; from about 11 to about 10 copies; from about 12 to about 10 copies; from about 13 to about 10 copies; from about 14 to about 10 copies; from about 15 to about 10 copies; from about 16 to about 10 copies; from about 17 to about 10 copies; from about 18 to about 10 copies; from about 19 to about 10 copies; from about 20 to about 10 copies; of the heterologous polynucleotide.

[00524] In yet other embodiments, the recombinant yeast cell can have from about 1 to about 5 copies; from about 2 to about 5 copies; from about 3 to about 5 copies; from about 4 to about 5 copies; from about 5 to about 5 copies; of the heterologous polynucleotide.

[00525] In some embodiments, a vector of the present disclosure can comprise multiple copies of: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof. For example, in some embodiments, the polynucleotide may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies of (i), (ii), or a combination thereof.

[00526] In some embodiments, a vector may have from about 1 to about 20 copies; from about 2 to about 20 copies; from about 3 to about 20 copies; from about 4 to about 20 copies; from about 5 to about 20 copies; from about 6 to about 20 copies; from about 7 to about 20 copies; from about 8 to about 20 copies; from about 9 to about 20 copies; from about 10 to about 20 copies; from about 11 to about 20 copies; from about 12 to about 20 copies; from about 13 to about 20 copies; from about 14 to about 20 copies; from about 15 to about 20 copies; from about 16 to about 20 copies; from about 17 to about 20 copies; from about 18 to about 20 copies; from about 19 to about 20 copies; of the heterologous polynucleotide.

[00527] In yet other embodiments, a vector can have from about 1 to about 10 copies; from about 2 to about 10 copies; from about 3 to about 10 copies; from about 4 to about 10 copies; from about 5 to about 10 copies; from about 6 to about 10 copies; from about 7 to about 10 copies; from about 8 to about 10 copies; from about 9 to about 10 copies; from about 10 to about 10 copies; from about 11 to about 10 copies; from about 12 to about 10 copies; from about 13 to about 10 copies; from about 14 to about 10 copies; from about 15 to about 10 copies; from about 16 to about 10 copies; from about 17 to about 10 copies; from about 18 to about 10 copies; from about 19 to about 10 copies; from about 20 to about 10 copies; of the heterologous polynucleotide.

[00528] In yet other embodiments, a vector can have from about 1 to about 5 copies; from about 2 to about 5 copies; from about 3 to about 5 copies; from about 4 to about 5 copies; from about 5 to about 5 copies; of the heterologous polynucleotide.

[00529] In some embodiments, a heterologous polynucleotide of the present disclosure can comprise multiple copies of: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof. For example, in some embodiments, the heterologous polynucleotide may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more copies of (i), (ii), or a combination thereof.

[00530] In some embodiments, a polynucleotide of the present disclosure can have from about 1 to about 20 copies; from about 2 to about 20 copies; from about 3 to about 20 copies; from about 4 to about 20 copies; from about 5 to about 20 copies; from about 6 to about 20 copies; from about 7 to about 20 copies; from about 8 to about 20 copies; from about 9 to about 20 copies; from about 10 to about 20 copies; from about 11 to about 20 copies; from about 12 to about 20 copies; from about 13 to about 20 copies; from about 14 to about 20 copies; from about 15 to about 20 copies; from about 16 to about 20 copies; from about 17 to about 20 copies; from about 18 to about 20 copies; from about 19 to about 20 copies; of the heterologous polynucleotide.

[00531] In yet other embodiments, a polynucleotide of the present disclosure can have from about 1 to about 10 copies; from about 2 to about 10 copies; from about 3 to about 10 copies; from about 4 to about 10 copies; from about 5 to about 10 copies; from about 6 to about 10 copies; from about 7 to about 10 copies; from about 8 to about 10 copies; from about 9 to about 10 copies; from about 10 to about 10 copies; from about 11 to about 10 copies; from about 12 to about 10 copies; from about 13 to about 10 copies; from about 14 to about 10 copies; from about 15 to about 10 copies; from about 16 to about 10 copies; from about 17 to about 10 copies; from about 18 to about 10 copies; from about 19 to about 10 copies; from about 20 to about 10 copies; of the heterologous polynucleotide.

[00532] In yet other embodiments, a polynucleotide of the present disclosure can have from about 1 to about 5 copies; from about 2 to about 5 copies; from about 3 to about 5 copies; from about 4 to about 5 copies; from about 5 to about 5 copies; of the heterologous polynucleotide.

[00533] Ratios of nucleotide sequences

[00534] In some embodiments, a polynucleotide of the present disclosure which is introduced into a host cell can comprise a heterologous polynucleotide comprising multiple copies of: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii). The heterologous polynucleotide can include multiple copies of (i) and (i) in varying ratios, for example, a ratio between the number of copies of (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), relative to the number of copies of (ii) a nucleotide sequence operable to encode a heterologous polypeptide: thus, the ratio of (i): (ii) describes the relationship in quantity between the copy number of (i) relative to the copy number of (ii).

[00535] In some embodiments, a heterologous polynucleotide comprising (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide, can have a ratio of (i) to (ii) varying from about from about 1 : 1 to about 1 :20 . [00536] In some embodiments, the heterologous polynucleotide may comprise a ratio of (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide, wherein the ratio of (i):(ii) is from about 1:1 to about 1:20; from about 1:2 to about 1:20; from about 1:3 to about 1:20; from about 1:4 to about 1:20; from about 1:5 to about 1:20; from about 1:6 to about 1:20; from about 1:7 to about 1:20; from about 1:8 to about 1:20; from about 1:9 to about 1:20; from about 1:10 to about 1:20; from about 1:11 to about 1:20; from about 1:12 to about 1:20; from about 1:13 to about 1:20; from about 1:14 to about 1:20; from about 1:15 to about 1:20; from about 1:16 to about 1:20; from about 1:17 to about 1:20; from about 1:18 to about 1:20; from about 1:19 to about 1:20; from about 1:20 to about 1:20; from about 2: 1 to about 20: 1 ; from about 3 : 1 to about 20: 1 ; from about 4: 1 to about 20: 1 ; from about 5:1 to about 20:1; from about 6:1 to about 20:1; from about 7:1 to about 20:1; from about 8: 1 to about 20: 1 ; from about 9: 1 to about 20: 1 ; from about 10:1 to about 20: 1 ; from about 11:1 to about 20:1; from about 12:1 to about 20:1; from about 13:1 to about 20:1; from about 14:1 to about 20:1; from about 15:1 to about 20:1; from about 16:1 to about 20:1; from about 17:1 to about 20:1; from about 18:1 to about 20:1; from about 19:1 to about 20:1; or about 20:1.

[00537] In some embodiments, the heterologous polynucleotide may comprise a ratio of (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide, wherein the ratio of (i):(ii) is about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10, about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9: 1 , or about 10:1.

[00538] In some embodiments, the heterologous polynucleotide may comprise a ratio of (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide, wherein the ratio is about 1:2 or about 1:3.

[00539] In some embodiments, the heterologous polynucleotide may comprise a ratio of (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide, wherein the ratio is about 1:1.

[00540] VECTORS

[00541] A vector of the present disclosure refers to a means for introducing one or more polynucleotides and heterologous polynucleotides into a host cell (e.g., a yeast cell). There are a variety of vectors available and cloning strategies known to those having ordinary skill in the art.

[00542] As used herein, the term “vector” refers to a carrier nucleic acid molecule into which a polynucleotide can be inserted for introduction into a cell (e.g., transformation), and where it can be replicated. In some embodiments, a vector may contain “vector elements,” e.g., and without limitation: an origin of replication (ORI); a gene or nucleotide sequence that allows for selection (e.g., a gene that confers antibiotic resistance or a nucleotide sequence that allows growth in defined media); multiple cloning sites; a promoter region; a primer binding site; and/or a combination thereof.

[00543] In some embodiments, some of the polynucleotides or nucleotide sequences inserted into a vector can be “heterologous” or “exogenous,” which means that it is foreign to the cell into which the vector is being introduced, or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. For example, in some embodiments, a recombinant yeast cell can be transformed with a vector comprising a heterologous polynucleotide comprising at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), wherein the heterologous SDH is homologous to the endogenous SDH (e.g., which has been at least partially inactivated), but is in a position within the host cell nucleic acid in which the endogenous SDH nucleotide sequence is ordinarily not found.

[00544] However, in other embodiments, a recombinant yeast cell can be transformed with a vector comprising a heterologous polynucleotide comprising at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), wherein the heterologous SDH is homologous to the endogenous SDH (which, e.g., has been at least partially inactivated), and is in the same position within the host cell nucleic acid in which the endogenous SDH nucleotide sequence is ordinarily found, but has been placed there by recombinant techniques (e.g., replacing the endogenous SDH gene with a heterologous SDH gene in the same location).

[00545] Vectors can be used both as a means to prepare the heterologous polynucleotides of the present disclosure, or to ultimately transform the cells used to generate a recombinant yeast cell and/or as a method to increase expression of a heterologous polypeptide.

[00546] In some embodiments, vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs). For example, in some embodiments, a vector can be a plasmid, which can introduce a heterologous polynucleotide and/or expression cassette into host cells to be transcribed and translated.

[00547] One having ordinary skill in the art would be well equipped to construct a vector through standard recombinant techniques, which are described in Sambrook et al., 1989 and Ausubel et al., 1996, both incorporated herein by reference in their entireties. [00548] In some embodiments, in addition to encoding heterologous polynucleotide, a vector may also encode a targeting molecule. A targeting molecule is one that directs the desired polynucleotide to a particular location.

[00549] In some embodiments, a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, can be inserted into any suitable vector, e.g., a plasmid, bacteriophage, or viral vector for amplification, and may thereby be propagated using methods known in the art, such as those described in Molecular Cloning A Laboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press: 1989), the disclosure of which is incorporated herein by reference in its entirety.

[00550] Expression cassettes

[00551] In addition to a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, additional DNA segments known as regulatory elements can be cloned into a vector that allow for enhanced expression of the heterologous polynucleotide. Examples of such regulatory elements include (1) promoters, terminators, and/or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements.

[00552] As described above, the combination of a DNA segment of interest (e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide) with any one of the foregoing cis-acting elements is called an “expression cassette.”

[00553] Thus, in some embodiments, these additional DNA segments known as regulatory elements can be operably linked and in any orientation with regard to (a) a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) a nucleotide sequence operable to encode a heterologous SDH; and/or (c) a nucleotide sequence operable to encode a heterologous polypeptide.

[00554] For example, in some embodiments, a vector can comprise an expression cassette, wherein the expression cassette comprises one or more (1) promoters, terminators, and/or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements, that allow for enhanced expression of the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00555] And, in some embodiments, the vector can comprise a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, wherein each of the (i) and (ii) has its own expression cassette comprising one or more (1) promoters, terminators, and/or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post-transcriptional regulatory elements, that allow for enhanced expression of (i) and (ii), respectively.

[00556] For example, in some embodiments, a vector can comprise a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, wherein either the heterologous polynucleotide as a whole, and/or each of (i) and (ii) has its own regulatory elements, e.g., each of (i) and (ii) is under the control of its own promotor and/or terminator. In such an example, where each of (i) and (ii) is under the control of its regulatory elements, then each of (i) and (ii) would be considered to have its own expression cassette.

[00557] In some embodiments, an expression cassette can contain one or more heterologous polynucleotides comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00558] In some embodiments, an expression cassette can contain one or more heterologous polynucleotides comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, and one or more additional regulatory elements such as: (1) promoters, terminators, and/or enhancer elements; (2) an appropriate mRNA stabilizing polyadenylation signal; (3) an internal ribosome entry site (IRES); (4) introns; and (5) post- transcriptional regulatory elements.

[00559] In some embodiments, a heterologous polynucleotide can comprise one or more expression cassettes.

[00560] In some embodiments, a vector can comprise one or more expression cassettes.

[00561] Cloning strategies

[00562] Insertion of the appropriate polynucleotide (e.g., a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide) into a vector can be performed by a variety of procedures.

[00563] In general, the DNA sequence is ligated to the desired position in the vector following digestion of the insert and the vector with appropriate restriction endonucleases. Alternatively, blunt ends in both the insert and the vector may be ligated. A variety of cloning techniques are disclosed in Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, Inc. 1997 and Sambrook et al., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory Press (1989); the disclosures of which are incorporated herein by reference in their entireties. Such procedures and others are deemed to be within the scope of those skilled in the art.

[00564] In some embodiments, a heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can be inserted into other commercially available plasmids and/or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a nonprofit plasmid repository); GenScript®; Takara®; Qiagen®; and Promega™

[00565] In some embodiments, a vector can be, for example, in the form of a plasmid, a viral particle, or a phage. In other embodiments, a vector can include chromosomal, non- chromosomal and synthetic DNA sequences, derivatives of SV40; bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus and pseudorabies.

[00566] In some embodiments, vectors compatible with eukaryotic cells, such as vertebrate cells, can be used. Eukaryotic cell vectors are well known in the art and are available from commercial sources. Contemplated vectors may contain both prokaryotic sequences (to facilitate the propagation of the vector in bacteria), and one or more eukaryotic transcription units that are functional in non-bacterial cells. Typically, such vectors provide convenient restriction sites for insertion of the desired recombinant DNA molecule. The pcDNAI, pSV2, pSVK, pMSG, pSVL, pPVV-l/PML2d and pTDTl (ATCC No. 31255) derived vectors are examples of mammalian vectors suitable for transfection of non-human cells. In some embodiments, some of the foregoing vectors may be modified with sequences from bacterial plasmids, such as pBR322, to facilitate replication and drug resistance selection in both prokaryotic and eukaryotic cells. Alternatively, derivatives of viruses such as the bovine papilloma virus (BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) may be used for expression of proteins in swine cells. The various methods employed in the preparation of the plasmids and the transformation of host cells are well known in the art.

[00567] In some embodiments, and in addition to a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous SDH, and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, a vector may include a signal sequence or a leader sequence for targeting membranes or secretion as well as expression regulatory elements, such as a promoter, an operator, an initiation codon, a stop codon, a poly adenylation signal, and/or an enhancer; and can be constructed in various forms depending on the purpose thereof. The initiation codon and stop codons are generally considered to be a portion of a nucleotide sequence coding for a target protein, are necessary to be functional in a subject to which a genetic construct has been administered, and must be in frame with the coding sequence.

[00568] In some embodiments, the promoter of the vector may be constitutive or inducible. In addition, expression vectors may include a selectable marker that allows the selection of host cells containing the vector, and replicable expression vectors include a replication origin. The vector may be self-replicable, or may be integrated into the host DNA. [00569] Use of promoters may not be required in cases in which transcriptionally active genes are targeted, if the design of the construct results in the marker being transcribed as directed by an endogenous promoter. Exemplary constructs and vectors for carrying out such targeted modification are described herein. However, other vectors that can be used in such approaches are known in the art and can readily be adapted for use in the invention.

[00570] In some embodiments, a targeting vector can be used. A basic targeting vector comprises a site-specific integration (SSI) sequence, e.g., 5’- and 3’- homology arms of sequence that is homologous to an endogenous DNA segment that is being targeted. [00571] In some embodiments, a targeting vector can also optionally include one or more positive and/or negative selection markers. In some embodiments, the selection markers can be used to disrupt gene function and/or to identify cells that have integrated targeting vector nucleotide sequences following transformation.

[00572] In some embodiments, the use of a targeting vector may utilize a heterologous polynucleotide comprising one or more mutations, in order to create restriction patterns that are distinguishable from the endogenous gene (if the transgene and endogenous gene are similar).

[00573] In some embodiments, during the introduction of the heterologous polynucleotide into the cell to be modified, the heterologous polynucleotide can be inserted into the locus of a similar endogenous gene, thereby knocking-out function of the similar endogenous gene. For example, in some embodiments, a nucleotide sequence operable to encode a heterologous SDH can be inserted into the locus of an endogenous SDH gene, thereby knocking-out function of the endogenous SDH gene.

[00574] Homology arms

[00575] Those having ordinary skill in the art will recognize that targeted gene modification requires the use of nucleic acid molecule vectors comprising regions of homology with a targeted gene (or flanking regions thereof), such that integration of the vector into the genome can be facilitated. Thus, a targeting vector is generally designed to contain three main regions: (1) a first region that is homologous to the locus to be targeted;

(2) a second region that is a heterologous polynucleotide sequence (e.g., comprising a polynucleotide operable to encode a protein of interest and/or encoding a selectable marker, such as an antibiotic resistance protein) that is to be inserted at a target locus and/or to specifically replace a portion of the targeted locus; and (3) a third region that, like the first region, is homologous to the targeted locus, but typically is not contiguous with the first region of the genome.

[00576] Homologous recombination between the targeting vector and the targeted endogenous or wild-type locus results in deletion of any locus sequences between the two regions of homology represented in the targeting vector and replacement of that sequence with, or insertion into that sequence of, a heterologous sequence that, for example, encodes the polynucleotide of interest and optionally one or more additional regulatory elements.

[00577] In order to facilitate homologous recombination, the first and third regions of the targeting vectors (see above) include sequences that exhibit substantial identity to the genes to be targeted (or flanking regions). By “substantially identical” is meant having a sequence that is at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably 100% identical to that of another sequence. Sequence identity is typically measured using BLAST® (Basic Local Alignment Search Tool) or BLAST® 2 with the default parameters specified therein (see, Altschul et al., J. Mol. Biol. 215: 403-410, 1990; Tatiana et al., FEMS Microbiol. Lett. 174: 247-250, 1999). These software programs match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Thus, sequences having at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98%, and even more preferably 100% sequence identity with the targeted gene loci can be used in the invention to facilitate homologous recombination.

[00578] The total size of the two regions of homology (i.e., the first and third regions noted above) can be, for example, approximately between 1-25 kilobases (kb) (for example, approximately between 2-20 kb, approximately between 5-15 kb, or approximately between 6-10 kb), and the size of the second region that replaces a portion of the targeted locus can be, for example, approximately between 0.5-5 kb (for example, approximately between 1-4 kb, approximately between 1-3 kb, approximately between 1-2 kb, or approximately between 3-4 kb).

[00579] In some embodiments, a targeting vector generally can comprise a selection marker and a site-specific integration (SSI) sequence. The SSI sequence can comprise a transgene of interest, e.g., a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; which is flanked with two genomic DNA fragments called “5’- and 3 ’-homology arms” or “5’ and 3’ arms” or “left and right arms” or “homology arms.” These homology arms recombine with the target genome sequence and/or endogenous gene of interest in the host organism in order to achieve successful genetic modification of the host organism’s chromosomal locus.

[00580] When designing the homology arms for a targeting vector, both the 5’- and 3’- arms should possess sufficient sequence homology with the endogenous sequence to be targeted in order to engender efficient in vivo pairing of the sequences, and cross-over formation. And, while homology arm length is variable, a homology covering at least 5-8 kb in total for both arms (with the shorter arm having no less than 1 kb in length), is a general guideline that can be followed to help ensure successful recombination. [00581] In some embodiments, the 5’- and/or 3’-homology arms may vary. For example, in some embodiments, different loci could be targeted by the 5’- and/or 3’- homology arms, e.g., either upstream and/or downstream from a homology arm described herein to exchange the sequence of interest at a different location.

[00582] Additional exemplary methods of vector design and in vivo homologous recombination can be found in U.S. Patent No. 5,464,764, entitled “Positive-negative selection methods and vectors” (filed 02/04/1993; assignee University of Utah Research Foundation, Salt Lake City, UT); U.S. Patent No. 5,733,761, entitled “Protein production and protein delivery” (filed 05/26/1995; assignee Transkaryotic Therapies, Inc., Cambridge, MA); U.S. Patent No. 5,789,215, entitled “Gene targeting in animal cells using isogenic DNA constructs” (filed 08/07/1997; assignee GenPharm International, San Jose, CA); U.S. Patent No. 6,090,554, entitled “Efficient construction of gene targeting vectors” (filed 10/31/1997; assignee Amgen, Inc., Thousand Oaks, CA); U.S. Patent No. 6,528,314, entitled “Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made” (filed 06/06/1995; assignee Institut, Pasteur);U.S. Patent No. 6,537,542, entitled “Targeted introduction of DNA into primary or secondary cells and their use for gene therapy and protein production (filed 04/14/2000; assignee Transkaryotic Therapies, Inc., Cambridge, MA); U.S. Patent No. 8,048,645, entitled “Method of producing functional protein domains (filed 08/01/2001; assignee Merck Serono SA); and U.S. Patent No. 8,173,394, entitled “Systems and methods for protein production” (filed 04/06/2009; assignee Wyeth LLC, Madison, NJ); the disclosures of which are incorporated herein by reference in their entirety.

[00583] Exemplary descriptions and methods concerning selection markers are provided in Wigler et al., Cell 11:223 (1977); Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992); Lowy et al., Cell 22:817 (1980); Wigler et al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981); Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981); Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926- 932 (1993); Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); Santerre et al., Gene 30:147 (1984); Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, N Y (1993); Kriegler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, N Y (1990); in Chapters 12 and 13, Dracopoli et al. (eds), Current Protocols in Human Genetics, John Wiley & Sons, N Y (1994); Colberre-Garapin et al., J. Mol. Biol.

150:1 (1981); U.S. Patent Nos. 6,548,285 (filed Apr. 3, 1997); 6,165,715 (filed June 22, 1998); and 6,110,707 (filed Jan. 17, 1997), the disclosures of which are incorporated by reference herein in their entireties.

[00584] Exemplary Vectors

[00585] In some embodiments, the present disclosure comprises, consists essentially of, or consists of, a vector comprising: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5 ’-homology arm, and a 3’ - homology arm, wherein said 5 ’-homology arm and said 3 ’-homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination- mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide.

[00586] In some embodiments, a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide can be cloned or inserted into a vector (e.g., a plasmid). In other embodiments, any of the components of the heterologous polynucleotide, or a complementary nucleotide sequence thereof, i.e., (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and/or (ii) a nucleotide sequence operable to encode a heterologous polypeptide, can be cloned or inserted into a vector.

[00587] In some embodiments, a recombinant yeast cell is transformed with a vector comprising, consisting essentially of, or consisting of, a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00588] In some embodiments, a heterologous polynucleotide comprising (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; or any component thereof (e.g., a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP)), can be cloned into a vector using a variety of cloning strategies, and commercial cloning kits and materials readily available to those having ordinary skill in the art.

[00589] For example, a heterologous polynucleotide and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be cloned into a vector using such strategies as the SnapFast; Gateway; TOPO; Gibson; LIC; InFusionHD; or Electra strategies.

[00590] There are numerous commercially available vectors that can be used to produce a vector of the present disclosure. For example, a heterologous polynucleotide and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be generated using polymerase chain reaction (PCR), and combined with a pCR II- TOPO vector, or a PCR™2.1-TOPO® vector (commercially available as the TOPO® TA Cloning ® Kit from Invitrogen) for 5 minutes at room temperature; the TOPO® reaction can then be transformed into competent cells, which can subsequently be selected based on color change (see Janke et al., A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast. 2004 Aug;

21(11):947-62; see also, Adams et al. Methods in Yeast Genetics. Cold Spring Harbor, NY, 1997, the disclosure of which is incorporated herein by reference in its entirety).

[00591] In some embodiments, a heterologous polynucleotide and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be cloned into a vector such as a plasmid, cosmid, virus (bacteriophage, animal viruses, and plant viruses), and/or artificial chromosome (e.g., YACs).

[00592] In some embodiments, a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be inserted into a vector, for example, a plasmid vector using E. coli as a host, by performing the following: digesting about 2 to 5 pg of vector DNA using the restriction enzymes necessary to allow the DNA segment of interest to be inserted, followed by overnight incubation to accomplish complete digestion (alkaline phosphatase may be used to dephosphorylate the 5 ’-end in order to avoid self-ligation/recircularization); gel purify the digested vector. Next, amplify the DNA segment of interest, for example, a polynucleotide encoding a heterologous SDH and a polynucleotide encoding a heterologous polypeptide (e.g., a CRP), via PCR, and remove any excess enzymes, primers, unincorporated dNTPs, short-failed PCR products, and/or salts from the PCR reaction using techniques known to those having ordinary skill in the art (e.g., by using a PCR clean-up kit). Ligate the DNA segment of interest to the vector by creating a mixture comprising: about 20 ng of vector; about 100 to 1,000 ng or DNA segment of interest; 2 pL lOx buffer (i.e., 30 mM Tris-HCl 4 mM MgCh, 26 pM NAD, 1 mM DTT, 50 pg/ml BSA, pH 8, stored at 25°C); 1 pL T4 DNA ligase; all brought to a total volume of 20 pL by adding H2O. The ligation reaction mixture can then be incubated at room temperature for 2 hours, or at 16°C for an overnight incubation. The ligation reaction (i.e., about 1 pL) can then be transformed to competent cell, for example, by using electroporation or chemical methods, and a colony PCR can then be performed to identify vectors containing the DNA segment of interest. [00593] In some embodiments, a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), along with other DNA segments together composing a heterologous polypeptide expression ORF can be designed for secretion from host yeast cells. An illustrative method of designing a heterologous polypeptide expression ORF is as follows: the ORF can begin with a signal peptide sequence, followed by a DNA sequence encoding a Kex2 cleavage site (Lysine- Arginine), and subsequently followed by the heterologous polynucleotide trans gene, with the addition of glycine-serine codons at the 5 ’-end, and finally a stop codon at the 3 ’-end. All these elements will then be expressed to a fusion peptide in yeast cells as a single open reading frame (ORF). An a-mating factor (aMF) signal sequence is most frequently used to facilitate metabolic processing of the recombinant insecticidal peptides through the endogenous secretion pathway of the recombinant yeast, i.e. the expressed fusion peptide will typically enter the Endoplasmic Reticulum, wherein the a - mating factor signal sequence is removed by signal peptidase activity, and then the resulting pro-insecticidal peptide will be trafficked to the Golgi Apparatus, in which the Lysine- Arginine dipeptide mentioned above is completely removed by Kex2 endoprotease, after which the mature, heterologous polypeptide (i.e., CRP), is secreted out of the cells.

[00594] In some embodiments, polypeptide expression levels in recombinant yeast cells can be enhanced by optimizing the codons based on the specific host yeast species. Naturally occurring frequencies of codons observed in endogenous open reading frames of a given host organism need not necessarily be optimized for high efficiency expression. Furthermore, different yeast species (for example, Kluyveromyces lactis, Pichia pastoris, Saccharomyces cerevisiae, etc.) have different optimal codons for high efficiency expression. Hence, codon optimization should be considered for the heterologous polypeptide expression ORF, including the sequence elements encoding the signal sequence, the Kex2 cleavage site and the heterologous polypeptide, because they are initially translated as one fusion peptide in the recombinant yeast cells.

[00595] In some embodiments, a codon-optimized heterologous polypeptide expression ORF can be ligated into a yeast-specific expression vectors for yeast expression. There are many expression vectors available for yeast expression, including episomal vectors and integrative vectors, and they are usually designed for specific yeast cells. One should carefully choose the appropriate expression vector in view of the specific yeast expression system which will be used for the peptide production. In some embodiments, integrative vectors can be used, which integrate into chromosomes of the transformed yeast cells and remain stable through cycles of cell division and proliferation. The integrative DNA sequences are homologous to targeted genomic DNA loci in the transformed yeast species, and such integrative sequences include pLAC4, 25S rDNA, pAOXl, and TRP2, etc. The locations of insecticidal peptide transgenes can be adjacent to the integrative DNA sequence (Insertion vectors) or within the integrative DNA sequence (replacement vectors).

[00596] In some embodiments, the expression vectors can contain E. coli elements for DNA preparation in E. coli, for example, E. coli replication origin, antibiotic selection marker, etc. In some embodiments, vectors can contain an array of the sequence elements needed for expression of the transgene of interest, for example, transcriptional promoters, terminators, yeast selection markers, integrative DNA sequences homologous to host yeast DNA, etc. There are many suitable yeast promoters available, including natural and engineered promoters, for example, yeast promoters such as pLAC4, pAOXl, pUPP, pADHl, pTEF, pGall, etc., and others, can be used in some embodiments.

[00597] In some embodiments, a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be inserted into a pJUSor plasmid (a modified pJUGalphKR plasmid; available from Biogrammatics). The pJUSor is designed to integrate sorbitol dehydrogenase and accomplish high-level expression of recombinant protein (e.g., a CRP) in the yeast Pichia Pastoris. The pJUSor plasmid can be linearized using the Pfol restriction enzyme, and integrates into the pUPP locus. [00598] In some embodiments, a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be inserted into a pLB10V5 plasmid. In some embodiments, endogenous SDH is first knocked-out of the yeast, for example the yeast K. lactis. In some embodiments, obtaining an SDH deficient cell is accomplished by using a pop-in/pop-out plasmid such as pKLD plasmid.

[00599] In some embodiments, a pKLD plasmid comprises a 5’- and 3 ’-homology arm corresponding to an endogenous target gene locus, followed by a repeated 3 ’-homology arm corresponding to the same target gene locus, which is repeated at the 5’ end of the plasmid. This repeated 3 ’-homology arm provides the opportunity for an integrated transgene marker (e.g., amdS) to out-recombine at a frequency that allows recovery a “pop-out” colony using a counter selection plate. Thus, while a typical integration vector may have one 3 ’-homology arm, and one 5 ’-homology arm for accurate insertion, a pKLD plasmid has an additional 3’- homology arm just upstream of the 5 ’-homology arm, and it is this repeated homology within the insertion vector that allows for the out recombination. The use of pop-in/pop-out are known in the art, and described herein.

[00600] In some embodiments, the pKLD plasmid can be linearized using the SacII restriction enzyme. Yeast, for example K. lactis, transformed with a pKLD plasmid replacing the endogenous SDH with an acetamidase selection marker gene (amdS), which allows transformed yeast cells to grow in YCB medium containing acetamide as its only nitrogen source. Once knocked out yeast colonies transformed with a pKLD plasmid, knocking out the endogenous SDH gene, are identified, they can be modified to incorporate a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP).

[00601] In some embodiments, a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be inserted into a pLB10V5 plasmid, or subcloned into a pLB10V5 plasmid subsequent to selection of yeast colonies transformed with pKLD plasmids knocking out the endogenous SDH gene. Yeast, for example K. lactis, transformed with a pLB10V5 plasmids ligated with a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be selected based on sorbitol dehydrogenase (lid or SDH), which allows transformed yeast cells to grow in medium containing sorbitol as its only carbon source.

[00602] In some embodiments, when the yeast to be transformed contains an endogenous SDH gene, the endogenous SDH gene would be inactivated or partially inactivated (e.g., with a pKLD plasmid), prior to incorporation of a polynucleotide encoding a heterologous SDH and a polynucleotide encoding a heterologous polypeptide (e.g., a CRP) (e.g., with a pLB10V5 plasmid).

[00603] In some embodiments, a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be inserted into other commercially available plasmids and/or vectors that are readily available to those having skill in the art, e.g., plasmids are available from Addgene (a non-profit plasmid repository); GenScript®; Takara®; Qiagen®; and Promega™

[00604] Following the preparation of a vector comprising a heterologous polynucleotide of the present disclosure, and/or a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and/or a nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), the vector is transformed into the yeast cell to produce a recombinant yeast cell of the present disclosure.

[00605] In some embodiments, a vector of the present disclosure comprises: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide.

[00606] In some embodiments, a vector can be a plasmid comprising an alpha-MF signal. [00607] In some embodiments, the alpha-MF signal is operable to express an alpha- MF signal peptide.

[00608] In some embodiments, the heterologous polypeptide is operably linked to the alpha-MF signal peptide.

[00609] Transformation and cell culture methods

[00610] In some embodiments, a yeast cell can be transformed using the following methods: electroporation; cell squeezing; microinjection; impalefection; the use of hydrostatic pressure; sonoporation; optical transfection; continuous infusion; lipofection; through the use of viruses such as adenovirus, adeno-associated virus, lentivirus, herpes simplex virus, and retrovirus; the chemical phosphate method; endocytosis via DEAE- dextran or polyethylenimine (PEI); protoplast fusion; hydrodynamic deliver; magnetofection; nucleoinfection; and/or others. Exemplary methods regarding transfection and/or transformation techniques can be found in Makrides (2003), Gene Transfer and Expression in Mammalian Cells, Elvesier; Wong, TK & Neumann, E. Electric field mediated gene transfer. Biochem. Biophys. Res. Commun. 107, 584-587 (1982); Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3; Kim & Eberwine, Mammalian cell transfection: the present and the future. Anal Bioanal Chem. 2010 Aug; 397(8): 3173-3178, each of these references are incorporated herein by reference in their entireties.

[00611] In some embodiments, multiple vectors, each comprising one or more of (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH) or a complementary nucleotide sequence thereof; and/or (c) a nucleotide sequence operable to encode a heterologous polypeptide, or a complementary nucleotide sequence thereof; can be used to transform a yeast host cell. In some embodiments, the transformation can happen simultaneously or concurrently.

[00612] For example, in some embodiments, a vector comprising (i) one or more nucleotide sequences operable to encode a heterologous sorbitol dehydrogenase (SDH) can be used to transform a yeast host cell; then, a vector comprising (ii) one or more nucleotide sequences operable to encode a heterologous polypeptide, can be subsequently used to transform the same yeast host cell — thereby resulting in a recombinant yeast cell comprising (i) one or more nucleotide sequences operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequences operable to encode a heterologous polypeptide.

[00613] In some embodiments, a vector comprising (ii) one or more nucleotide sequences operable to encode a heterologous polypeptide can be used to transform a yeast host cell; then, a vector comprising (i) one or more nucleotide sequences operable to encode a heterologous sorbitol dehydrogenase (SDH), can be subsequently used to transform the same yeast host cell — thereby resulting in a recombinant yeast cell comprising (i) one or more nucleotide sequences operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequences operable to encode a heterologous polypeptide.

[00614] Electroporation is a technique in which electricity is applied to cells causing the cell membrane to become permeable; this in turn allows exogenous DNA to be introduced into the cells. Electroporation is readily known to those having ordinary skill in the art, and the tools and devices required to achieve electroporation are commercially available (e.g., Gene Pulser Xcell™ Electroporation Systems, Bio-Rad®; Neon® Transfection System for Electroporation, Thermo-Fisher Scientific; and other tools and/or devices). Exemplary methods of electroporation are illustrated in Potter & Heller, Transfection by Electroporation. Curr Protoc Mol Biol. 2003 May; CHAPTER: Unit-9.3; Saito (2015) Electroporation Methods in Neuroscience. Springer press; Pakhomov et al., (2017) Advanced Electroporation Techniques in Biology and Medicine. Taylor & Francis; the disclosure of which is incorporated herein by reference in its entirety.

[00615] In some embodiments, a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, or a vector comprising the same, can be cloned into a plasmid, and transformed a host cell (e.g., a yeast cell).

[00616] In some embodiments, one or more expression vectors comprising a heterologous polynucleotide comprising (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can be transformed into yeast cells as follows. First, the expression vectors are usually linearized by specific restriction enzyme cleavage to facilitate chromosomal integration via homologous recombination. The linear expression vector is then transformed into yeast cells by a chemical or electroporation method of transformation and integrated into the targeted locus of the yeast genome by homologous recombination. The integration can happen at the same chromosomal locus multiple times; therefore, the genome of a transformed yeast cell can contain multiple copies of the heterologous polynucleotide comprising (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide. The successfully transformed yeast cells can be identified using growth conditions that favor a selective marker engineered into the expression vector and co-integrated into yeast chromosomes with the heterologous polynucleotide. In some embodiments, the heterologous SDH integrated into the yeast cell serves as a selection marker.

[00617] In some embodiments, electroporation can be used to transform a yeast host cell. For example, in some embodiments, a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, or a vector comprising the same, can be cloned into a plasmid, and transformed a host cell (e.g., a yeast cell), via electroporation can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) with a suitable yeast species, for example, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Pichia pastoris, etc., and incubate on a shaker at 30°C until the early exponential phase of yeast culture (e.g. about 0.6 to 2 x 10⁸ cells/mL); harvesting the yeast in sterile centrifuge tube and centrifuging at 3000 rpm for 5 minutes at 4°C (note: keep cells chilled during the procedure) washing cells with 40 mL of ice cold, sterile deionized water, and pelleting the cells a 23,000 rpm for 5 minutes; repeating the wash step, and the resuspending the cells in 100 mL of 40% fermentable sugar, e.g. sorbitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold 40% fermentable sugar, e.g. sorbitol, to final cell density of 3xl0⁹ cell/mL; (1.5xl0⁹ cell/mL to 6xl0⁹ cell/mL are acceptable cell densities); mixing 40 pL of the yeast suspension with about 1-4 pL (at a concentration of 100-300ng/pL) of the vector containing a linear polynucleotide encoding a heterologous SDH and a heterologous polypeptide (e.g., a CRP) (~1 pg) in a prechilled 0.2 cm electroporation cuvette (note: ensure the sample is in contact with both sides of the aluminum cuvette); providing a single pulse at 2000 V, for optimal time constant of 5 ms of the RC circuit, the cells was then let recovered in 0.5 ml YED and 0.5 mL 40% fermentable sugar, e.g. sorbitol, and then spreading onto selective plates.

[00618] In some embodiments, electroporation can be used to introduce a vector comprising a heterologous polynucleotide comprising (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can be cloned into a plasmid, and transformed into a K. lactis cell via electroporation.

[00619] For example, in some embodiments, electroporation can be used to introduce a vector comprising a heterologous polynucleotide comprising (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can be cloned into a plasmid, and transformed into a K. lactis cell via electroporation can be accomplished by inoculating about 10-200 mL of yeast extract peptone dextrose (YEPD) incubating on a shaker at 30°C until the early exponential phase of yeast culture (e.g. about 0.6 to 2 x 10⁸ cells/mL); harvesting the yeast in sterile centrifuge tube and centrifuging at 3000 rpm for 5 minutes at 4°C (note: keep cells chilled during the procedure) washing cells with 40 mL of ice cold, sterile deionized water, and pelleting the cells a 23,000 rpm for 5 minutes; repeating the wash step, and the resuspending the cells in 100 mL of 40% fermentable sugar, e.g. sorbitol, followed by spinning down at 3,000 rpm for 5 minutes; resuspending the cells with proper volume of ice cold 40% fermentable sugar, e.g. sorbitol, to final cell density of 3xl0⁹ cell/mL; mixing 40 pL of the yeast suspension with about 1-4 pL of the vector containing a linear polynucleotide encoding a heterologous SDH and a heterologous polypeptide (e.g., a CRP) (~1 pg) in a prechilled 0.2 cm electroporation cuvette (note: ensure the sample is in contact with both sides of the aluminum cuvette); providing a single pulse at 2000 V, for optimal time constant of 5 ms of the RC circuit, the cells was then let recovered in 0.5 ml YED and 0.5 mL 40% fermentable sugar, e.g. sorbitol, and then spreading onto selective plates. In some embodiments, yeast cell fermentation media comprises a sole carbon source that is sorbitol.

[00620] Due to the influence of unpredictable and variable factors — such as epigenetic modification of genes and networks of genes, and variation in the number of integration events that occur in individual cells in a population undergoing a transformation procedure — individual yeast colonies of a given transformation process will differ in their capacities to produce a heterologous polypeptide (e.g., a CRP). Further, the media (e.g., having a sole carbon source) upon which the recombinant yeast cells are grown in has a significant effect in growth and yield production. Therefore, recombinant yeast colonies carrying the heterologous SDH and heterologous polypeptide (e.g., a CRP) trans genes can be screened for heterologous SDH incorporation and/or high (CRP) yield cells.

[00621] Heterologous polynucleotide incorporation analysis

[00622] Incorporation of a heterologous polynucleotide into the host cell genome can be accomplished in various ways known in the art. In one exemplary embodiment, the method comprises determining the integration of: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP), can be analyzed by methods known in the art. For example, in some embodiments, quantitative PCR (qPCR) and paralog ratio test (PRT) can be used to determine if the heterologous polynucleotide has been incorporated. In some embodiments, qPCR is used to confirm the integration of the heterologous sorbitol dehydrogenase and/or the nucleotide sequence encoding the CRP, into the recombinant yeast cell.

[00623] Quantitative PCR (qPCR) has been utilized for the analysis of gene expression and quantification of copy number variation by real-time PCR. qPCR involves amplification of a test locus with unknown copy number and a reference locus with known copy number. There are two approaches to the assay: fluorescent dyes and intercalating dyes. In either approach, fluorescence doubles with every cycle of PCR, and the amount of starting template can be determined from the number of cycles required to achieve a specified threshold level of fluorescence. The actual qPCR experiment takes half a day after sample preparation. Commonly used methods for qPCR data analysis are absolute quantification by relating the PCR signal to a standard curve and relative quantification that relates the PCR signal of the target transcript in one group to another.

[00624] To measure DNA copy number, the amplicon should be located either within an exon or intron with sequences unique to that gene. A control gene with a known number of copies should also be included. A master mix containing all of the components is prepared and distributed in 96 or 384-well plate. Template and/or primers are added for each reaction. The assay is performed on a qPCR instrument and data are collected in real time.

[00625] An exemplary qPCR quantification method regarding incorporation in P. Pastoris is as follows: a heterologous polynucleotide encoding a heterologous SDH and a heterologous polypeptide (e.g., a CRP) can be inserted into the expression vector, pJUSor, and transformed into the P. Pastoris strain, BG10. Once the heterologous polypeptide (e.g., a CRP) transgenes were cloned into pJUSor and transformed into BG10, their expression was controlled by the pUPP promoter. The resulting transformed colonies expressed SDH and the heterologous polypeptide (e.g., a CRP). The yeast cell BG10, which contains no native sorbitol dehydrogenase gene is then compared with transformed strain JUSor2al using actin as the endogenous reference gene via qPCR. When the SDH CT values are close to the CT values of endogenous reference gene (e.g., actin) it suggests a similar number of gene copies as the reference.

[00626] High yield strains

[00627] An exemplary method of yeast transformation is as follows: the expression vectors carrying a heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide are transformed into yeast cells.

[00628] First, the expression vectors are usually linearized by specific restriction enzyme cleavage to facilitate chromosomal integration via homologous recombination. The linear expression vector is then transformed into yeast cells by a chemical or electroporation method of transformation and integrated into the targeted locus of the yeast genome by homologous recombination. The integration can happen at the same chromosomal locus multiple times; therefore, the genome of a transformed yeast cell can contain multiple copies of a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00629] The successfully transformed yeast cells can be identified using growth conditions that favor a selective marker engineered into the expression vector and cointegrated into yeast chromosomes with the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; examples of such markers include, but are not limited to, acetamide prototrophy, zeocin resistance, geneticin resistance, nourseothricin resistance, and uracil prototrophy.

[00630] Due to the influence of unpredictable and variable factors — such as epigenetic modification of genes and networks of genes, and variation in the number of integration events that occur in individual cells in a population undergoing a transformation procedure — individual yeast colonies of a given transformation process will differ in their capacities to produce a heterologous polypeptide. Therefore, transgenic yeast colonies carrying the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, should be screened for high yield strains.

[00631] Two effective methods for such screening — each dependent on growth of small-scale cultures of the transgenic yeast to provide conditioned media samples for subsequent analysis — use reverse-phase HPLC or housefly injection procedures to analyze conditioned media samples from the positive transgenic yeast colonies.

[00632] The transgenic yeast cultures can be obtained, e.g., using 14 mL round bottom polypropylene culture tubes with 5 to 10 mL defined medium added to each tube, or in 48- well deep well culture plates with 2.2 mL defined medium added to each well. The defined medium, not containing crude proteinaceous extracts or by-products such as yeast extract or peptone, is used for the cultures to reduce the protein background in the conditioned media harvested for the later screening steps. The cultures are performed at the optimal temperature, for example, 23.5°C for K. lactis, for about 5-6 days, until the maximum cell density is reached. The heterologous polypeptide (e.g., CRPs) will now be produced by the transformed yeast cells and secreted out of cells to the growth medium. To prepare samples for the screening, cells are removed from the cultures by centrifugation and the supernatants are collected as the conditioned media, which are then cleaned by filtration through 0.22 pm filter membrane and then made ready for strain screening.

[00633] In some embodiments, positive yeast colonies transformed with the nucleotide sequence operable to encode heterologous polypeptide (e.g., a CRP) can be screened via reverse-phase HPLC (rpHPLC) screening of putative yeast colonies. In this screening method, an HPLC analytic column with bonded phase of C18 can be used. Acetonitrile and water are used as mobile phase solvents, and a UV absorbance detector set at 220 nm is used for the peptide detection. Appropriate amounts of the conditioned medium samples are loaded into the rpHPLC system and eluted with a linear gradient of mobile phase solvents. The corresponding peak area of the insecticidal peptide in the HPLC chromatograph is used to quantify the heterologous polypeptide (e.g., a CRP) concentrations in the conditioned media. Known amounts of pure heterologous polypeptide (e.g., a CRP) are run through the same rpHPLC column with the same HPLC protocol to confirm the retention time of the peptide and to produce a standard peptide HPLC curve for the quantification.

[00634] An exemplary reverse-phase HPLC screening process of positive K. lactis cells is as follows: a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can be inserted into an expression vector, e.g., pLB10V5, and transformed into the K. lactis cell, e.g., a wild-type YCT306. Once the nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP) were cloned into pLB10V5 plasmid and transformed into YCT306, the expression of the nucleotide sequence operable to encode the heterologous polypeptide can be controlled by a LAC4 promoter. The resulting transformed colonies produced pre-propeptides comprising an a-mating factor signal peptide, a Kex2 cleavage site and mature heterologous polypeptides (e.g., CRPs). The a-Mating factor signal peptide guides the pre-propeptides to enter the endogenous secretion pathway, and mature heterologous polypeptides (e.g., CRPs) are released into the growth media.

[00635] In some embodiments, the yeast, Pichia pastoris, can be transformed with a heterologous polynucleotide comprises: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide. An exemplary method for transforming P. pastoris is as follows: yeast vectors can be used to transform a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide into P. pastoris. The vectors can be obtained from commercial vendors known to those having ordinary skill in the art.

[00636] In some embodiments, the vectors can be integrative vectors, and may use the uracil phosphoribosyltransferase promoter (pUPP) to enhance the heterologous transgene expression. In some embodiments, the vectors may offer a selection strategies; e.g., sorbitol dehydrogenase incorporation. In some embodiments, pairs of complementary oligonucleotides, encoding the heterologous SDH and heterologous polypeptide (e.g., a CRP) may be designed and synthesized into a yeast expression vector (e.g., pJUG plasmid). Hybridization reactions can be performed by mixing the corresponding complementary oligonucleotides to a final concentration of 20 pM in 30 mM NaCl, 10 mM Tris-Cl (all final concentrations), pH 8, and then incubating at 95 °C for 20 min, followed by a 9-hour incubation starting at 92°C and ending at 17°C, with 3°C drops in temperature every 20 min. The hybridization reactions will result in DNA fragments encoding heterologous polypeptide (e.g., a CRP). The pJUG vector can be linearized with Pfol restriction enzyme. Following verification of the sequences of the subclones, plasmid aliquots can be transfected by electroporation into aP. pastoris strain (e.g., BglO). [00637] Methods of protein purification are well-known in the art, and any known method can be employed to purify and/or recover heterologous polypeptides (e.g., CRPs) of the present disclosure. For example, in some embodiments, the following procedures are exemplary of suitable purification procedures: fractionation on immunoaffinity or ionexchange columns; ethanol precipitation; reverse phase HPLC; chromatography on silica, or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; and the like. In some embodiments, proteins of the present disclosure can be purified using one of the following; affinity chromatography; ion exchange chromatography; filtration; electrophoresis; hydrophobic interaction chromatography; gel filtration chromatography; reverse phase chromatography; concanavalin A chromatography; and differential solubilization.

[00638] Exemplary methods of protein purification are provided in: U.S. Patent Nos. 6,339,142; 7,585,955; 8,946,395; 9,067,990; 10,246,484; and Marshak et al., “Strategies for Protein Purification and Characterization — A Laboratory Course Manual” CSHL Press (1996); the disclosures of which are incorporated herein by reference in their entireties.

[00639] Yield screening and evaluation

[00640] Peptide yield can be determined by any of the methods known to those of skill in the art (e.g., capillary gel electrophoresis (CGE), Western blot analysis, and the like). Activity assays, as described herein and known in the art, can also provide information regarding peptide yield. In some embodiments, these or any other methods known in the art can be used to evaluate peptide yield.

[00641] Quantification assays

[00642] In some embodiments, and without limitation, heterologous SDH and heterologous polypeptide (e.g., a CRP) yield can be measured using: HPLC; Mass spectrometry (MS) and related techniques; LC/MS/MS; reverse phase protein arrays (RPPA); immunohistochemistry; ELISA; suspension bead array, mass spectrometry; dot blot; SDS- PAGE; capillary gel electrophoresis (CGE); Western blot analysis; Bradford assay; measuring UV absorption at 260nm; Lowry assay; Smith copper/bicinchoninic assay; a secretion assay; Pierce protein assay; Biuret reaction; and the like. Exemplary methods of protein quantification are provided in Stoscheck, C. 1990 “Quantification of Protein” Methods in Enzymology , 182:50-68; Lowry, O. Rosebrough, A., Farr, A. and Randall, R. 1951 J. Biol. Chem . 193:265; Smith, P. et al., (1985) Anal. Biochem. 150:76-85; Bradford, M. 1976 “A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding” Anal. Biochem. 72:248- 254; Cabib, E. and Polacheck, I. 1984 “Protein assay for dilute solutions.” Methods in Enzymology, 104:318-328; Turcanu, Victor; Williams, Neil A. (2001). ’’Cell identification and isolation on the basis of cytokine secretion: A novel tool for investigating immune responses.” Nature Medicine. 7 (3): 373-376; U.S. Patent NO. 6,391,649; the disclosures of which are incorporated herein by reference in their entireties.

[00643] In other embodiments, heterologous SDH and heterologous polypeptide (e.g., a CRP) yield can be quantified and/or assessed using methods that include, without limitation: recombinant protein mass per volume of culture (e.g., gram or milligrams protein per liter culture); percent or fraction of recombinant protein insoluble precipitate obtained after cell lysis determined in (e.g., recombinant protein extracted supernatant in an amount/amount of protein in the insoluble components); percentage or fraction of active protein (e.g., an amount/analysis of the active protein for use in protein amount); total cell protein (tcp) percentage or fraction; and/or the amount of protein/ cell and the dry biomass of a percentage or ratio.

[00644] In some embodiments, wherein yield is expressed in terms of culture volume, the culture cell density may be taken into account, particularly when yields between different cultures are being compared.

[00645] In some embodiments, the present disclosure provides a method of producing a heterologous polypeptide that is at least about 5%, at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, or greater of total cell protein (tcp). “Percent total cell protein” is the amount of heterologous polypeptide in the host cell as a percentage of aggregate cellular protein. The determination of the percent total cell protein is well known in the art.

[00646] “Total cell protein (tcp)” or “Percent total cell protein (% tcp)” is the amount of protein or polypeptide in the host cell as a percentage of aggregate cellular protein. Methods for the determination of the percent total cell protein are well known in the art.

[00647] In some embodiments, HPLC can be used to quantify peptide yield. For example, in some embodiments, heterologous polypeptide (e.g., a CRP) yield can be evaluated using an Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, Cl 8 reverse-phase analytical HPLC column and an auto-injector. An illustrative use of the Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto-injector is as follows: filtered conditioned media samples from transformed K. lactis cells are analyzed using Agilent 1100 HPLC system equipped with an Onyx monolithic 4.5 x 100 mm, C18 reverse-phase analytical HPLC column and an auto-injector by analyzing HPLC grade water and acetonitrile containing 0.1% trifluoroacetic acid, constituting the two mobile phase solvents used for the HPLC analyses; the peak areas of the heterologous polypeptide (e.g., a CRP) are analyzed using HPLC chromatographs, and then used to calculate the peptide concentration in the conditioned media, which can be further normalized to the corresponding final cell densities (as determined by OD600 measurements) as normalized peptide yield.

[00648] Peptide yields are not always sufficient to accurately compare the strain production rate. Individual strains may have different growth rates, hence when a culture is harvested, different cultures may vary in cell density. A culture with a high cell density may produce a higher concentration of the peptide in the media, even though the peptide production rate of the strain is lower than another strain which has a higher production rate. Accordingly, the term “normalized yield” is created by dividing the peptide yield with the cell density in the corresponding culture and this allows a better comparison of the peptide production rate between strains. The cell density is represented by the light absorbance at 600 nm with a unit of “A” (Absorbance unit).

[00649] Screening yeast colonies that have undergone a transformation with a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, can identify the high yield yeast cells from hundreds of potential colonies. These strains can be fermented in bioreactor to achieve at least up to 4 g/L or at least up to 3 g/L or at least up to 2 g/L yield of the heterologous polypeptide (e.g., a CRP) when using optimized fermentation media and fermentation conditions described herein. The higher rates of production (expressed in mg/L) can be anywhere from about 100 mg/L to about 100,000 mg/L; or from about 100 mg/L to about 90, 000 mg/L; or from about 100 mg/L to about 80,000 mg/L; or from about 100 mg/L to about 70,000 mg/L; or from about 100 mg/L to about 60,000 mg/L; or from about 100 mg/L to about 50,000 mg/L; or from about 100 mg/L to about 40,000 mg/L; or from about 100 mg/L to about 30,000 mg/L; or from about 100 mg/L to about 20,000 mg/L; or from about 100 mg/L to about 17,500 mg/L; or from about 100 mg/L to about 15,000 mg/L; or from about 100 mg/L to about 12,500 mg/L; or from about 100 mg/L to about 10,000 mg/L; or from about 100 mg/L to about 9,000 mg/L; or from about 100 mg/L to about 8,000 mg/L; or from about 100 mg/L to about 7,000 mg/L; or from about 100 mg/L to about 6,000 mg/L; or from about 100 mg/L to about 5,000 mg/L; or from about 100 mg/L to about 3,000 mg/L; or from about 100 mg/L to 2,000 mg/L; or from about 100 mg/L to 1,500 mg/L; or from about 100 mg/L to 1,000 mg/L; or from about 100 mg/L to 750 mg/L; or from about 100 mg/L to 500 mg/L; or from about 150 mg/L to 100,000 mg/L; or from about 200 mg/L to 100,000 mg/L; or from about 300 mg/L to 100,000 mg/L; or from about 400 mg/L to 100,000 mg/L; or from about 500 mg/L to 100,000 mg/L; or from about 750 mg/L to 100,000 mg/L; or from about 1,000 mg/L to 100,000 mg/L; or from about 1,250 mg/L to 100,000 mg/L; or from about 1,500 mg/L to 100,000 mg/L; or from about 2,000 mg/L to 100,000 mg/L; or from about 2,500 mg/L to 100,000 mg/L; or from about 3,000 mg/L to 100,000 mg/L; or from about 3,500 mg/L to 100,000 mg/L; or from about 4,000 mg/L to 100,000 mg/L; or from about 4,500 mg/L to 100,000 mg/L; or from about 5,000 mg/L to 100,000 mg/L; or from about 6,000 mg/L to 100,000 mg/L; or from about 7,000 mg/L to 100,000 mg/L; or from about 8,000 mg/L to 100,000 mg/L; or from about 9,000 mg/L to 100,000 mg/L; or from about 10,000 mg/L to 100,000 mg/L; or from about 12,500 mg/L to 100,000 mg/L; or from about 15,000 mg/L to 100,000 mg/L; or from about 17,500 mg/L to 100,000 mg/L; or from about 20,000 mg/L to 100,000 mg/L; or from about 30,000 mg/L to 100,000 mg/L; or from about 40,000 mg/L to 100,000 mg/L; or from about 50,000 mg/L to 100,000 mg/L; or from about 60,000 mg/L to 100,000 mg/L; or from about 70,000 mg/L to 100,000 mg/L; or from about 80,000 mg/L to 100,000 mg/L; or from about 90,000 mg/L to 100,000 mg/L; or any range of any value provided or even greater yields than can be achieved with a peptide before conversion, using the same or similar production methods that were used to produce the peptide before conversion.

[00650] Any of the foregoing methods can be used to produce heterologous polypeptide of the present disclosure. For example, any of the foregoing methods can be used to produce, generate, make, express, transcribe, translate, synthesize or otherwise create, any of the heterologous polypeptide (e.g., a CRP) described herein, including, without limitation, ACTX peptides (e g., U-ACTX-Hvla; U+2-ACTX-Hvla; rU-ACTX-Hvla; rU-ACTX- Hvlb; K-ACTX-Hvla; K+2-ACTX-Hvla; ®-ACTX-Hvla; and/or ®+2-ACTX-Hvla); ctenitoxin (CNTX); T-CNTX-Pnla; Ul-agatoxin-Talb; TVPs; Av2; Av3; AVPs; sea anemone toxins; and/or Phoneutria toxins.

[00651] CULTURE AND FERMENTATION CONDITIONS

[00652] Cell culture techniques are well-known in the art. In some embodiments, the culture method and/or materials will necessarily require adaption based on the host cell selected (e.g., modifying pH, temperature, medium contents, and the like). In some embodiments, the medium culture contains a sole carbon source (e.g., sorbitol). In some embodiments, any known culture technique may be employed to produce a recombinant yeast cell of the present disclosure.

[00653] Exemplary culture methods are provided in U.S. Patent Nos.

3,933,590; 3,946,780; 4,988,623; 5,153,131; 5,153,133; 5,155,034; 5,316,905; 5,330,908; 6,159,724; 7,419,801; 9,320,816; 9,714,408; and 10,563,169; the disclosures of which are incorporated herein by reference in their entireties.

[00654] Yeast culture

[00655] Yeast cell culture techniques are well known to those having ordinary skill in the art. Exemplary methods of yeast cell culture can be found in Evans, Yeast Protocols.

Springer (1996); Bill, Recombinant Protein Production in Yeast. Springer (2012); Hagan et al., Fission Yeast: A Laboratory Manual, CSH Press (2016); Konishi et al., Improvement of the transformation efficiency of Saccharomyces cerevisiae by altering carbon sources in preculture. Biosci Biotechnol Biochem. 2014; 78(6): 1090-3; Dymond, Saccharomyces cerevisiae growth media. Methods Enzymol. 2013; 533:191-204; Looke et al., Extraction of genomic DNA from yeasts for PCR-based applications. Biotechniques. 2011 May; 50(5):325- 8; and Romanos et al., Culture of yeast for the production of heterologous proteins. Curr Protoc Cell Biol. 2014 Sep 2; 64:20.9.1-16, the disclosure of which is incorporated herein by reference in its entirety.

[00656] Yeast can be cultured in a variety of media, e.g., in some embodiments, yeast can be cultured in minimal medium; yeast synthetic drop-out medium; Yeast Nitrogen Base (YNB with or without amino acids); YEPD medium; ADE D medium; ADE DS" medium; LEU D medium; HIS D medium; or Mineral salts medium.

[00657] In some embodiments, the media contains a sole carbon source. In some embodiments, the sole carbon source is sorbitol.

[00658] In some embodiments, yeast can be cultured in minimal medium having a sole carbon source. In some embodiments, minimal medium ingredients can comprise: 4% Alcohol Sugar (e.g., sorbitol); Phosphate Buffer, pH 6.0; Magnesium Sulfate; Calcium Chloride; Ammonium Sulfate; Sodium Chloride; Potassium Chloride; Copper Sulfate;

Manganese Sulfate; Zinc Chloride; Potassium Iodide; Cobalt Chloride; Sodium Molybdate; Boric Acid; Iron Chloride; Biotin; Calcium pantothenate; Thiamine; Myo-inositol; Nicotinic Acid; and Pyridoxine.

[00659] In some embodiments, Kluyveromyces lactis are grown in minimal media supplemented with 4% sorbitol, as the sole carbon source. Cultures are incubated at 30°C until mid-log phase (24-48 hours) for [3-galactosidase measurements, or for up to 4-10 days at 23.5°C for heterologous protein expression.

[00660] In some embodiments, yeast cells can be cultured in 48-well Deep-well plates, sealed after inoculation with sterile, air-permeable cover. Colonies of yeast, for example, K. lactis cultured on plates can be picked and inoculated the deep-well plates with 2.2 mL media per well, composed of defined medium comprising sorbitol (DMSor). Inoculated deep-well plates can be grown for 4-10 days at 23.5°C with 280 rpm shaking in a refrigerated incubatorshaker. In one example, on day 6 post-inoculation, conditioned media should be harvested by centrifugation at 4000 rpm for 10 minutes, followed by filtration using filter plate with 0.22 pM membrane, with filtered media are subject to HPLC analyses.

[00661] In some embodiments, yeast species such as Kluyveromyces lactis, Saccharomyces cerevisiae, Pichia pastoris, and others, can be used as the yeast to be modified using the methods described herein.

[00662] Temperature and pH conditions will vary depending on the stage of culture and the yeast cell species selected. Variables such as temperature and pH in cell culture are readily known to those having ordinary skill in the art.

[00663] The pH level is important in the culturing of yeast. One of skill in the art will appreciate that the culturing process includes not only the start of the yeast culture but the maintenance of the culture as well. The yeast culture may be started at any pH level, however, since the media of a yeast culture tends to become more acidic (i.e., lowering the pH) over time, care must be taken to monitor the pH level during the culturing process.

[00664] In some embodiments of the invention, the yeast is grown in a medium at a pH level that is dictated based on the species of yeast used, the stage of culture, and/or the temperature. Thus, in some embodiments, the pH level can fall within a range from about 2 to about 10. Those having ordinary skill in the art will recognize that the optimum pH for most microorganisms is near the neutral point (pH 7.0). However, in some embodiments, some fungal species prefer an acidic environment: accordingly, in some embodiments, the pH can range from 2 to 6.5. In some embodiments, the pH can range from about 4 to about 4.5. Some fungal species (e.g., molds) can grow can grow in a pH of from about 2 to about 8.5, but favor an acid pH. See Mountney & Gould, Practical food microbiology and technology. 1988. Ed. 3; and Pena et al., Effects of high medium pH on growth, metabolism and transport in Saccharomyces cerevisiae. FEMS Yeast Res. 2015 Mar;15(2):fou005.

[00665] In other embodiments, the pH is about 5.7 to 5.9, 5.8 to 6.0, 5.9 to 6.1, 6.0 to 6.2, 6.1 to 6.3, 6.2 to 6.5, 6.4 to 6.7, 6.5 to 6.8, 6.6 to 6.9, 6.7 to 7.0, 6.8 to 7.1, 6.9 to 7.2, 7.0 to 7.3, 7.1 to 7.4, 7.2 to 7.5, 7.3 to 7.6, 7.4 to 7.7, 7.5 to 7.8, 7.6 to 7.9, 7.7 to 8.0, 7.8 to 8.1, 7.9 to 8.2, 8.0 to 8.3, 8.1 to 8.4, 8.2 to 8.5, 8.3 to 8.6, 8.4 to 8.7, or 8.5 to 8.8.

[00666] In some embodiments, the pH of the medium can be at least 5.5. In other aspects, the medium can have a pH level of about 5.5. In other aspects, the medium can have a pH level of between 4 and 8. In some cases, the culture is maintained at a pH level of between 5.5 and 8. In other aspects, the medium has a pH level of between 6 and 8. In some cases, medium has a pH level that is maintained at a pH level of between 6 and 8. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.1 and 8.1. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.2 and

8.2, In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.3 and 8.3. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.4 and 8.4. In some embodiments, the yeast is grown and/or maintained at a pH level of between 5.5 and 8.5. In some embodiments, the yeast is grown and/or maintained at a pH level of between 6.5 and 8.5. In some embodiments, the yeast is grown at a pH level of about

5.6, 5.7, 5.8 or 5.9. In some embodiments, the yeast is grown at a pH level of about 6. In some embodiments, the yeast is grown at a pH level of about 6.5. In some embodiments, the yeast is grown at a pH level of about 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 or 7.0. In some embodiments, the yeast is grown at a pH level of about 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,

7.7, 7.8, 7.9, or 8.0. In some embodiments, the yeast is grown at a level of above 8.

[00667] In some embodiments, the pH of the medium can range from a pH of 2 to 8.5. In certain embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1,

5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2,

7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[00668] Exemplary methods of yeast culture can be found in U.S. Patent No. 5,436,136, entitled “Repressible yeast promoters” (filed 12/20/1991; assignee Ciba-Geigy Corporation); U.S. Patent No. 6,645,739, entitled “Yeast expression systems, methods of producing polypeptides in yeast, and compositions relating to same” (filed 07/26/2001; assignee Phoenix Pharmacologies, Inc., Lexington, KY); and U.S. Patent No. 10,023,836, entitled “Medium for yeasts” (filed 08/23/2013; assignee Yamaguchi University); the disclosures of which are incorporated herein by reference in their entirety.

[00669] Fermentation

[00670] The present disclosure contemplates the culture of yeast cells in any fermentation format. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes may be employed herein. [00671] Fermentation may be performed at any scale. The methods and techniques contemplated according to the present disclosure are useful for recombinant protein expression at any scale. Thus, in some embodiments, e.g., microliter-scale, milliliter scale, centiliter scale, and deciliter scale fermentation volumes may be used, and 1 liter scale and larger fermentation volumes can be used.

[00672] In some embodiments, the fermentation volume is at or above about 1 liter. For example, in some embodiments, the fermentation volume is about 1 liter to about 100 liters. In some embodiments, the fermentation volume is about 1 liter, about 2 liters, about 3 liters, about 4 liters, about 5 liters, about 6 liters, about 7 liters, about 8 liters, about 9 liters, or about 10 liters. In some embodiments, the fermentation volume is about 1 liter to about 5 liters, about 1 liter to about 10 liters, about 1 liter to about 25 liters, about 1 liter to about 50 liters, about 1 liter to about 75 liters, about 10 liters to about 25 liters, about 25 liters to about 50 liters, or about 50 liters to about 100 liters.

[00673] In other embodiments, the fermentation volume is at or above 5 liters, 10 liters, 15 liters, 20 liters, 25 liters, 50 liters, 75 liters, 100 liters, 200 liters, 500 liters, 1,000 liters, 2,000 liters, 5,000 liters, 10,000 liters, or 50,000 liters.

[00674] In some embodiments, the fermentation medium can be a nutrient solution used for growing and or maintaining cells. Without limitation, this solution ordinarily provides at least one component from one or more of the following categories: (1) an energy source, usually in the form of a carbon source; (2) all essential amino acids, and usually the basic set of twenty amino acids; (3) vitamins and/or other organic compounds required at low concentrations; (4) free fatty acids or lipids, for example linoleic acid; and (5) trace elements, where trace elements are defined as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, usually in the micromolar range.

[00675] In some embodiments, the fermentation medium can be the same as the cell culture medium or any other media described herein. In some embodiments, the fermentation medium can be different from the cell culture medium. In some embodiments, the fermentation medium can be modified in order to accommodate the large-scale production of proteins.

[00676] In some embodiments, the fermentation medium can be supplemented electively with one or more components from any of the following categories: (1) hormones and other growth factors such as, serum, insulin, transferrin, and the like; (2) salts, for example, magnesium, calcium, and phosphate; (3) buffers, such as HEPES; (4) nucleosides and bases such as, adenosine, thymidine, etc.; (5) protein and tissue hydrolysates, for example peptone or peptone mixtures which can be obtained from purified gelatin, plant material, or animal byproducts; (6) antibiotics, such as gentamycin; and (7) cell protective agents, for example pluronic polyol.

[00677] In some embodiments, the pH of the fermentation medium can be maintained using pH buffers and methods known to those of skill in the art. Control of pH during fermentation can also can be achieved using aqueous ammonia. In some embodiments, the pH of the fermentation medium will be selected based on the preferred pH of the organism used. Thus, in some embodiments, and depending on the host cell and temperature, the pH can range from about to 1 to about 10.

[00678] In some embodiments, the pH of the fermentation medium can range from a pH of 2 to 8.5. In certain embodiments, the pH is about 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7,

4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8,

6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, or 8.8.

[00679] In other embodiments, the pH is about 5.7 to about 5.9, 5.8 to about 6.0, 5.9 to about 6.1, 6.0 to about 6.2, 6.1 to about 6.3, 6.2 to about 6.5, 6.4 to about 6.7, 6.5 to about

6.8, 6.6 to about 6.9, 6.7 to about 7.0, 6.8 to about 7.1, 6.9 to about 7.2, 7.0 to about 7.3, 7.1 to about 7.4, 7.2 to about 7.5, 7.3 to about 7.6, 7.4 to about 7.7, 7.5 to about 7.8, 7.6 to about

7.9, 7.7 to about 8.0, 7.8 to about 8.1, 7.9 to about 8.2, 8.0 to about 8.3, 8.1 to about 8.4, 8.2 to about 8.5, 8.3 to about 8.6, 8.4 to about 8.7, or 8.5 to about 8.8.

[00680] In other embodiments, e.g., where a yeast cell is used, the pH can range from about 4.0 to about 8.0.

[00681] In some embodiments, neutral pH, i.e., a pH of about 7.0 can be used.

[00682] Those having ordinary skill in the art will recognize that during fermentation, the pH levels may drift as result of conversion and production of substrates and metabolic compounds.

[00683] In some embodiments, the fermentation medium can be supplemented with a buffer or other chemical in order to avoid changes to the pH. For example, in some embodiments, the addition of Ca(OH)2, CaCOs. NaOH, or NH4OH can be added to the fermentation medium to neutralize the production of acidic compounds that occur, e.g., in some yeast species during industrial processes.

[00684] Temperature is another important consideration in the fermentation process; and, like pH considerations, temperature will depend on the type of host cell selected.

[00685] In some embodiments, the fermentation temperature is maintained at about 4°C to about 42°C. In certain embodiments, the fermentation temperature is about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, about 15°C, about 16°C, about 17°C, about 18°C, about 19°C, about 20°C, about 21°C, about 22°C, about 23°C, about 24°C, about 25°C, about 26°C, about 27°C, about 28°C, about 29°C, about 30°C, about 31 °C, about 32°C, about 33°C, about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, about 39°C, about 40°C, about 41°C, or about 42°C. [00686] In other embodiments, the fermentation temperature is maintained at about 25°C to about 27°C, about 25°C to about 28°C, about 25°C to about 29°C, about 25°C to about 30°C, about 25°C to about 31 °C, about 25°C to about 32°C, about 25°C to about 33°C, about 26°C to about 28°C, about 26°C to about 29°C, about 26°C to about 30°C, about 26°C to about 31 °C, about 26°C to about 32°C, about 27°C to about 29°C, about 27°C to about 30°C, about 27°C to about 31 °C, about 27°C to about 32°C, about 26°C to about 33°C, about 28°C to about 30°C, about 28°C to about 31 °C, about 28°C to about 32°C, about 29°C to about 31°C, about 29°C to about 32°C, about 29°C to about 33°C, about 30°C to about 32°C, about 30°C to about 33°C, about 31°C to about 33°C, about 31°C to about 32°C, about 30°C to about 33°C, or about 32°C to about 33°C.

[00687] In other embodiments, the temperature is changed during fermentation, e.g., depending on the stage of fermentation.

[00688] Fermentation can be achieved with a variety of microorganisms known to those having ordinary skill in the art. Suitable yeasts for up-scaled production of a recombinant yeast cell of the present disclosure include any yeast listed herein. In some embodiments, non-limiting examples of yeast include those belonging to the genus Saccharomyces spp. (including, but not limited to, 5. cerevisiae (baker's yeast), 5. distaticus, S. uvarum); the genus Kluyveromyces, (including, but not limited to, K. marxianus, K. fragilis),' the genus Candida (including, but not limited to, C. pseudotropicalis , and C. brassicae , Pichia stipitis (a relative of Candida shehatae),' the genus Clavispora (including, but not limited to, C. lusitaniae and C. opuntiae . the genus Pachysolen (including, but not limited to, P. tannophilus),' the genus Bretannomyces (including, but not limited to, e.g., B. clausenii. Other suitable microorganisms include, for example, Moniliella pollinis, Moniliella megachiliensis, Yarrowia lipolytica, Aureobasidium sp., Trichosporonoides sp., Trigonopsis variabilis, Trichosporon sp., Moniliellaacetoabutans sp., Candida magnolias, Ustilaginomycetes sp., Pseudozyma tsukubaensis , yeast species of genera Zygosaccharomyces, Debaryomyces, Hansenula and Pichia, and fungi of the dematioid genus Torula. See, e.g., Philippidis, G. P., 1996, Cellulose bioconversion technology, in Handbook on Bioethanol: Production and Utilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C., 179-212.

[00689] Fermentation medium may be selected depending on the host cell and/or needs of the end-user. Any necessary supplements besides, e.g., carbon, may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source.

[00690] Yeast Fermentation

[00691] Fermentation methods using yeast are well known to those having ordinary skill in the art. In some embodiments, batch fermentation can be used according to the methods provided herein; in other embodiments, continuous fermentation procedures can be used.

[00692] In some embodiments, the batch method of fermentation can be used to practice the present disclosure. Briefly, the batch method of fermentation refers to a type of fermentation that is performed with a closed system, wherein the composition of the medium is determined at the beginning of the fermentation and is not subject to artificial alterations during the fermentation (i.e., the medium is inoculated with one or more yeast cells at the start of fermentation, and fermentation is allowed to proceed, uninterrupted by the user). Typically, in batch fermentation systems, the metabolite and biomass compositions of the system change constantly up to the time the fermentation is stopped. Within batch cultures, yeast cells pass through a static lag phase to a high growth log phase, and, finally, to a stationary phase, in which the growth rate is diminished or stopped. If untreated, yeast cells in the stationary phase will eventually die. In a batch method, yeast cells in log phase generally are responsible for the bulk of synthesis of end product.

[00693] In some embodiments, fed-batch fermentation can be used to practice the present disclosure. Briefly, fed-batch fermentation is similar to typical batch method (described above), however, the substrate in the fed-batch method is added in increments as the fermentation progresses. Fed-batch fermentation is useful when catabolite repression may inhibit yeast cell metabolism, and when it is desirable to have limited amounts of substrate in the medium. Generally, the measurement of the substrate concentration in a fed-batch system is estimated on the basis of the changes of measurable factors reflecting metabolism, such as pH, dissolved oxygen, the partial pressure of waste gases (e.g., CO2), and the like.

[00694] In some embodiments, the fed-batch fermentation procedure can be used to culture recombinant yeast cells as follows: culturing a production organism (e.g., a modified yeast cell) in a 10 L bioreactor sparged with an N2/CO2 mixture, using 5 L broth containing 5 g/L potassium phosphate, 2.5 g/L ammonium chloride, 0.5 g/L magnesium sulfate, and 30 g/L com steep liquor, and a sole carbon source concentration of 20 g/L. As the recombinant yeast cells grow and utilize the carbon source, additional 70% carbon source mixture is then fed into the bioreactor at a rate approximately balancing carbon source consumption. The temperature of the bioreactor is generally maintained at 30° C. Growth continues for approximately 24 hours or more, and the heterologous peptides reach a desired concentration, e.g., with the cell density being between about 5 and 10 g/L. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit such as a centrifuge to remove cells and cell debris, and the fermentation broth can be transferred to a product separations unit. Isolation of the heterologous peptides can take place by standard separations procedures well known in the art.

[00695] In some embodiments, continuous fermentation can be used to practice the present disclosure. Briefly, continuous fermentation refers to fermentation with an open system, wherein a fermentation medium is added continuously to a bioreactor, and an approximately equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a high density, in which yeast cells are primarily in log phase growth. Typically, continuous fermentation methods are performed to maintain steady state growth conditions, and yeast cell loss, due to medium withdrawal, should be balanced against the cell growth rate in the fermentation.

[00696] In some embodiments, the continuous fermentation method can be used as follows: a modified yeast cell can be cultured using a bioreactor apparatus and a medium composition, albeit where the initial carbon source is about, e.g., 30-50 g/L. When the carbon source is exhausted, feed medium of the same composition is supplied continuously at a rate of between about 0.5 L/hr and 1 L/hr, and liquid is withdrawn at the same rate. The heterologous peptide concentration in the bioreactor generally remains constant along with the cell density. Temperature is generally maintained at 30° C., and the pH is generally maintained at about 4.5 using concentrated NaOH and HC1, as required.

[00697] In some embodiments, when producing the heterologous polypeptides (e.g., a CRP), the bioreactor can be operated continuously, for example, for about one month, with samples taken every day or as needed to assure consistency of the target chemical compound concentration. In continuous mode, fermenter contents are constantly removed as new feed medium is supplied. The exit stream, containing cells, medium, and heterologous peptides, can then be subjected to a continuous product separations procedure, with or without removing cells and cell debris, and can be performed by continuous separations methods well known in the art to separate organic products from peptides of interest.

[00698] In some embodiments, a recombinant yeast cell of the present disclosure can be grown, e.g., using a fed batch process in aerobic bioreactor. Briefly, reactors are filled to about 20% to about 70% capacity with medium comprising a carbon source and other reagents. Temperature and pH is maintained using one or more chemicals as described herein. Oxygen level is maintained by sparging air intermittently in concert with agitation.

[00699] For example, in some embodiments, the present disclosure provides a method of using a fed batch process in aerobic bioreactor, wherein the reactor is filled to about 20%;

21%; 22%; 23%; 24%; 25%; 26%; 27%; 28%; 29%; 30%; 31%; 32%; 33%; 34%; 35%; 36%;

37%; 38%; 39%; 40%; 41%; 42%; 43%; 44%; 45%; 46%; 47%; 48%; 49%; 50%; 51%; 52%;

53%; 54%; 55%; 56%; 57%; 58%; 59%; 60%; 61%; 62%; 63%; 64%; 65%; 66%; 67%; 68%;

69%; or 70% capacity.

[00700] In some embodiments, the present disclosure provides a fed batch fermentation method using an aerobic bioreactor to produce the heterologous polypeptides (e.g., CRPs), wherein the medium is a rich culture medium. For example, in some embodiments, the carbon source can be sorbitol.

[00701] In some embodiments, the amount of sorbitol can be about 2 g/L; 3 g/L; 4 g/L;

5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L;

29 g/L; or 30 g/L of the medium.

[00702] In some embodiments, the present disclosure provides a fed batch fermentation method using an aerobic bioreactor, wherein the medium is supplemented with one or more of phosphoric acid, calcium sulfate, potassium sulfate, magnesium sulfate heptahydrate, potassium hydroxide, and/or com steep liquor.

[00703] In some embodiments, the medium can be supplemented with phosphoric acid in an amount of about 2 g/L; 3 g/L; 4 g/L; 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L;

12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; or 30 g/L to the medium.

[00704] In some embodiments, the medium can be supplemented with calcium sulfate in an amount of about 0.05 g/L; 0.15 g/L; 0.25 g/L; 0.35 g/L; 0.45 g/L; 0.55 g/L; 0.65 g/L;

0.75 g/L; 0.85 g/L; 0.95 g/L; 1.05 g/L; 1.15 g/L; 1.25 g/L; 1.35 g/L; 1.45 g/L; 1.55 g/L; 1.65 g/L; 1.75 g/L; 1.85 g/L; 1.95 g/L; 2.05 g/L; 2.15 g/L; 2.25 g/L; 2.35 g/L; 2.45 g/L; 2.55 g/L; 2.65 g/L; 2.75 g/L; 2.85 g/L; or 2.95 g/L to the medium. [00705] In some embodiments, the medium can be supplemented with potassium sulfate in an amount of about 2 g/L; 2.5 g/L; 3 g/L; 3.5 g/L; 4 g/L; 4.5 g/L; 5 g/L; 5.5 g/L; 6 g/L; 6.5 g/L; 7 g/L; 7.5 g/L; 8 g/L; 8.5 g/L; 9 g/L; 9.5 g/L; 10 g/L; 10.5 g/L; 11 g/L; 11.5 g/L; 12 g/L; 12.5 g/L; 13 g/L; 13.5 g/L; 14 g/L; 14.5 g/L; 15 g/L; 15.5 g/L; 16 g/L; 16.5 g/L; 17 g/L; 17.5 g/L; 18 g/L; 18.5 g/L; 19 g/L; 19.5 g/L; or 20 g/L to the medium.

[00706] In some embodiments, the medium can be supplemented with magnesium sulfate heptahydrate in an amount of about 0.25 g/L; 0.5 g/L; 0.75 g/L; 1 g/L; 1.25 g/L; 1.5 g/L; 1.75 g/L; 2 g/L; 2.25 g/L; 2.5 g/L; 2.75 g/L; 3 g/L; 3.25 g/L; 3.5 g/L; 3.75 g/L; 4 g/L; 4.25 g/L; 4.5 g/L; 4.75 g/L; 5 g/L; 5.25 g/L; 5.5 g/L; 5.75 g/L; 6 g/L; 6.25 g/L; 6.5 g/L; 6.75 g/L; 7 g/L; 7.25 g/L; 7.5 g/L; 7.75 g/L; 8 g/L; 8.25 g/L; 8.5 g/L; 8.75 g/L; 9 g/L; 9.25 g/L; 9.5 g/L; 9.75 g/L; 10 g/L; 10.25 g/L; 10.5 g/L; 10.75 g/L; 11 g/L; 11.25 g/L; 11.5 g/L; 11.75 g/L;

12 g/L; 12.25 g/L; 12.5 g/L; 12.75 g/L; 13 g/L; 13.25 g/L; 13.5 g/L; 13.75 g/L; 14 g/L; 14.25 g/L; 14.5 g/L; 14.75 g/L; or 15 g/L to the medium.

[00707] In some embodiments, the medium can be supplemented with potassium hydroxide in an amount of about 0.25 g/L; 0.5 g/L; 0.75 g/L; 1 g/L; 1.25 g/L; 1.5 g/L; 1.75 g/L; 2 g/L; 2.25 g/L; 2.5 g/L; 2.75 g/L; 3 g/L; 3.25 g/L; 3.5 g/L; 3.75 g/L; 4 g/L; 4.25 g/L; 4.5 g/L; 4.75 g/L; 5 g/L; 5.25 g/L; 5.5 g/L; 5.75 g/L; 6 g/L; 6.25 g/L; 6.5 g/L; 6.75 g/L; or 7 g/L to the medium.

[00708] In some embodiments, the medium can be supplemented with com steep liquor in an amount of about 5 g/L; 6 g/L; 7 g/L; 8 g/L; 9 g/L; 10 g/L; 11 g/L; 12 g/L; 13 g/L; 14 g/L; 15 g/L; 16 g/L; 17 g/L; 18 g/L; 19 g/L; 20 g/L; 21 g/L; 22 g/L; 23 g/L; 24 g/L; 25 g/L; 26 g/L; 27 g/L; 28 g/L; 29 g/L; 30 g/L; 31 g/L; 32 g/L; 33 g/L; 34 g/L; 35 g/L; 36 g/L;

37 g/L; 38 g/L; 39 g/L; 40 g/L; 41 g/L; 42 g/L; 43 g/L; 44 g/L; 45 g/L; 46 g/L; 47 g/L; 48 g/L; 49 g/L; 50 g/L; 51 g/L; 52 g/L; 53 g/L; 54 g/L; 55 g/L; 56 g/L; 57 g/L; 58 g/L; 59 g/L;

60 g/L; 61 g/L; 62 g/L; 63 g/L; 64 g/L; 65 g/L; 66 g/L; 67 g/L; 68 g/L; 69 g/L; or 70 g/L to the medium.

[00709] In some embodiments, the temperature of the reactor can be maintained between about 15°C and about 45°C. In some embodiments, the reactor can have a temperature of about 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[00710] In some embodiments, the pH can have a level of about 3 to about 6. In some embodiments, the pH can be 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, or 6.0. [00711] In some embodiments, the pH can be maintained at a constant level via the addition of one or more chemicals. For example, in some embodiments, ammonium hydroxide can be added to maintain pH. In some embodiments, ammonium hydroxide can be added to a level of ammonium hydroxide in the medium that is about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, of ammonium hydroxide [00712] In some embodiments, oxygen levels can be maintained by sparging. For example, in some embodiments, dissolved oxygen can be maintained at a constant level by sparging air between 0.5-1.5 volume/volume/min and by increasing agitation to maintain a set point of 10-30%.

[00713] In some embodiments, inoculation of the reactor can be accomplished based on an overnight seed culture comprising from about 2.5 g/L to about 50 g/L of a sole carbon source, e.g., sorbitol. In some embodiments, the overnight seed culture can comprise com steep liquor, e.g., from about 2.5 g/L to about 50 g/L of com steep liquor.

[00714] In some embodiments, the inoculation percentage can range from about 5-20% of initial fill volume. Following inoculation, the reactor can be fed with from about a 50% to about an 80% solution of the selected carbon source up until the reactor is filled and/or the desired supernatant peptide concentration is achieved. In some embodiments, the time required to fill the reactor can range from about 86 hours to about 160 hours. In some embodiments, the quantity required to reach the desired peptide concentration can range from about 0.8 g/L to about 1.2 g/L. Upon completion of the fermentation, the contents can be passed through a cell separation unit and optionally concentrated, depending on intended use of the material.

[00715] Additional recipes for yeast fermentation media are well known in the art, and are provided herein.

[00716] In some embodiments, recipes for yeast cell fermentation media and stocks can be as follows: (1) MSM media recipe: 2 g/L sodium citrate dihydrate; 1 g/L calcium sulfate dihydrate (0.79 g/L anhydrous calcium sulfate); 42.9g/L potassium phosphate monobasic; 5.17g/L ammonium sulfate; 14.33 g/L potassium sulfate; 11.7 g/L magnesium sulfate heptahydrate; 2 mL/L PTMltrace salt solution; 0.4 ppm biotin (from 500X, 200 ppm stock); 1-2% pure sorbitol. (2) PTM1 trace salts solution: Cupric sulfate-5H2O 6.0 g; Sodium iodide 0.08 g; Manganese sulfate-H2O 3.0 g; Sodium molybdate-2H2O 0.2 g; Boric Acid 0.02 g; Cobalt chloride 0.5 g; Zinc chloride 20.0 g; Ferrous sulfate-7 H2O 65.0 g; Biotin 0.2 g; Sulfuric Acid 5.0 ml; add Water to a final volume of 1 liter. [00717] In some embodiments, an exemplary K. lactis defined medium comprising sorbitol (DMSor) is as follows: 11.83 g/L KH₂PO₄, 2.299 g/L K₂HPO₄, 40 g/L of a fermentable sugar alcohol, for example, sorbitol, 1 g/L MgSO₄.7H₂O, 10 g/L (NH₄)SO₄, 0.33 g/L CaCl₂.2H₂O, 1 g/L NaCl, 1 g/L KC1, 5 mg/L CuSO₄.5H₂O, 30 mg/L MnSO₄.H₂O, 10 mg/L, ZnCl₂, 1 mg/L KI, 2 mg/L COC1₂.6H₂O, 8mg/L Na₂MoO₄.2H₂O, 0.4 mg/L H₃BO₃,15 mg/L FeCl₃.6H₂O, 0.8 mg/L biotin, 20 mg/L Ca-pantothenate, 15 mg/L thiamine, 16 mg/L myo-inositol, 10 mg/L nicotinic acid, and 4 mg/L pyridoxine.

[00718] Any of the foregoing methods can be used to practice the present disclosure, e.g., growing recombinant yeast and/or increasing the expression of a heterologous polypeptide in a recombinant yeast cell. And, any of the foregoing methods can be used to produce heterologous polypeptide of the present disclosure (e.g., one or more CRPs). For example, any of the foregoing methods can be used to produce, generate, make, express, transcribe, translate, synthesize or otherwise create, any of the heterologous polypeptide (e.g., a CRP) described herein, including, without limitation, ACTX peptides (e.g., U-ACTX- Hvla; U+2-ACTX-Hvla; rU-ACTX-Hvla; rU-ACTX-Hvlb; K-ACTX-Hvla; K+2-ACTX- Hvla; ®-ACTX-Hvla; and/or ®+2-ACTX-Hvla); ctenitoxin (CNTX); T-CNTX-Pnla; Ul- agatoxin-Talb; TVPs; Av2; Av3; AVPs; sea anemone toxins; and/or Phoneutria toxins.

[00719] Detecting heterologous polypeptide degradation

[00720] Proteins, polypeptides, and peptides degrade in both biological samples and in solution (e.g., cell culture and/or during fermentation). Methods of detecting peptide degradation (e.g., degradation of a heterologous polypeptide (e.g., a CRP)) are well known in the art. Any of the well-known methods of detecting peptide degradation (e.g., during fermentation) may be employed here.

[00721] In some embodiments, peptide degradation can be detected using isotope labeling techniques; liquid chromatography/mass spectrometry (LC/MS); HPLC; radioactive amino acid incorporation and subsequent detection, e.g., via scintillation counting; the use of a reporter protein, e.g., a protein that can be detected (e.g., by fluorescence, spectroscopy, luminometry, etc.); fluorescent intensity of one or more bioluminescent proteins and/or fluorescent proteins and/or fusions thereof; pulse-chase analysis (e.g., pulse-labeling a cell with radioactive amino acids and following the decay of the labeled protein while chasing with unlabeled precursor, and arresting protein synthesis and measuring the decay of total protein levels with time); cycloheximide-chase assays;

[00722] In some embodiments, an assay can be used to detect peptide degradation, wherein a sample is contacted with a non-fluorescent compound that is operable to react with free primary amine in said sample produced via the degradation of a peptide, and which then produces a fluorescent signal that can be quantified and compared to a standard. Examples of non-fluorescent compounds that can be utilized as fluorescent tags for free amines according to the present disclosure are 3-(4-carboxybenzoyl) quinoline-2-carboxaldehyde (CBQCA), fluorescamine, and o-phthaldialdehyde.

[00723] In some embodiments, the method to determine the readout signal from the reporter protein depends from the nature of the reporter protein. For example, for fluorescent reporter proteins, the readout signal corresponds to the intensity of the fluorescent signal. The readout signal may be measured using spectroscopy-, fluorometry-, photometry-, and/or luminometry-based methods and detection systems, for example. Such methods and detection systems are well known in the art.

[00724] In some embodiments, standard immunological procedures known to those having ordinary skill in the art can be used to detect peptide degradation. For example, in some embodiments, peptide degradation can be detected in a sample using immunoassays that employ a detectable antibody. Such immunoassays include, for example, agglutination assays, ELISA, Pandex microfluorimetric assay, flow cytometry, serum diagnostic assays, and immunohistochemical staining procedures, all of which are well- known in the art. In some embodiments, the levels (e.g., of fluorescence) in one sample can be compared to a standard. An antibody can be made detectable by various means well known in the art. For example, a detectable marker can be directly or indirectly attached to the antibody. Useful markers include, for example, radionucleotides, enzymes, fluorogens, chromogens and chemiluminescent labels.

[00725] Exemplary methods of detecting peptide degradation is provided in U.S. Patent Nos. 5,766,927; 7,504,253; 9,201,073; 9,429,566; United States Patent Application 20120028286; Eldeeb et al., A molecular toolbox for studying protein degradation in mammalian cells. J Neurochem. 2019 Nov;151(4):520-533; and Buchanan et al., Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae. J Vis Exp. 2016; (110): 53975, the disclosures of which are incorporated herein by reference in their entireties.

[00726] INACTIVATING ENDOGENOUS SDH

[00727] In some embodiments, if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence (e.g., an endogenous SDH gene), said endogenous SDH nucleotide sequence is at least partially inactivated. In some embodiments, a yeast species having an endogenous SDH nucleotide sequence has that endogenous SDH nucleotide sequence at least partially inactivated, for example, essentially completely inactivated.

[00728] In some embodiments, inactivating the endogenous SDH nucleotide sequence ensures a level of control over the recombinant system, and/or allows for a correct ratio of (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), to (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, to be practiced by the user.

[00729] Methods of inactivating endogenous genes are known in the art. For example, inactivating or at least partially inactivating genes, such as endogenous SDH, may be accomplished using one or more of the following techniques: in vivo homologous recombination, RNAi; microRNAs (miRNAs); methylation-based transcriptional silencing; acetylation-based transcriptional silencing; small interfering RNA (siRNA); DNA-directed RNA interference (ddRNAi); Piwi-interacting RNA (piRNA); short hairpin RNA (shRNA); small-temporal RNA (stRNA); morphilinos; zinc finger nuclease (ZFN); transcription activation-like effector nuclease (TALEN); CRISPR/Cas system, and the like.

[00730] In some embodiments, in vivo homologous recombination can be used to inactivate an endogenous SDH gene.

[00731] Typically, the transformation of cells utilizes the power of homologous recombination. Homologous recombination generally describes a process in which nucleotide sequences are exchanged between similar or homologous DNA sequences. Homologous recombination is an intrinsic property of many cells, and is used by cells in certain circumstances to repair DNA damage; homologous recombination also occurs during meiosis, resulting in new combinations of DNA sequences. The molecular machinery underpinning the process of homologous recombination can be harnessed to practice the present disclosure in order to modify an organism’s genome and/or DNA sequences.

[00732] For example, by harnessing the process of homologous recombination, one or more polynucleotides, e.g., a gene (or part of a gene) contained within an organism’s genome, can be removed or replaced with a heterologous polynucleotide (also referred to as a “transgene”) or allele created in vitro. Indeed, the process is so precise, and can be reproduced with such fidelity, that the only genetic difference between the initial organism and the organism post-modification, is the modification itself.

[00733] Homologous recombination can also be used to modify genes via the attachment of an epitope tag (e.g., FLAG, myc, or HA); alternatively, a gene of interest can be operably linked to the coding sequence of a fluorescent protein, e.g., green fluorescent protein (GFP). And, because a given epitope tag or fusion is created within the context of the organism and/or its genome, said gene of interest is subjected to the inherent regulatory elements and regulatory events that normally would occur in host organism — both spatially and temporally. Accordingly, tagged transgenes (e.g., a heterologous polynucleotide of interest tagged with an epitope tag or operably linked to GFP) can be compared to an isogenic wild-type organism in order to examine gene function, peptide localization, and/or regulation. [00734] In some embodiments, a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, can be integrated into a host cell’s genome through homologous recombination.

[00735] Exemplary descriptions of in vivo homologous recombination, and methods regarding the same, can be found in U.S. Patent No. 5,789,215, entitled “Gene targeting in animal cells using isogenic DNA constructs” (filed 08/07/1997; assignee GenPharm International, San Jose, CA); U.S. Patent No. 6,528,314, entitled “Procedure for specific replacement of a copy of a gene present in the recipient genome by the integration of a gene different from that where the integration is made” (filed 06/06/1995; assignee Institut, Pasteur); U.S. Patent No. 6,537,542, entitled “Targeted introduction of DNA into primary or secondary cells and their use for gene therapy and protein production (filed 04/14/2000; assignee Transkaryotic Therapies, Inc., Cambridge, MA); U.S. Patent No. 8,048,645, entitled “Method of producing functional protein domains (filed 08/01/2001; assignee Merck Serono SA); and U.S. Patent No. 8,173,394, entitled “Systems and methods for protein production” (filed 04/06/2009; assignee Wyeth LLC, Madison, NJ); the disclosures of which are incorporated herein by reference in their entirety.

[00736] Exemplary descriptions of RNAi, and methods of using the same, are provided in U.S. Patent No. 6,506,559, the disclosures of which are incorporated herein by reference in its entirety.

[00737] Exemplary descriptions of microRNAs, and methods of using the same, are provided in U.S. Patent Nos. 8,288,356; 8,014,956; 8,846,350; and 9,695,475; the disclosures of which are incorporated herein by reference in their entireties.

[00738] Exemplary descriptions of methylation-based transcriptional silencing, and methods of using the same, are provided in U.S. Patent Nos. 5,840,497 and 9,914,936; and U.S. Patent Application Publication No. 20040006036; the disclosures of which are incorporated herein by reference in their entireties. [00739] Exemplary descriptions of acetylation-based transcriptional silencing, and methods of using the same, are provided in U.S. Patent No. 8,518,635 and U.S. Patent Application Publication No. 20030232782; the disclosures of which are incorporated herein by reference in their entireties.

[00740] Exemplary descriptions of small interfering RNA (siRNA), and methods of using the same, are provided in U.S. Patent Nos. 7,691,997; 7,807,819; 7,855,186; 8,457,902; 9,315,808; and 9,334,497; the disclosures of which are incorporated herein by reference in their entireties.

[00741] Exemplary descriptions of DNA-directed RNA interference (ddRNAi), and methods of using the same, are provided in U.S. Patent Nos. 8,008,468; 9,518,260; and 10,898,505; the disclosures of which are incorporated herein by reference in their entireties.

[00742] Exemplary descriptions of Piwi -interacting RNA (piRNA), and methods of using the same, are provided in U.S. Patent No. 10,767,178; and U.S. Patent Application Publication No. 20090062228; the disclosures of which are incorporated herein by reference in their entireties.

[00743] Exemplary descriptions of short hairpin RNA (shRNA), and methods of using the same, are provided in U.S. Patent Nos. 8,779,115; 9,290,763; 9,556,431; 10,329,562; and 10,968,451; the disclosures of which are incorporated herein by reference in their entireties.

[00744] Exemplary descriptions of zinc finger nuclease (ZFN), and methods of using the same, are provided in U.S. Patent Nos. 8,524,221; 9,388,426; 9,434,776; and 10,918,668; the disclosures of which are incorporated herein by reference in their entireties.

[00745] Exemplary descriptions of transcription activation-like effector nuclease (TALEN), and methods of using the same, are provided in U.S. Patent Nos. 9,040,677; 9,181,535; and 10,227,581; the disclosures of which are incorporated herein by reference in their entireties.

[00746] Exemplary descriptions of CRISPR/Cas systems, and methods of using the same, are provided in U.S. Patent Nos. 8,871,445; 8,932,814; 8,945,839;

10,808,245; 10,995,327; and 11,060,114; the disclosures of which are incorporated herein by reference in their entireties.

[00747] COPY LOSS AND COPY MAINTENANCE

[00748] Multi-copy integrations of polynucleotides operable to encode heterologous peptides exhibit copy number loss as a result of the repeated regions of homology in close proximity. Copy number can correlate positively with peptide expression, and the loss of copies can result in a decrease in yield. Furthermore, if a copy loss event (e.g., “out- recombination”) is accompanied by a fitness benefit in growth, selection can promote the increase in frequency of the lower copy number cells in a mixed culture over time, thus reducing yield further.

[00749] The recombinant yeast cells of the present disclosure advantageously eliminate, prevent, and/or reduce the rate of copy number loss and/or the number of copy loss events in cells expressing heterologous peptides, when grown in a sole carbon source that is sorbitol.

[00750] Methods of determining copy number maintenance and/or copy number loss are well known in the art. For example, in some embodiments, copy number maintenance or copy number loss can be determined by determining the initial copy number of a nucleotide sequences operable to encode a heterologous peptide, in a recombinant cell, and comparing said initial copy number to a copy number after the cell at a later time, e.g., after the cell has been replicated once, twice, or for multiple generations.

[00751] In some embodiments, copy number(s) of specific genes (and/or their open reading frames ORFs) can be determined via qPCR. An exemplary qPCR copy number screening process of K. lactis cells is as follows: a heterologous polynucleotide operable to encode a heterologous SDH and a heterologous polypeptide (e.g., a CRP) ORF can be inserted into the expression vector, pLB10V5, and transformed into the T. lactis strain, YCT306, from New England Biolabs, Ipswich, MA, USA. Once the heterologous SDH and heterologous polypeptide (e.g., a CRP) transgenes were cloned into pLB10V5 plasmid and transformed into YCT306, their expression was controlled by the LAC4 promoter. The resulting transformed colonies were screened for integration of the number of expression cassettes that were integrated into these strains were estimated via qPCR amplification of the sorbitol dehydrogenase (SDH or “lid”) gene using ura3 as the reference gene.

[00752] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein the vector comprises 2 or more copies of the heterologous polynucleotide. [00753] In some embodiments, the vector comprises 10 or more copies of the heterologous polynucleotide.

[00754] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least

60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least

67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least

74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least

81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least 14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least 18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least 22 generations, at least 23 generations, at least 24 generations, at least 25 generations, at least 26 generations, at least 27 generations, at least 28 generations, at least 29 generations, at least 30 generations, at least 31 generations, at least 32 generations, at least 33 generations, at least 34 generations, at least 35 generations, at least 36 generations, at least 37 generations, at least 38 generations, at least 39 generations, at least 40 generations, at least 41 generations, at least 42 generations, at least 43 generations, at least 44 generations, at least 45 generations, at least 46 generations, at least 47 generations, at least 48 generations, at least 49 generations, at least 50 generations, at least 51 generations, at least 52 generations, at least 53 generations, at least 54 generations, at least 55 generations, at least 56 generations, at least 57 generations, at least 58 generations, at least 59 generations, at least 60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least 64 generations, at least 65 generations, at least 66 generations, at least 67 generations, at least 68 generations, at least 69 generations, at least 70 generations, at least 71 generations, at least 72 generations, at least 73 generations, at least 74 generations, at least 75 generations, at least 76 generations, at least 77 generations, at least 78 generations, at least 79 generations, at least 80 generations, at least 81 generations, at least 82 generations, at least 83 generations, at least 84 generations, at least 85 generations, at least 86 generations, at least 87 generations, at least 88 generations, at least 89 generations, at least 90 generations, at least 91 generations, at least 92 generations, at least 93 generations, at least 94 generations, at least 95 generations, at least 96 generations, at least 97 generations, at least 98 generations, at least 99 generations, or at least

100 generations.

[00755] In some embodiments, the recombinant yeast cells have a least 1 copy of a nucleotide sequence operable to encode a heterologous SDH. In some embodiments, the recombinant yeast cells have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 copies of a nucleotide sequence operable to encode a heterologous SDH. In some embodiments, the recombinant yeast cells have a least 1 copy of a nucleotide sequence operable to encode a heterologous SDH integrated immediately after transformation (e.g., at least 1 initial copy). [00756] In some embodiments, the recombinant yeast cells have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 copies of a nucleotide sequence operable to encode a heterologous SDH integrated immediately after transformation of the yeast cell with the heterologous polynucleotide (e.g., 1-16 initial copies). [00757] In some embodiments, the recombinant yeast cells have 10, 11, or 12 copies of a nucleotide sequence operable to encode a heterologous SDH integrated immediately after transformation (e.g., 10, 11, or 12 initial copies).

[00758] In some embodiments, the recombinant yeast cells have 10 copies of a nucleotide sequence operable to encode a heterologous SDH integrated immediately after transformation (e.g., 10 initial copies).

[00759] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the one or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00760] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 10 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00761] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 10 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00762] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 5 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 5 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00763] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 5 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 5 copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. [00764] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 3 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 3 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00765] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 3 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 3 copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00766] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 2 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00767] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 2 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00768] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00769] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 10 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00770] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 10 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00771] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 5 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 5 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00772] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 5 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 5 copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00773] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 3 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 3 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00774] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 3 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 3 copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00775] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 2 or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00776] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 2 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00777] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the one or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00778] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 10 or more or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00779] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 10 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. [00780] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 5 or more or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 5 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00781] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 5 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 5 copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00782] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 3 or more or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 3 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00783] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 3 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 3 copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00784] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 2 or more or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 2 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00785] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising: (a) providing a vector comprising 2 copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol; and wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 2 copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00786] Growing yeast cells in sorbitol

[00787] The recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers and/or prevent copy loss of the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide.

[00788] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers and/or prevent copy loss of the heterologous nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH).

[00789] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers and/or prevent copy loss of the nucleotide sequence operable to encode a heterologous polypeptide.

[00790] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers of a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, wherein copy number maintenance of the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide can be at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least 14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least 18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least 22 generations, at least 23 generations, at least 24 generations, at least 25 generations, at least 26 generations, at least 27 generations, at least 28 generations, at least 29 generations, at least 30 generations, at least 31 generations, at least 32 generations, at least 33 generations, at least 34 generations, at least 35 generations, at least 36 generations, at least 37 generations, at least 38 generations, at least 39 generations, at least 40 generations, at least 41 generations, at least 42 generations, at least 43 generations, at least 44 generations, at least 45 generations, at least 46 generations, at least 47 generations, at least 48 generations, at least 49 generations, at least 50 generations, at least 51 generations, at least 52 generations, at least 53 generations, at least 54 generations, at least 55 generations, at least 56 generations, at least 57 generations, at least 58 generations, at least 59 generations, at least 60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least 64 generations, at least 65 generations, at least 66 generations, at least 67 generations, at least 68 generations, at least 69 generations, at least 70 generations, at least 71 generations, at least 72 generations, at least 73 generations, at least 74 generations, at least 75 generations, at least 76 generations, at least 77 generations, at least 78 generations, at least 79 generations, at least 80 generations, at least 81 generations, at least 82 generations, at least 83 generations, at least 84 generations, at least 85 generations, at least 86 generations, at least 87 generations, at least 88 generations, at least 89 generations, at least 90 generations, at least 91 generations, at least 92 generations, at least 93 generations, at least 94 generations, at least 95 generations, at least 96 generations, at least 97 generations, at least 98 generations, at least 99 generations, or at least 100 generation: [00791] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers of a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, wherein copy number maintenance of the nucleotide sequence operable to encode a heterologous SDH can be at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least 14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least 18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least 22 generations, at least 23 generations, at least 24 generations, at least 25 generations, at least 26 generations, at least 27 generations, at least 28 generations, at least 29 generations, at least 30 generations, at least 31 generations, at least 32 generations, at least 33 generations, at least 34 generations, at least 35 generations, at least 36 generations, at least 37 generations, at least 38 generations, at least 39 generations, at least 40 generations, at least 41 generations, at least 42 generations, at least 43 generations, at least 44 generations, at least 45 generations, at least 46 generations, at least 47 generations, at least 48 generations, at least 49 generations, at least 50 generations, at least 51 generations, at least 52 generations, at least 53 generations, at least 54 generations, at least 55 generations, at least 56 generations, at least 57 generations, at least 58 generations, at least 59 generations, at least 60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least 64 generations, at least 65 generations, at least 66 generations, at least 67 generations, at least 68 generations, at least 69 generations, at least 70 generations, at least 71 generations, at least 72 generations, at least 73 generations, at least 74 generations, at least 75 generations, at least 76 generations, at least 77 generations, at least 78 generations, at least 79 generations, at least 80 generations, at least 81 generations, at least 82 generations, at least 83 generations, at least 84 generations, at least 85 generations, at least 86 generations, at least 87 generations, at least 88 generations, at least 89 generations, at least 90 generations, at least 91 generations, at least 92 generations, at least 93 generations, at least 94 generations, at least 95 generations, at least 96 generations, at least 97 generations, at least 98 generations, at least 99 generations, or at least 100 generations.

[00792] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers of a heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, wherein copy number maintenance of the nucleotide sequence operable to encode a heterologous polypeptide can be at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least

62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least

69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least

76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least

83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least

90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least

97%, at least 98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least

14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least

18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least

22 generations, at least 23 generations, at least 24 generations, at least 25 generations, at least

26 generations, at least 27 generations, at least 28 generations, at least 29 generations, at least

30 generations, at least 31 generations, at least 32 generations, at least 33 generations, at least

34 generations, at least 35 generations, at least 36 generations, at least 37 generations, at least

38 generations, at least 39 generations, at least 40 generations, at least 41 generations, at least

42 generations, at least 43 generations, at least 44 generations, at least 45 generations, at least

46 generations, at least 47 generations, at least 48 generations, at least 49 generations, at least

50 generations, at least 51 generations, at least 52 generations, at least 53 generations, at least

54 generations, at least 55 generations, at least 56 generations, at least 57 generations, at least

58 generations, at least 59 generations, at least 60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least 64 generations, at least 65 generations, at least

66 generations, at least 67 generations, at least 68 generations, at least 69 generations, at least

70 generations, at least 71 generations, at least 72 generations, at least 73 generations, at least

74 generations, at least 75 generations, at least 76 generations, at least 77 generations, at least

78 generations, at least 79 generations, at least 80 generations, at least 81 generations, at least

82 generations, at least 83 generations, at least 84 generations, at least 85 generations, at least

86 generations, at least 87 generations, at least 88 generations, at least 89 generations, at least

90 generations, at least 91 generations, at least 92 generations, at least 93 generations, at least

94 generations, at least 95 generations, at least 96 generations, at least 97 generations, at least

98 generations, at least 99 generations, or at least 100 generations.

[00793] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can prevent copy loss of the heterologous polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide, wherein growing the recombinant yeast cells in sorbitol prevents copy loss of least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least

60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least

67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least

74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least

81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least 14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least 18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least 22 generations, at least 23 generations, at least 24 generations, at least 25 generations, at least 26 generations, at least 27 generations, at least 28 generations, at least 29 generations, at least 30 generations, at least 31 generations, at least 32 generations, at least 33 generations, at least 34 generations, at least 35 generations, at least 36 generations, at least 37 generations, at least 38 generations, at least 39 generations, at least 40 generations, at least 41 generations, at least 42 generations, at least 43 generations, at least 44 generations, at least 45 generations, at least 46 generations, at least 47 generations, at least 48 generations, at least 49 generations, at least 50 generations, at least 51 generations, at least 52 generations, at least 53 generations, at least 54 generations, at least 55 generations, at least 56 generations, at least 57 generations, at least 58 generations, at least 59 generations, at least 60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least 64 generations, at least 65 generations, at least 66 generations, at least 67 generations, at least 68 generations, at least 69 generations, at least 70 generations, at least 71 generations, at least 72 generations, at least 73 generations, at least 74 generations, at least 75 generations, at least 76 generations, at least 77 generations, at least 78 generations, at least 79 generations, at least 80 generations, at least 81 generations, at least 82 generations, at least 83 generations, at least 84 generations, at least 85 generations, at least 86 generations, at least 87 generations, at least 88 generations, at least 89 generations, at least 90 generations, at least 91 generations, at least 92 generations, at least 93 generations, at least 94 generations, at least 95 generations, at least 96 generations, at least 97 generations, at least 98 generations, at least 99 generations, or at least

100 generations.

[00794] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can prevent copy loss of the heterologous nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH), wherein growing the recombinant yeast cells in sorbitol prevents copy loss of least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least

63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least

70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least

77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least 14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least 18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least 22 generations, at least 23 generations, at least 24 generations, at least 25 generations, at least 26 generations, at least 27 generations, at least 28 generations, at least 29 generations, at least 30 generations, at least 31 generations, at least 32 generations, at least 33 generations, at least 34 generations, at least 35 generations, at least 36 generations, at least 37 generations, at least 38 generations, at least 39 generations, at least 40 generations, at least 41 generations, at least 42 generations, at least 43 generations, at least 44 generations, at least 45 generations, at least 46 generations, at least 47 generations, at least 48 generations, at least 49 generations, at least 50 generations, at least 51 generations, at least 52 generations, at least 53 generations, at least 54 generations, at least 55 generations, at least 56 generations, at least 57 generations, at least 58 generations, at least 59 generations, at least 60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least 64 generations, at least 65 generations, at least 66 generations, at least 67 generations, at least 68 generations, at least 69 generations, at least 70 generations, at least 71 generations, at least 72 generations, at least 73 generations, at least 74 generations, at least 75 generations, at least 76 generations, at least 77 generations, at least 78 generations, at least 79 generations, at least 80 generations, at least 81 generations, at least 82 generations, at least 83 generations, at least 84 generations, at least 85 generations, at least 86 generations, at least 87 generations, at least 88 generations, at least 89 generations, at least 90 generations, at least 91 generations, at least 92 generations, at least 93 generations, at least 94 generations, at least 95 generations, at least 96 generations, at least 97 generations, at least 98 generations, at least 99 generations, or at least 100 generations.

[00795] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can prevent copy loss of the nucleotide sequence operable to encode a heterologous polypeptide, wherein growing the recombinant yeast cells in sorbitol prevents copy loss of least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least

59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least

66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least

73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least

80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least

87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least

94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, of the initial copy number, after least 1 generation, at least 2 generations, at least 3 generations, at least 4 generations, at least 5 generations, at least 6 generations, at least 7 generations, at least 8 generations, at least 9 generations, at least 10 generations, at least 11 generations, at least 12 generations, at least 13 generations, at least 14 generations, at least 15 generations, at least 16 generations, at least 17 generations, at least 18 generations, at least 19 generations, at least 20 generations, at least 21 generations, at least 22 generations, at least 23 generations, at least

24 generations, at least 25 generations, at least 26 generations, at least 27 generations, at least

28 generations, at least 29 generations, at least 30 generations, at least 31 generations, at least

32 generations, at least 33 generations, at least 34 generations, at least 35 generations, at least

36 generations, at least 37 generations, at least 38 generations, at least 39 generations, at least

40 generations, at least 41 generations, at least 42 generations, at least 43 generations, at least

44 generations, at least 45 generations, at least 46 generations, at least 47 generations, at least

48 generations, at least 49 generations, at least 50 generations, at least 51 generations, at least

52 generations, at least 53 generations, at least 54 generations, at least 55 generations, at least

56 generations, at least 57 generations, at least 58 generations, at least 59 generations, at least

60 generations, at least 61 generations, at least 62 generations, at least 63 generations, at least

64 generations, at least 65 generations, at least 66 generations, at least 67 generations, at least

68 generations, at least 69 generations, at least 70 generations, at least 71 generations, at least

72 generations, at least 73 generations, at least 74 generations, at least 75 generations, at least

76 generations, at least 77 generations, at least 78 generations, at least 79 generations, at least

80 generations, at least 81 generations, at least 82 generations, at least 83 generations, at least

84 generations, at least 85 generations, at least 86 generations, at least 87 generations, at least

88 generations, at least 89 generations, at least 90 generations, at least 91 generations, at least

92 generations, at least 93 generations, at least 94 generations, at least 95 generations, at least

96 generations, at least 97 generations, at least 98 generations, at least 99 generations, or at least 100 generations.

[00796] In some embodiments, the recombinant yeast cells of the present disclosure, when grown in media comprising a sole carbon source that is sorbitol, can advantageously maintain copy numbers and/or prevent copy loss of at least 90% of copies of the heterologous polynucleotide after at least 10, 15, 20, 25, or 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of copies of the heterologous polynucleotide after at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 50% of copies of the heterologous polynucleotide after at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, or 65 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00797] In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol. In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00798] EXEMPLARY EMBODIMENTS

[00799] In some embodiments, the present disclosure provides a recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated.

[00800] In some embodiments, the recombinant yeast cell comprises from about 1 to about 15 copies of the heterologous polynucleotide.

[00801] In some embodiments, the recombinant yeast cell comprises from about 1 to about 5 copies of the heterologous polynucleotide.

[00802] In some embodiments, the heterologous polynucleotide has a ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, wherein said ratio is 1 : 1 to 1 : 10, or any ratio in between.

[00803] In some embodiments, the ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, is 1:2 or 1:3. [00804] In some embodiments, the heterologous polypeptide is a Cysteine Rich Peptide (CRP).

[00805] In some embodiments, the CRP is a Ul-agatoxin-Talb peptide; a Ul- agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX).

[00806] In some embodiments, the Ul-agatoxin-Talb peptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 195.

[00807] In some embodiments, the Ul-agatoxin-Talb peptide has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 195.

[00808] In some embodiments, the TVP has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218- 222, 225-234, 236-245, or 247-248.

[00809] In some embodiments, the TVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236- 245, or 247-248.

[00810] In some embodiments, the sea anemone toxin is an Av2 toxin, or an Av3 toxin.

[00811] In some embodiments, the Av2 toxin has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 457.

[00812] In some embodiments, the Av2 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 457.

[00813] In some embodiments, the Av3 toxin has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 453.

[00814] In some embodiments, the Av3 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 453.

[00815] In some embodiments, the AVP has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

[00816] In some embodiments, the AVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

[00817] In some embodiments, the CRP is a ctenitoxin (CNTX).

[00818] In some embodiments, the CNTX is T-CNTX-Pnla.

[00819] In some embodiments, the T-CNTX-Pnla has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 193. [00820] In some embodiments, the T-CNTX-Pnla has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 193.

[00821] In some embodiments, the CRP is an ACTX.

[00822] In some embodiments, the ACTX is a U-ACTX peptide, Omega- ACTX peptides, or Kappa- ACTX peptide.

[00823] In some embodiments, the ACTX is a U-ACTX-Hvla, a U+2-ACTX-Hvla, a rU-ACTX-Hvla, a rU-ACTX-Hvlb, aK-ACTX-Hvla, aK+2-ACTX-Hvla, a co-ACTX- Hvla, or a co+2-ACTX-Hvla.

[00824] In some embodiments, the ACTX has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00825] In some embodiments, the ACTX has an amino acid sequence according an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00826] In some embodiments, the recombinant yeast cell is a species selected from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces .

[00827] In some embodiments, the recombinant yeast cell is a species selected from the group consisting of: Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

[00828] In some embodiments, the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence.

[00829] In some embodiments, the recombinant yeast cell is Kluyveromyces lactis, Kluyveromyces marxianus, or Saccharomyces cerevisiae.

[00830] In some embodiments, the endogenous SDH nucleotide sequence is at least partially inactivated via in vivo homologous recombination.

[00831] In some embodiments, the recombinant yeast cell is selected from a yeast species that does not have an endogenous SDH nucleotide sequence.

[00832] In some embodiments, the recombinant yeast cell is Pichia pastoris.

[00833] In some embodiments, the recombinant yeast cell comprises 2 or more copies of the heterologous polynucleotide.

[00834] In some embodiments, the recombinant yeast cell comprises 10 or more copies of the heterologous polynucleotide.

[00835] In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00836] In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00837] In some embodiments, growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00838] In some embodiments, the present disclosure provides a method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising (a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; (b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and (c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00839] In some embodiments, the vector comprises from about 1 to about 15 copies of the heterologous polynucleotide.

[00840] In some embodiments, the vector comprises from about 1 to about 5 copies of the heterologous polynucleotide.

[00841] In some embodiments, the heterologous polynucleotide has a ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, wherein said ratio 1 : 1 to 1 : 10, or any ratio in between.

[00842] In some embodiments, the ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide is 1:2 or 1:3.

[00843] In some embodiments, the heterologous polypeptide is a Cysteine Rich Peptide (CRP).

[00844] In some embodiments, the CRP is a Ul-agatoxin-Talb peptide; a Ul- agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX).

[00845] In some embodiments, the Ul-agatoxin-Talb peptide has an amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 195.

[00846] In some embodiments, the Ul-agatoxin-Talb peptide has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 195.

[00847] In some embodiments, the TVP has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218- 222, 225-234, 236-245, or 247-248.

[00848] In some embodiments, the TVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236- 245, or 247-248.

[00849] In some embodiments, the sea anemone toxin is an Av2 toxin, or an Av3 toxin.

[00850] In some embodiments, the Av2 toxin has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 457.

[00851] In some embodiments, the Av2 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 457.

[00852] In some embodiments, the Av3 toxin has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 453.

[00853] In some embodiments, the Av3 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 453.

[00854] In some embodiments, the AVP has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 454-456. [00855] In some embodiments, the AVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

[00856] In some embodiments, the CRP is a ctenitoxin (CNTX).

[00857] In some embodiments, the CNTX is T-CNTX-Pnla.

[00858] In some embodiments, the T-CNTX-Pnla has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in SEQ ID NO: 193.

[00859] In some embodiments, the T-CNTX-Pnla has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 193.

[00860] In some embodiments, the CRP is an ACTX.

[00861] In some embodiments, the ACTX is a U-ACTX peptide, Omega- ACTX peptides, or Kappa- ACTX peptide.

[00862] In some embodiments, the ACTX is a U-ACTX-Hvla, a U+2-ACTX-Hvla, a rU-ACTX-Hvla, a rU-ACTX-Hvlb, aK-ACTX-Hvla, aK+2-ACTX-Hvla, a co-ACTX- Hvla, or a co+2-ACTX-Hvla.

[00863] In some embodiments, the ACTX has an amino acid sequence that is at least 90% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00864] In some embodiments, the ACTX has an amino acid sequence according an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

[00865] In some embodiments, the yeast host cell is a species selected from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces .

[00866] In some embodiments, the yeast host cell is a species selected from the group consisting of: Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

[00867] In some embodiments, the yeast host cell is selected from a yeast species having an endogenous SDH nucleotide sequence.

[00868] In some embodiments, the yeast host cell is Kluyveromyces lactis, Kluyveromyces marxianus, or Saccharomyces cerevisiae.

[00869] In some embodiments, the endogenous SDH nucleotide sequence is at least partially inactivated via in vivo homologous recombination.

[00870] In some embodiments, the yeast host cell is selected from a yeast species that does not have an endogenous SDH nucleotide sequence.

[00871] In some embodiments, the yeast host cell is Pichia pastoris.

[00872] In some embodiments, the vector comprises 2 or more copies of the heterologous polynucleotide. [00873] In some embodiments, the vector comprises 10 or more copies of the heterologous polynucleotide.

[00874] In some embodiments, growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00875] In some embodiments, growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00876] In some embodiments, growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

[00877] In some embodiments, the level of expression of the heterologous polypeptide provides a yield of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L, at least 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least 5,500 mg/L, at least at least 6,000 mg/L, at least 6,500 mg/L, at least 7,000 mg/L, at least 7,500 mg/L, at least 8,000 mg/L, at least 8,500 mg/L, at least 9,000 mg/L, at least 9,500 mg/L, at least 10,000 mg/L, at least 11,000 mg/L, at least 12,000 mg/L, at least 12,500 mg/L, at least 13,000 mg/L, at least 14,000 mg/L, at least 15,000 mg/L, at least 16,000 mg/L, at least

17,000 mg/L, at least 17,500 mg/L, at least 18,000 mg/L, at least 19,000 mg/L, at least

20,000 mg/L, at least 25,000 mg/L, at least 30,000 mg/L, at least 40,000 mg/L, at least

50,000 mg/L, at least 60,000 mg/L, at least 70,000 mg/L, at least 80,000 mg/L, at least

90,000 mg/L, or at least 100,000 mg/L of heterologous polypeptide per liter of yeast culture medium. [00878] In some embodiments, the present disclosure provides a vector comprising: (a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising: (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) a nucleotide sequence operable to encode a heterologous polypeptide; (b) a 5’- homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3’- homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide.

[00879] In some embodiments, the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 to 1:10, or any ratio in between.

[00880] In some embodiments, the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3.

[00881] In some embodiments, said vector comprises a nucleic acid sequence as set forth in SEQ ID NO: 458, or a complementary nucleotide sequence thereof.

[00882] In some embodiments, the present disclosure provides a polynucleotide comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof.

[00883] In some embodiments, the at least one nucleotide sequence operable to encode a SDH has a nucleotide sequence that is at least 90% identical to the nucleotide sequence set forth in SEQ ID NO: 1.

[00884] In some embodiments, the at least one nucleotide sequence operable to encode a SDH has a nucleotide sequence according to the nucleotide sequence set forth in SEQ ID NO: 1 and the at least one nucleotide sequence operable to encode a heterologous polypeptide has a nucleotide sequence that is at least 90% identical to the nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 186-191, 193, 195-216, 218- 222, 225-234, 236-245, 247-248, or 453-457.

[00885] In some embodiments, the at least one nucleotide sequence operable to encode a SDH has a nucleotide sequence according to the nucleotide sequence set forth in SEQ ID NO: 1 operably linked to the at least one nucleotide sequence operable to encode a heterologous polypeptide having a nucleotide sequence that is the nucleotide sequence encoding the amino acid sequence set forth in any one of SEQ ID NO: 186-191, 193, 195- 216, 218-222, 225-234, 236-245, 247-248, or 453-457.

[00886] In some embodiments, the present disclosure provides a method of increasing expression of a Cysteine Rich Peptide (CRP) in a recombinant Kluyveromyces lactis cell, said method comprising: (a) inactivating or at least partially inactivating an endogenous sorbitol dehydrogenase (SDH) nucleotide sequence; (b) providing a vector comprising one or more copies of a heterologous polynucleotide, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) a nucleotide sequence operable to encode a heterologous SDH; and (ii) a nucleotide sequence operable to encode a CRP; (c) creating a recombinant Kluyveromyces lactis cell by transforming the vector into a Kluyveromyces lactis host cell; and (d) growing the recombinant Kluyveromyces lactis cell in a medium comprising a sole carbon source that is sorbitol; wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3; wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); aPhoneutria toxin; or an Atracotoxin (ACTX); and wherein growing the recombinant Kluyveromyces lactis cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the CRP, relative to a level of expression of the CRP when growing the recombinant Kluyveromyces lactis cell in a medium comprising a sole carbon source that is not sorbitol.

[00887] In some embodiments, the present disclosure provides a method of increasing expression of a Cysteine Rich Peptide (CRP) in a recombinant Pichia pastoris cell, said method comprising: (a) providing a vector comprising one or more copies of a heterologous polynucleotide, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation: (i) a nucleotide sequence operable to encode a heterologous SDH; and (ii) a nucleotide sequence operable to encode a CRP; (b) creating a recombinant Pichia pastoris cell by transforming the vector into a Pichia pastoris host cell; and (c) growing the recombinant Pichia pastoris cell in a medium comprising a sole carbon source that is sorbitol; wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3; wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX); and wherein growing the recombinant Pichia pastoris cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the CRP, relative to a level of expression of the CRP when growing the recombinant Pichia pastoris cell in a medium comprising a sole carbon source that is not sorbitol.

[00888] In some embodiments, the present disclosure provides a recombinant Kluyveromyces lactis cell comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) two or more nucleotide sequences operable to encode a Cysteine Rich Peptide (CRP); wherein the ratio of (i) to (ii) is at least 1:2; wherein the recombinant Kluyveromyces lactis has an endogenous SDH nucleotide sequence that has been inactivated or at least partially inactivated; and wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a Phoneutria toxin; or an Atracotoxin (ACTX). [00889] In some embodiments, the present disclosure provides a recombinant Pichia pastoris cell comprising: (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) two or more nucleotide sequences operable to encode a Cysteine Rich Peptide (CRP); wherein the ratio of (i) to (ii) is at least 1:2; and wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a. Phoneutria toxin; or an Atracotoxin (ACTX).

EXAMPLES

[00890] The Examples in this specification are not intended to, and should not be used to, limit the invention; they are provided only to illustrate the invention.

[00891] Example 1: SDH Introduction into Pichia pastoris

[00892] Pichia pastoris does not have an endogenous sorbitol dehydrogenase gene and exhibits only weak latent utilization of sorbitol. This example shows that incorporation of K. lactis sorbitol dehydrogenase gene facilitates the utilization of sorbitol as a sole carbon source by Pichia.

[00893] A yeast host cell, i.e., a BG10 (commercially available from Atumbio; Catalog No. AKA PPS-9010) was obtained. The BG10 yeast strain is a wild type P. pastoris strain with no known sorbitol dehydrogenase. Next, sorbitol dehydrogenase derived from a non-U pastoris species was transformed into the BG10 strain.

[00894] First, a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase was obtained. Here, the nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase was derived from the K. lactis sorbitol dehydrogenase (also known as “Lid” or “lid,” which is an abbreviation for L-iditol 2-dehydrogenase) having a nucleotide sequence of SEQ ID NO: 1. The nucleotide sequence operable to encode a heterologous SDH or “lid” was then inserted into a vector, i.e., the plasmid “pJUSor.” FIG.

1. The pJUSor plasmid is designed to integrate the nucleotide sequence operable to encode a heterologous SDH derived from T. lactis derived (SEQ ID NO: 1), at the pUPP locus in the Pichia pastoris wild type strain BG10. Expression of the sorbitol dehydrogenase gene was driven by the A. gossyppi TEF promoter.

[00895] Next, to transform the yeast host cell BG10, the pJUSor plasmid was linearized with a Pfol restriction enzyme digestion, and transformed into electrocompetent BG10 cells via electroporation.

[00896] Transformed yeast colonies that showed growth on defined media sorbitol (DMSor) selection plates, with no other carbon sources present (Media Recipe given in Table 4), were selected for further analysis. Integration of the K. lactis sorbitol dehydrogenase was subsequently confirmed by qPCR comparing the amplification of actin, of which Pichia pastoris is known to have one copy of, to the amplification of the nucleotide sequence operable to encode the sorbitol dehydrogenase (Table 5). The qPCR primer sequences were: [00897] Actin qPCR primers:

GGGTCAAAAGGACTCCTTCGTCGG (Forward) (SEQ ID NO: 459)/ GCCAGACGCAACTCGTTGTAGAAGG (Reverse) (SEQ ID NO: 460) [00898] Lid qPCR primers:

TAAACGACAGCAGGCACCAAAGG (Forward) (SEQ ID NO: 461) / AGCCTCTTGCTGTTGGTGTTCAGC (Reverse) (SEQ ID NO: 462) [00899] Table 4: Sorbitol selection media plate recipe

[00900] Table 5: qPCR analysis for successful knock-in of SDH in Pichia pastoris

CT: Cycle threshold.

[00901] The yeast host cell, BG10, containing no native SDH, was compared with the transformed strain, JUSor2, using actin as the endogenous reference gene. Here, when target (i.e. , SDH) CT values are close to the CT values of endogenous reference gene (actin), it suggests a similar number of target copies and reference copies (BG10 is known to have one copy of actin). CT levels are shown in Table 5.

[00902] The ability for the JUSor2 strain to utilize sorbitol was determined by assessing growth in DMSor selection media. The DMSor media used is identical to the sorbitol selection media plate recipe of Table 3 (albeit without agar). Growth rate was measured by assessing culture media OD600, haphazardly, throughout about 88 hours of growth at 27°C in 14 mL culture tubes.

[00903] Transforming wild type P. pastoris (BG10) with a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase derived from K. lactis results in a significant increase of growth in defined media comprising sorbitol as the sole carbon source (4% sorbitol defined media). FIG. 2.

[00904] As shown in FIG. 2, there is a clear signal that the transformed JUSor2, containing the K. lactis sorbitol dehydrogenase gene, significantly improved growth in media where sorbitol was the sole carbon source.

[00905] Example 2: Knock-out and knock-in of SDH in K. Lactis

[00906] The ability to (1) inactivate or partially inactivate endogenous SDH in K. lactis (knock-out); and (2) transform K. lactis with a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase; were evaluated. In addition, the ability of sorbitol dehydrogenase as a selection marker to recover successful transformation events of K. lactis cells transformed with the heterologous polynucleotide of the present disclosure, was also evaluated.

[00907] First, a sorbitol dehydrogenase deficient K. lactis strain was created using the pop-in/ pop-out plasmid, pKlDlid (SEQ ID NO: 463). FIG. 3. The pKlDlid plasmid comprises a 5’- and 3 ’-homology arm corresponding to the endogenous SDH gene locus. Additionally, the pKIDlid plasmid comprises a repeated 3 ’-homology arm corresponding to the endogenous SDH gene locus, which is repeated at the 5’ end of the plasmid. FIG. 3. This repeated 3’- homology arm provides the opportunity for the integrated amdS transgene to out recombine at a frequency that allows recovery a “pop-out” colony using a counter selection plate. Thus, while a typical integration vector has one 3 ’-homology arm, and one 5 ’-homology arm for accurate insertion, pKIDlid has an additional 3 ’-homology arm just upstream of the 5’- homology arm, and it is this repeated homology within the insertion vector that allows for the out recombination. Consequently, a nucleotide sequence operable to encode a heterologous amdS transgene is integrated at the endogenous SDH gene loci in the K. lactis chromosome — thereby inactivating the endogenous SDH gene. And, an acetamidase selection marker gene (amdS) is inserted in the locus of the endogenous SDH gene, thereby allowing the transformants to utilize acetamide as a nitrogen source.

[00908] Next the pKIDlid plasmid was transformed into wild type K. lactis cells. Yeast with a successful inactivation of the endogenous sorbitol dehydrogenase gene were recovered by plating the transformants on Yeast Carbon Base media with 5 mM acetamide (New England Biolabs; Catalog No. NEB- B9017S).

[00909] As mentioned above, because the heterologous DNA fragment from the vector that is integrated into the yeast host cell chromosome contains homology at the 3’ integration portion and just before the 5’ integration portion, the heterologous amdS transgene is frequently out-recombined; the end result of this out-recombination is that the transformants have an inactivated or partially inactivated endogenous sorbitol dehydrogenase sequence, and lack an amdS selection marker. FIG. 3.

[00910] After ~3 days of growth in non-selective media, colonies derived from cells that out-recombined the amdS selection marker were recovered by growing the cells in the presence of Fluoro-acetamide. Only cells that successfully out-recombined the amdS selection marker were able to grow to form colonies in the presence of Fluoro-acetamide, which is metabolized into toxic byproducts by a functional amdS gene.

[00911] Inactivation or partial inactivation of the endogenous SDH nucleotide sequence, was demonstrated by comparing transformants to a wild-type K. lactis strain (YCT-306), when grown on defined media with sorbitol as the only carbon source. FIG. 4. [00912] Briefly, transformants il - i4 (which have partial inactivation of the endogenous SDH nucleotide sequence) and a wild-type K. lactis cell having one copy of endogenous SDH nucleotide sequence (YCT-306), were grown on defined media comprising 4% sorbitol without com steep liquor (CSL), wherein sorbitol is the sole carbon source. An exemplary description of the YCT-306 strain is provided in PCT Patent Publication No. WO2013134734A2, the disclosure of which is incorporated herein by reference in its entirety.

[00913] As shown in FIG. 4, transformants il - i4 (which have partial inactivation of the endogenous SDH nucleotide sequence) were compared to the wild-type K. lactis cell having one copy of endogenous SDH nucleotide sequence (YCT-306). Here, significant growth differences were observed between transformants il - i4 (which have partial inactivation of the endogenous SDH nucleotide sequence) and YCT-306, after 4 days.

[00914] Successful transformation of wild-type K. lactis with the pKlDlid vector, and subsequent inactivation of the endogenous SDH nucleotide sequence and out-recombination of amdS, was confirmed as shown in FIG. 5. As shown in FIG. 5, strain VSTLB1 Oil shows no amplification of the endogenous SDH nucleotide sequence, and about 1 copy of the amdS transgene. FIG. 5. The qPCR primers used to detect and quantify the endogenous SDH nucleotide sequence and amdS transgene, are given below in Table 6.

[00915] Table 6. qPCR primers.

[00916] Successful out-recombination of the amdS transgene is shown in strain VSTLB10, as confirmed by a lack of amplification of both the endogenous SDH nucleotide sequence, and a lack of amplification of the amdS transgene. FIG. 5. Consequently, the VSTLB10 strain served as a strain deficient in endogenous SDH nucleotide sequences for future experiments.

[00917] Example 3. Selection of recombinant cells using SDH

[00918] To determine if a nucleotide sequence operable to encode a heterologous SDH could be used to select recombinant cells comprising a nucleotide sequence operable to encode a heterologous polypeptide, the VSTLB10 strain (described in Example 2), a strain having a partially inactivated endogenous SDH nucleotide sequence (and an out-recombined amdS transgene) was transformed with a pLB10V5DS plasmid. FIG. 6.

[00919] The pLB10V5DS plasmid (SEQ ID NO: 458) comprises a heterologous polynucleotide of the present disclosure, i.e., a heterologous polynucleotide comprising: (i) one or more nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequence operable to encode a heterologous polypeptide. FIG. 6.

[00920] As shown in FIG. 6, the pLB10V5DS plasmid comprises, inter alia, (i) a heterologous nucleotide sequence operable to encode a heterologous SDH (as indicated by the segment labeled “lid (SorDH)” (SEQ ID NO: 1); and (ii) two nucleotide sequences operable to encode a heterologous polypeptide. Here, two nucleotide sequence operable to encode a heterologous polypeptide encode the ACTX peptide, “Hybrid+2-ACTX-Hvla,” having an amino acid sequence set forth in SEQ ID NO. 187.

[00921] The pLB10V5DS plasmid comprises 5’- and 3’-homology arms designed to allow integration of the heterologous polynucleotide comprising: (i) one or more nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequence operable to encode a heterologous polypeptide into the endogenous LAC4 promoter (pLAC4) locus. Accordingly, when the pLB10V5DS plasmid is transformed into a yeast host cell, in vivo homologous-recombination-mediated integration of the heterologous polynucleotide occurs at the endogenous yeast host cell pLAC4 locus.

Expression of the (ii) two nucleotide sequences operable to encode Hybrid+2-ACTX-Hvla, was driven by an intact LAC4 promoter. FIG. 6.

[00922] To create a recombinant yeast cell, the pLB10V5DS plasmids were linearized via SacII digestion, and transformed into the yeast host cell, i.e., VSTLB10 cells, using electroporation. Briefly, about 10-200 mL of yeast extract peptone dextrose (YEPD) and VSTLB10 cells were incubated on a shaker at 30°C until the early exponential phase of yeast culture (e.g. about 0.6 to 2 x 10⁸ cells/mL). Next, the yeast were harvested in a sterile centrifuge tube and centrifuged at 3000 rpm for 5 minutes at 4°C (while keeping cells chilled during the procedure), followed by washing cells with 40 mL of ice cold, sterile deionized water, and pelleting the cells a 23,000 rpm for 5 minutes. The wash step was repeated, and the cells were resuspended in 100 mL of 40% sorbitol, followed by spinning down at 3,000 rpm for 5 minutes; the cells were then resuspended with a proper volume of ice cold 40% sorbitol, to final cell density of 3xl0⁹ cell/mL (1.5xl0⁹ cell/mL to 6xl0⁹ cell/mL are acceptable cell densities). Next, 40 pL of the yeast suspension was mixed with about 1-4 pL (at a concentration of 100-300 ng/pL) of the vector comprising a linear heterologous polynucleotide comprising (i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequence operable to encode a heterologous polypeptide (e.g., a CRP) (~1 pg) in a prechilled 0.2 cm electroporation cuvette. A single pulse at 2000 V was then provided, for optimal time constant of 5 ms of the RC circuit, and the cells were allowed to recover in 0.5 ml YED and 0.5 mL 40% sorbitol, followed by spreading onto selective plates.

[00923] Transformed recombinant yeast cells were selected from colonies that grew on defined media comprising 4% sorbitol selection media plates (Media of Table 3). Copy number of the heterologous polynucleotide that integrated into the yeast host cell was estimated based on qPCR amplification of the nucleotide sequence operable to encode a heterologous SDH, using ura3 as the reference gene (primers targeting SDH were those primers described in Example 1; ura3 primers are shown below).

[00924] Seven recombinant yeast cells were identified. FIGs. 7-8. Successful transformation was confirmed, and estimated copy numbers of the heterologous polynucleotide were measured, via qPCR, based on the presence of an endogenous SDH nucleotide sequence (in the wild-type strain), and the presence of a nucleotide sequence operable to encode a heterologous SDH. As shown in FIG. 8, the agar plate comprising 4% sorbitol as the sole carbon source, has been inoculated with the recombinant yeast cells; and these recombinant yeast cells are able to grow on the plate. Here, larger colonies of the recombinant yeast cells indicate a significant improvement of sorbitol utilization, suggesting successful integration of the nucleotide sequence operable to encode a heterologous SDH into VSTLB10 (SDH deficient strain).

[00925] The seven recombinant yeast cell strains were shown to have at least one copy of the heterologous polynucleotide, based on the presence of a nucleotide sequence operable to encode a heterologous SDH. FIG. 7. As shown in FIG. 7, the YCT306 strain, a wild-type K. lactis strain, has a single copy of a nucleotide sequence operable to encode a SDH (i.e., the endogenous SDH gene). The yeast host cell, VSTLB10 (background strain), shows no copies of a nucleotide sequence operable to encode a heterologous SDH. The recombinant yeast cell named lid-Hl possessed an estimated 2.5 copies of the nucleotide sequence operable to encode a heterologous SDH. The recombinant yeast cell named lid-H2 likewise possessed an estimated 2.5 copies of the nucleotide sequence operable to encode a heterologous SDH. The recombinant yeast cells named lid-H3, lid-Ml, lid-M2, and lid-Ll possessed about 1 copy of the nucleotide sequence operable to encode a heterologous SDH. Finally, the recombinant yeast cell named lid-L2 possessed an estimated 2 copies of the nucleotide sequence operable to encode a heterologous SDH. FIG. 7.

[00926] Expression of the heterologous polypeptide, i.e., Hybrid+2-ACTX-Hvla, HPLC was employed, using caffeine as an internal standard. Briefly, 44 additional recombinant yeast cell colonies that grew on the defined media with 4% sorbitol selection plates were grown in deep-well plates in 2.2 mL of defined media with 4% sorbitol + 0.2% com steep liquor (CSL). After 6 days of growth at 23.5°C in shaker incubators, 250 pL of supernatant was combined with 50 pL of 0.1 mg/mL caffeine + 0.3% Trifluoroacetic acid (TFA). All 44 cells showed a 6300 peak 1 at 3.787 mins via HPLC, and showed expression of the heterologous polypeptide, i.e., Hybrid+2-ACTX-Hvla. FIG. 9. This result suggests that the nucleotide sequence operable to encode a heterologous SDH can be used to identify and select colonies with successful integration of the heterologous polynucleotide and for the expression of a heterologous polypeptide. FIG. 9.

[00927] Example 4: Increasing expression and copy number maintenance

[00928] The recombinant yeast cells of the present disclosure were evaluated to determine their ability to maintain copy numbers of heterologous polynucleotides when grown in sorbitol.

[00929] Recombinant yeast cells were created according to the methods described in Examples 2 and 3 above. Briefly the VSTLB10 strain described above, i.e., aK lactis strain having an inactivated or partially inactivated endogenous SDH nucleotide sequence, was transformed with the pLB10V5DS plasmid, which comprises a heterologous polynucleotide comprising: (i) one or more nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequence operable to encode a heterologous polypeptide. FIG. 6.

[00930] As shown in FIG. 6, and described above, the pLB10V5DS comprises a heterologous polynucleotide comprising: (i) a heterologous nucleotide sequence operable to encode a heterologous SDH (SEQ ID NO: 1); and (ii) two nucleotide sequences operable to encode the CRP, Hybrid+2-ACTX-Hvla (having an amino acid sequence set forth in SEQ ID NO. 187); thus, the heterologous polynucleotide has a ratio of SDH:CRP of 1:2.

[00931] A recombinant yeast cell comprising 5 copies of the heterologous polynucleotide (i.e., a ratio of SDH: CRP that is 5:10) was created and subsequently used to evaluate copy number maintenance, when grown in a medium comprising a sole carbon source that is sorbitol.

[00932] Two replicates of the recombinant yeast cell, named “lidlOa” and “lidlOb,” were created, and then cultured in media containing either glucose or sorbitol as the sole carbon source.

[00933] For the recombinant yeast cells grown in sorbitol, the cells were grown in defined medium (DM) containing 4% sorbitol, with no com steep liquor (CSL) (same ingredients as those of Table 4, but without agar). For the recombinant yeast cells grown in glucose, the cells were grown in DM containing 4% glucose, with no CSL (same recipe as above, but with 4% glucose instead of sorbitol, and likewise without agar). Here, CSL was not included in the media to ensure that sorbitol and glucose were the only carbon sources available. The recombinant yeast cells were then grown for about 48 hours in 20 mL of their respective media, in 150 mL shake flasks, at 27°C in shaker incubators. After 48 hours in culture, samples were collected for assessment.

[00934] To assess copy number, qPCR was performed using ura3 as control gene, and pLAC4 to assess copy number of the nucleotide sequence operable to encode a heterologous SDH (AACT calculated from YCT306 unmodified base strain).

[00935] The pLB10V5DS vector (FIG. 6) has a 5’-homology arm and a 3’-homology arm corresponding to loci in the LAC4 promoter (pLAC4). Thus, the pLB10V5DS vector integrates the heterologous polynucleotide into the endogenous pLAC4 loci. Here, heterologous polynucleotide comprises an expression cassette that includes an intact heterologous pLAC4, which drives expression of the two copies of the Hybrid+2-ACTX- Hvla in the heterologous polynucleotide; copy numbers of the pLAC4 region (using ura3 as endogenous control) are therefore equal to the number of expression cassettes (each comprising 2 copies of Hybrid+2-ACTX-Hvla) present minus 1 copy for the native pLAC4 gene present in the VSTLB10 background.

[00936] Generations of the recombinant yeast cells were estimated using OD600 measurements on spectrophotometer upon inoculation, and after 48 hours. Following the 48- hour period, and after obtaining OD600 measurements, each culture was flipped and reinoculated. The cultures were inoculated with cells from the previous 48-hour culture at an OD = 0.1. Accordingly, enough cells from the previous culture were inoculated into the fresh culture media to achieve an OD = 0.1, and were used to continue the growth for more generations beyond the carrying capacity of the initial 20 mL of medium. This process was repeated for 6 cycles, and OD600 measurements were used to estimate the number of generations for each group based on their respective media types.

[00937] Copy number estimates of recombinant yeast cells grown in sorbitol as the sole carbon source, vs. glucose as the sole carbon source, were compared to determine if the nucleotide sequence operable to encode a heterologous SDH prevented copy loss of the heterologous polynucleotide, or provided any advantage to the maintenance of copy number. FIG. 10. The lidlO strain, which contains 5 copies of sorbitol dehydrogenase, maintained a higher average copy number throughout 65 generations of growth, when grown in media where sorbitol is the sole carbon source, compared to when grown in media where glucose was the sole carbon source.

[00938] Example 5: Patterns of higher copy number maintenance

[00939] Multi-copy integrations of heterologous peptides exhibit copy number loss as a result of the repeated regions of homology in close proximity. Copy number is known to correlate positively with peptide expression and the loss of copies results in a decrease in yield. Furthermore, if a copy loss event is accompanied by a fitness benefit in growth, selection can promote the increase in frequency of the lower copy number cells in a mixed culture over time thus reducing yield further.

[00940] Four strains of recombinant yeast cells were generated according to the methods described above. Here, the recombinant yeast cells varied in the number of copies of the heterologous polynucleotide they possessed. The four recombinant yeast cell strains were assessed for growth over 130 hours by assessing OD600 haphazardly throughout the growth process.

[00941] Average instantaneous growth and maximum instantaneous growth over this time course was compared between strains to predict the ability of the nucleotide sequence operable to encode a heterologous SDH to maintain high copies of the heterologous polynucleotide in these strains grown in media comprising sorbitol as its sole carbon source. [00942] Recombinant yeast cells containing 0, 1, 3, and 5 copies of the heterologous polynucleotide were created as described above, and tested to estimate growth rate in media containing sorbitol as the sole carbon source. Copy number was estimated by qPCR using primers that amplified the nucleotide sequence operable to encode a heterologous SDH, and comparing the amplification to the URA3 reference gene. FIG. 11.

[00943] Recombinant yeast cells containing 0 (VSTLB10), 1, 3, and 5 copies of the heterologous polynucleotide were then grown in either DM 4% sorbitol with no CSL, or DM 4% glucose with no CSL. Growth in these cultures was assessed by measuring the OD600 throughout 130 hours of growth in shake flask containing 20 mL of each media type. All recombinant yeast cell strains were inoculated at a starting OD600 = 0.1. OD600 measurements that assessed multiple time points during lag, exponential, and stationary phases were used to fit a growth curve model using IGOR Pro Version 6.3.7.2. Instantaneous growth rates were estimated every 6 minutes from hour 2 to hour 100 of growth (at which time all strains had reached saturation) from the fitted model for each strain in each media type. Two metrics of competitive growth were assessed, maximum instantaneous growth and average instantaneous growth over the 98-hour growth period. [00944] FIG. 12 shows the growth rates of the recombinant yeast cells when grown in media comprising either sorbitol or glucose as the sole carbon source. FIG. 12A shows the instant growth rate of recombinant yeast cell strains comprising 0, 1, 3, or 5 copies of the heterologous polynucleotide in media with sorbitol as the sole carbon source. FIG. 12B shows the instant growth rate of recombinant yeast cell strains comprising 0, 1, 3, or 5 copies of the heterologous polynucleotide in media with glucose as the sole carbon source. FIG. 12C shows the maximum instantaneous growth rate of recombinant yeast cell strains comprising 0, 1, 3, or 5 copies of the heterologous polynucleotide in media with sorbitol as the sole carbon source (“SOR growth) or in media with glucose as the sole carbon source (“GLU growth) over 98 hours of growth. FIG. 12D shows the average instantaneous growth rate of recombinant yeast cell strains comprising 0, 1, 3, or 5 copies of the heterologous polynucleotide in media with sorbitol as the sole carbon source (“SOR growth) or in media with glucose as the sole carbon source (“GLU growth).

[00945] The instant example was used to predict the effect of copy number maintenance of a heterologous polynucleotide comprising: (i) one or more nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and (ii) one or more nucleotide sequence operable to encode a heterologous polypeptide, in recombinant yeast cells, cultured in media containing either sorbitol or glucose as the sole carbon source. [00946] If maximum instantaneous growth rate (FIG. 12C) predicts competitive fitness outcomes, the data here would suggest that the recombinant yeast cells comprising 3 copies of the nucleotide sequence operable to encode a heterologous SDH would outcompete all other cells when grown in glucose media, and cells comprising 1 copy of the nucleotide sequence operable to encode a heterologous SDH would outcompete other cells in sorbitol media. For example, as shown in FIG. 12C, the highest maximum instantaneous growth rate was in the cells comprising 3 copies of the nucleotide sequence operable to encode a heterologous SDH when grown in glucose, and the highest maximum instantaneous growth rate was in the cells comprising 1 copy of the nucleotide sequence operable to encode a heterologous SDH when grown in sorbitol. Thus, it is expected that the average copy number of a culture that has copy number variation would stabilize at about 3 copies when grown in glucose media, and would stabilize at about 1 copy when grown in sorbitol media. This metric would predict that sorbitol selection marker strains would maintain more copies in glucose than sorbitol.

[00947] Alternatively, if average instantaneous growth rate best predicts competitive fitness outcomes, the present example would suggest that there is little difference in average instantaneous growth rate between copy number variants in glucose. FIG. 12D. Selection in glucose media should not promote the increase in frequency of cells that have experienced copy number loss as growth in not improved in any strain with fewer sorbitol dehydrogenase genes. However, in sorbitol media, having 1-copy of the nucleotide sequence operable to encode a heterologous SDH provides a significant boost to growth, whereas having greater than 1 copy gives less of a growth benefit. FIG. 12D. Using average instantaneous growth rate as the metric, it is expected that selection would actively maintain at least 1 copy when cells are grown in sorbitol, but have little effect on the frequency of copy number variation when cells are grown in glucose. Thus, it is expected that selection may drive copy number down in sorbitol (to at least 1 copy, but not to 0 copies); while the frequency change in glucose would be largely driven by drift, where copy number seemingly has little effect on the growth of these transformants when grown in glucose.

[00948] These present examples demonstrate that a benefit to copy number maintenance is observed when multi-copy strains are grown in the presence of sorbitol as the sole carbon source; and, the unexpected result that this benefit (i. e. , copy number maintenance) when using a nucleotide sequence operable to encode a heterologous SDH as a selection marker, is not predicted by growth benefits from having more copies of sorbitol dehydrogenase.

[00949] It is widely known in the art that that having more copies of a heterologous polynucleotide translates into higher yield of the heterologous peptide. As such, the data presented here shows that pairing the nucleotide sequence operable to encode a heterologous SDH as the selection marker in fermentations where sorbitol is the sole carbon source, the culture should maintain a higher copy number throughout fermentation resulting in higher yield at the end of fermentation. Furthermore, this effect of higher copy number maintenance is not predicted by the growth rate changes of having more copies of sorbitol dehydrogenase making the observation of higher copy number maintenance an unexpected finding.

[00950] Example 6: Yield per copy number data for Av3 mutants

[00951] To evaluate the relationship between copy number and yield, two recombinant yeast cell strains were made: a first recombinant yeast cell strain named 103b, transformed with a heterologous polynucleotide comprising two nucleotide sequences operable to encode the sea anemone toxin variant protein, Av3103b. The protein Av3103b has the amino acid sequence: “KSCCPCYWGGCPWGQNCYPEGCSGPK” (SEQ ID NO: 484). The second recombinant yeast cell strain, named 165, was transformed with a heterologous polynucleotide comprising two nucleotide sequences operable to encode the sea anemone toxin variant protein, Av3165. The protein Av3165 has the amino acid sequence: “KSCCPCYWGGCPWGQNCYPEGCSGPKVG” (SEQ ID NO: 485).

[00952] The recombinant yeast cells described above were created by transforming host cells with their respective vectors (i. e. , comprising either the heterologous nucleotides operable to encode Av3103b, or Av3165). Briefly, two copies of either (1) a nucleotide sequence operable to encode the sea anemone toxin variant protein, Av3103b (SEQ ID NO: 484); or (2) a nucleotide sequence operable to encode the sea anemone toxin variant protein, Av3165 (SEQ ID NO: 485), were cloned into a pKLACl vector (New England Biolabs, Ipswich, MA, USA).

[00953] As shown in FIG. 13, the resulting vectors, named pLB103bBD and pLB103bM165BD, comprised a 5’-homology arm and a 3’-homology arm corresponding to loci in the LAC4 promoter (pLAC4) (allowing integration of the dual expression cassettes into the endogenous pLAC4 loci), and a heterologous polynucleotide comprising a dual expression cassette. Here, the dual expression cassettes, i.e., cassette #1 and cassette #2, comprised following: either an intact heterologous pLAC4 (cassette #1) or pLAC12 (cassette #2); a nucleotide sequence operable to encode a heterologous polypeptide (i.e., either Av3103b for the pLB103bBD vector, or Av3165 for the pLB103bM165BD vector); and an alpha-MF signal sequence. For each of the two copies of the expression cassette, there was a single copy of an amdS transgene (note: the relationship between copy number and yield is independent of the selection marker used in the vector). In addition, the vectors comprised the following elements: a Kex2 cleavage site; a multiple cloning site; a LAC4 terminator (cassette #1) or a LAC12 terminator (cassette #2); and ADH1 promoter; a [3-lactamase (bla) gene; and an origin of replication site. FIG. 13 shows the pLB103bM165BD vector, comprising the nucleotide sequence operable to encode the Av3165 heterologous polypeptide.

[00954] These vectors were transformed into the K. lactis strain, YCT306, (New England Biolabs, Ipswich, MA, USA), resulting in recombinant yeast cell strains possessing a varied number of copies of the heterologous polynucleotide. Individual recombinant yeast cells, each with a potentially different number of copies of a heterologous polynucleotide, were screened for expression of the heterologous polypeptide after being grown in DM comprising 4% sorbitol as the sole carbon source, with 0.2% CSL, for 6 days at 23.5°C. [00955] After 6 days, the supernatant of the deep well cultures were assessed for the level of expression of either Av3165 or Av3103b, via HPLC. The recombinant yeast cells then had gDNA extracted using an Invitrogen yeast gDNA extraction kit (ThermoFisher; Catalog No. 78870) and qPCR was used to estimate the number of copies of the nucleotide sequence operable to encode Av3165 or Av3103b that were integrated.

[00956] Here, qPCR primers for ura3 were used as an endogenous control, and copies of pLAC4 were used to estimate the number of copies of heterologous polynucleotide. pLAC4 primers amplify both the pLAC4 copy driving expression cassette #1 and the pLAC12 region driving expression of expression cassette #2. Number of copies of the nucleotide sequences operable to encode the sea anemone toxin variant proteins that had been integrated into the host cell genome, were determined according to the following formula:

Formula (IV)

[00957] Wherein RQ is relative quantification. Here, each transformant has one native pLAC4 sequence and one Av3 sequence for each additional pLAC4 sequence beyond the native sequence.

[00958] Results

[00959] The recombinant yeast cells 103b and 165 had R² values of 0.8502 and 0.8333, respectively, amounting to -84% of the variation in peptide expression. This variation in peptide expression is due to variation in copy number in the respective recombinant yeast cell strains — with higher strains having higher copy number expressing more peptide. FIG. 14.

[00960] The qPCR analysis demonstrated a strong positive relationship between copy number and yield. Thus, a recombinant yeast cell that experiences less copy number loss, or minimizes the effect of any copy number loss event (i.e. hindering the ability for that copy number loss to increase in frequency in the population driving down the average copy number of said culture) results in recombinant yeast cells with a higher yield of heterologous polypeptide expression, relative to those that do experience copy number loss, or fail to minimize the effect of any copy number loss event.

Claims

1. A recombinant yeast cell comprising a heterologous polynucleotide, wherein the heterologous polynucleotide comprises:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; wherein if the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence, then said endogenous SDH nucleotide sequence is at least partially inactivated.

2. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell comprises from about 1 to about 15 copies of the heterologous polynucleotide.

3. The recombinant yeast cell of claim 2, wherein the recombinant yeast cell comprises from about 1 to about 5 copies of the heterologous polynucleotide.

4. The recombinant yeast cell of any one of claims 1-3, wherein the heterologous polynucleotide has a ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, wherein said ratio is 1:1 to 1:10, or any ratio in between.

5. The recombinant yeast cell of claim 4, wherein the ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, is 1:2 or 1:3.

6. The recombinant yeast cell of claim 1, wherein the heterologous polypeptide is a Cysteine Rich Peptide (CRP).

7. The recombinant yeast cell of claim 6, wherein the CRP is a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); aPhoneutria toxin; or an Atracotoxin (ACTX).

233

8. The recombinant yeast cell of claim 7, wherein the CRP is a Ul-agatoxin-Talb peptide.

9. The recombinant yeast cell of claim 8, wherein the Ul-agatoxin-Talb peptide has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence as set forth in SEQ ID NO: 195.

10. The recombinant yeast cell of claim 9, wherein the Ul-agatoxin-Talb peptide has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 195.

11. The recombinant yeast cell of claim 7, wherein the CRP is a TVP.

12. The recombinant yeast cell of claim 11, wherein the TVP has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

13. The recombinant yeast cell of claim 12, wherein the TVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

14. The recombinant yeast cell of claim 7, wherein the CRP is a sea anemone toxin.

15. The recombinant yeast cell of claim 14, wherein the sea anemone toxin is an Av2 toxin, or an Av3 toxin.

16. The recombinant yeast cell of claim 15, wherein the Av2 toxin has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in SEQ ID NO: 457.

17. The recombinant yeast cell of claim 16, wherein the Av2 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 457.

18. The recombinant yeast cell of claim 15, wherein the Av3 toxin has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in SEQ ID NO: 453.

19. The recombinant yeast cell of claim 18, wherein the Av3 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 453.

20. The recombinant yeast cell of claim 7, wherein the CRP is an AVP.

21. The recombinant yeast cell of claim 20, wherein the AVP has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

22. The recombinant yeast cell of claim 21, wherein the AVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

23. The recombinant yeast cell of claim 7, wherein the CRP is a ctenitoxin (CNTX).

24. The recombinant yeast cell of claim 23, wherein the CNTX is a T-CNTX-Pnla.

25. The recombinant yeast cell of claim 24, wherein the T-CNTX-Pnla has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in SEQ ID NO: 193.

26. The recombinant yeast cell of claim 25, wherein the T-CNTX-Pnla has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 193.

27. The recombinant yeast cell of claim 7, wherein the CRP is an ACTX.

28. The recombinant yeast cell of claim 27, wherein the ACTX has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

29. The recombinant yeast cell of claim 28, wherein the ACTX has an amino acid sequence according an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

30. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell is a species selected from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces .

31. The recombinant yeast cell of claim 30, wherein the recombinant yeast cell is a species selected from the group consisting of: Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

32. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence.

33. The recombinant yeast cell of claim 32, wherein the recombinant yeast species having an endogenous SDH nucleotide sequence is Kluyveromyces lactis, Kluyveromyces marxianus, or Saccharomyces cerevisiae.

34. The recombinant yeast cell of any one of claims 32-33, wherein the endogenous SDH nucleotide sequence is at least partially inactivated via in vivo homologous recombination.

35. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell is selected from a yeast species that does not have an endogenous SDH nucleotide sequence.

36. The recombinant yeast cell of claim 35, wherein the yeast species that does not have an endogenous SDH nucleotide sequence is Pichia pastoris.

37. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell comprises 10 or more copies of the heterologous polynucleotide.

38. The recombinant yeast cell of claim 37, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

39. The recombinant yeast cell of claim 37, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

40. The recombinant yeast cell of claim 37, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

41. The recombinant yeast cell of claim 1, wherein the recombinant yeast cell comprises 2 or more copies of the heterologous polynucleotide.

42. The recombinant yeast cell of claim 41, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

43. The recombinant yeast cell of claim 41, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

44. The recombinant yeast cell of claim 41, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of the 2 or more copies of the heterologous polynucleotide after at least 50 generations,

237 relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

45. A method of increasing expression of a heterologous polypeptide in a recombinant yeast cell, said method comprising:

(a) providing a vector comprising one or more copies of a heterologous polynucleotide, or a complementary nucleotide sequence thereof, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) at least one nucleotide sequence operable to encode a heterologous polypeptide;

(b) creating the recombinant yeast cell by transforming the vector into a yeast host cell, wherein, if the yeast host cell has an endogenous SDH nucleotide sequence, then the endogenous SDH nucleotide sequence is at least partially inactivated; and

(c) growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol; wherein growing the recombinant yeast cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the heterologous polypeptide, relative to a level of expression of the heterologous polypeptide when growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

46. The method of claim 45, wherein the vector comprises from about 1 to about 15 copies of the heterologous polynucleotide.

47. The method of claim 46, wherein the vector comprises from about 1 to about 5 copies of the heterologous polynucleotide.

48. The method of any one of claims 45-47, wherein the heterologous polynucleotide has a ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, wherein said ratio 1: 1 to 1:10, or any ratio in between.

238

49. The method of claim 48, wherein the ratio of the nucleotide sequence operable to encode the heterologous sorbitol dehydrogenase (SDH), to the nucleotide sequence operable to encode the heterologous polypeptide, is 1:2 or 1:3.

50. The method of claim 45, wherein the heterologous polypeptide is a Cysteine Rich Peptide (CRP).

51. The method of claim 50, wherein the CRP is a Ul-agatoxin-Talb peptide; a Ul- agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (AVP); a Phoneutria toxin; or an Atracotoxin (ACTX).

52. The method of claim 51, wherein the CRP is a Ul-agatoxin-Talb peptide.

53. The method of claim 52, wherein the Ul-agatoxin-Talb peptide has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 195.

54. The method of claim 53, wherein the Ul-agatoxin-Talb peptide has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 195.

55. The method of claim 51, wherein the CRP is a TVP.

56. The method of claim 55, wherein the TVP has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

57. The method of claim 56, wherein the TVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 196-216, 218-222, 225-234, 236-245, or 247-248.

58. The method of claim 51, wherein the CRP is a sea anemone toxin.

59. The method of claim 58, wherein the sea anemone toxin is an Av2 toxin, or an Av3 toxin.

239

60. The method of claim 59, wherein the Av2 toxin has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in SEQ ID NO: 457.

61. The method of claim 60, wherein the Av2 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 457.

62. The method of claim 59, wherein the Av3 toxin has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in SEQ ID NO: 453.

63. The method of claim 62, wherein the Av3 toxin has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 453.

64. The method of claim 51, wherein the CRP is an AVP.

65. The method of claim 64, wherein the AVP has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

66. The method of claim 65, wherein the AVP has an amino acid sequence according to the amino acid sequence set forth in any one of SEQ ID NOs: 454-456.

67. The method of claim 51, wherein the CRP is a ctenitoxin (CNTX).

68. The method of claim 67, wherein the CNTX is a T-CNTX-Pnla.

69. The method of claim 68, wherein the T-CNTX-Pnla has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in SEQ ID NO: 193.

70. The method of claim 69, wherein the T-CNTX-Pnla has an amino acid sequence according to the amino acid sequence set forth in SEQ ID NO: 193.

240

71. The method of claim 51, wherein the CRP is an ACTX.

72. The method of claim 71, wherein the ACTX has an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

73. The method of claim 72, wherein the ACTX has an amino acid sequence according an amino acid sequence set forth in any one of SEQ ID NOs: 186-191.

74. The method of claim 45, wherein the yeast host cell is a species selected from the genera Saccharomyces, Pichia, Kluyveromyces, Hansenula, Yarrowia or Schizosaccharomyces .

75. The method of claim 74, wherein the recombinant yeast cell is a species selected from the group consisting of: Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia pastoris.

76. The method of claim 45, wherein the recombinant yeast cell is selected from a yeast species having an endogenous SDH nucleotide sequence.

77. The method of claim 76, wherein the recombinant yeast species having an endogenous SDH nucleotide sequence is Kluyveromyces lactis, Kluyveromyces marxianus, or Saccharomyces cerevisiae.

78. The method of any one of claims 76-77, wherein the endogenous SDH nucleotide sequence is at least partially inactivated via in vivo homologous recombination.

79. The method of claim 45, wherein the recombinant yeast cell is selected from a yeast species that does not have an endogenous SDH nucleotide sequence.

80. The method of claim 79, wherein the yeast species that does not have an endogenous SDH nucleotide sequence is Pichia pastoris.

241

81. The method of claim 45, wherein the recombinant yeast cell comprises 10 or more copies of the heterologous polynucleotide.

82. The method of claim 81, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

83. The method of claim 81, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 10 or more copies of the heterologous polynucleotide after at least 30 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

84. The method of claim 81, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of the 10 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

85. The method of claim 45, wherein the recombinant yeast cell comprises 2 or more copies of the heterologous polynucleotide.

86. The method of claim 85, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 or more copies of the heterologous polynucleotide after at least 10 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

87. The method of claim 85, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 90% of the 2 or more copies of the heterologous polynucleotide after at least 30 generations, relative to

242 growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

88. The method of claim 85, wherein growing the recombinant yeast cell in a medium comprising a sole carbon source that is sorbitol, results in maintaining at least 60% of the 2 or more copies of the heterologous polynucleotide after at least 50 generations, relative to growing the recombinant yeast cell in a medium comprising a sole carbon source that is not sorbitol.

89. The method of claim 45, wherein the vector is a plasmid comprising an alpha-MF signal.

90. The method of claim 89, wherein the alpha-MF signal is operable to express an alpha- MF signal peptide.

91. The method claim 90, wherein the heterologous polypeptide is operably linked to the alpha-MF signal peptide.

92. The method of claim 91, wherein the heterologous polypeptide is secreted into the growth medium.

93. The method of claim 45, wherein the level of expression of the heterologous polypeptide provides a yield of: at least 70 mg/L, at least 80 mg/L, at least 90 mg/L, at least 100 mg/L, at least 110 mg/L, at least 120 mg/L, at least 130 mg/L, at least 140 mg/L, at least 150 mg/L, at least 160 mg/L, at least 170 mg/L, at least 180 mg/L, at least 190 mg/L, at least 200 mg/L, at least 500 mg/L, at least 750 mg/L, at least 1,000 mg/L, at least 1,250 mg/L, at least 1,500 mg/L, at least 1,750 mg/L, at least 2,000 mg/L, at least 2,500 mg/L, at least 3,000 mg/L, at least 3,500 mg/L, at least 4,000 mg/L, at least 4,500 mg/L, at least 5,000 mg/L, at least 5,500 mg/L, at least at least 6,000 mg/L, at least 6,500 mg/L, at least 7,000 mg/L, at least 7,500 mg/L, at least 8,000 mg/L, at least 8,500 mg/L, at least 9,000 mg/L, at least 9,500 mg/L, at least 10,000 mg/L, at least 11,000 mg/L, at least 12,000 mg/L, at least 12,500 mg/L, at least 13,000 mg/L, at least 14,000 mg/L, at least 15,000 mg/L, at least 16,000 mg/L, at least 17,000 mg/L, at least 17,500 mg/L, at least 18,000 mg/L, at least 19,000 mg/L, at least

243 20,000 mg/L, at least 25,000 mg/L, at least 30,000 mg/L, at least 40,000 mg/L, at least 50,000 mg/L, at least 60,000 mg/L, at least 70,000 mg/L, at least 80,000 mg/L, at least 90,000 mg/L, or at least 100,000 mg/L of heterologous polypeptide per liter of yeast culture medium.

94. A vector comprising:

(a) a heterologous polynucleotide, or a complementary nucleotide sequence thereof, comprising:

(i) a nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) a nucleotide sequence operable to encode a heterologous polypeptide;

(b) a 5 ’-homology arm, and a 3’- homology arm, wherein said 5 ’-homology arm and said 3 ’-homology arm are located upstream and downstream of the heterologous polynucleotide, respectively; wherein said vector is operable to allow a homologous-recombination-mediated integration of the heterologous polynucleotide into an endogenous yeast host cell pLAC4 locus; and wherein said homologous-recombination-mediated integration results in a replacement of an endogenous yeast host cell pLAC4 DNA segment with the heterologous polynucleotide.

95. The vector of claim 94, wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 to 1:10, or any ratio in between.

96. The vector of claim 95, wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3.

97. The vector of claim 94, wherein the nucleotide sequence operable to encode a heterologous polypeptide is operable to encode a heterologous polypeptide having an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence as set forth in any one of SEQ ID NOs: 186-191, 193, 195-216, 218-222, 225-234, 236-245, 247-248, or 453-457

98. A polynucleotide comprising:

244 (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) at least one nucleotide sequence operable to encode a heterologous polypeptide; or a complementary nucleotide sequence thereof, wherein (i) is operably linked to (ii); or a complementary nucleotide sequence thereof.

99. The polynucleotide of claim 98, wherein the at least one nucleotide sequence operable to encode a SDH has a nucleotide sequence that is operable to encode an SDH having an amino acid sequence that is at least 80%, 85%, 90%, or at least 95% identical to an amino acid sequence set forth in any one of SEQ ID NOs: 2-6.

100. The polynucleotide of claim 98, wherein the at least one nucleotide sequence operable to encode a heterologous polypeptide has a nucleotide sequence that is operable to encode a heterologous polypeptide that is at least 80%, 85%, 90%, or at least 95% identical to an amino sequence set forth in any one of SEQ ID NO: 186-191, 193, 195-216, 218-222, 225- 234, 236-245, 247-248, or 453-457.

101. A method of increasing expression of a Cysteine Rich Peptide (CRP) in a recombinant Kluyveromyces lactis cell, said method comprising:

(a) at least partially inactivating an endogenous sorbitol dehydrogenase (SDH) nucleotide sequence;

(b) providing a vector comprising one or more copies of a heterologous polynucleotide, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation:

(i) a nucleotide sequence operable to encode a heterologous SDH; and

(ii) a nucleotide sequence operable to encode a CRP;

(c) creating a recombinant Kluyveromyces lactis cell by transforming the vector into a Kluyveromyces lactis host cell; and

(d) growing the recombinant Kluyveromyces lactis cell in a medium comprising a sole carbon source that is sorbitol; wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3;

245 wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a Phoneutria toxin; or an Atracotoxin (ACTX); and wherein growing the recombinant Kluyveromyces lactis cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the CRP, relative to a level of expression of the CRP when growing the recombinant Kluyveromyces lactis cell in a medium comprising a sole carbon source that is not sorbitol.

102. A method of increasing expression of a Cysteine Rich Peptide (CRP) in a recombinant Pichia pastoris cell, said method comprising:

(a) providing a vector comprising one or more copies of a heterologous polynucleotide, said heterologous polynucleotide comprising the following nucleotide sequences, operably linked and in any orientation:

(i) a nucleotide sequence operable to encode a heterologous SDH; and

(ii) a nucleotide sequence operable to encode a CRP;

(b) creating a recombinant Pichia pastoris cell by transforming the vector into a

Pichia pastoris host cell; and

(c) growing the recombinant Pichia pastoris cell in a medium comprising a sole carbon source that is sorbitol; wherein the heterologous polynucleotide has a ratio of (i) to (ii) that is 1:2 or 1:3; wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a. Phoneutria toxin; or an Atracotoxin (ACTX); and wherein growing the recombinant Pichia pastoris cell in the medium comprising the sole carbon source that is sorbitol, results in an increased level of expression of the CRP, relative to a level of expression of the CRP when growing the recombinant Pichia pastoris cell in a medium comprising a sole carbon source that is not sorbitol.

103. A recombinant Kluy ver omyces lactis cell comprising:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) at least one nucleotide sequence operable to encode a Cysteine Rich Peptide (CRP); wherein the ratio of (i) to (ii) is at least 1 :2;

246 wherein the recombinant Kluyveromyces lactis has an endogenous SDH nucleotide sequence that has been at least partially inactivated; and wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a Phoneutria toxin; or an Atracotoxin (ACTX).

104. A recombinant Pichia pastoris cell comprising:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) at least one nucleotide sequence operable to encode a Cysteine Rich Peptide (CRP); wherein the ratio of (i) to (ii) is at least 1 :2; and wherein the CRP is: a Ul-agatoxin-Talb peptide; a Ul-agatoxin-Talb Variant Polypeptide (TVP); a sea anemone toxin; an Av3 Variant Polypeptide (A VP); a. Phoneutria toxin; or an Atracotoxin (ACTX).

105. A recombinant Kluy ver omyces lactis cell comprising:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) two or more nucleotide sequences operable to encode a U+2-ACTX-Hvla peptide having an amino acid sequence set forth in SEQ ID NO: 187. wherein the recombinant Kluyveromyces lactis has an endogenous SDH nucleotide sequence that has been at least partially inactivated.

106. A recombinant Kluy ver omyces lactis cell comprising:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) two or more nucleotide sequences operable to encode an AVP having an amino acid sequence set forth in any one of SEQ ID NOs: 484 or 485. wherein the recombinant Kluyveromyces lactis has an endogenous SDH nucleotide sequence that has been at least partially inactivated.

107. A recombinant Pichia pastoris cell comprising:

247 (i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) two or more nucleotide sequences operable to encode a U+2-ACTX-Hvla peptide having an amino acid sequence set forth in SEQ ID NO: 187. A recombinant Pichia pastoris cell comprising:

(i) at least one nucleotide sequence operable to encode a heterologous sorbitol dehydrogenase (SDH); and

(ii) two or more nucleotide sequences operable to encode an AVP having an amino acid sequence set forth in any one of SEQ ID NOs: 484 or 485.

248