WO2019070246A1 - Souches modifiées pour la production de soie recombinante - Google Patents

Souches modifiées pour la production de soie recombinante Download PDF

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WO2019070246A1
WO2019070246A1 PCT/US2017/054997 US2017054997W WO2019070246A1 WO 2019070246 A1 WO2019070246 A1 WO 2019070246A1 US 2017054997 W US2017054997 W US 2017054997W WO 2019070246 A1 WO2019070246 A1 WO 2019070246A1
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microorganism
yps1
gene
seq
protein
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PCT/US2017/054997
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English (en)
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Matthew Scott Gamboa
Joshua Tyler KITTLESON
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Bolt Threads, Inc .
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Priority to CN201780095487.8A priority Critical patent/CN111315763A/zh
Priority to AU2017434920A priority patent/AU2017434920B2/en
Priority to EP17928005.2A priority patent/EP3692054A4/fr
Priority to MX2020003362A priority patent/MX2020003362A/es
Priority to KR1020207011698A priority patent/KR102558303B1/ko
Priority to PCT/US2017/054997 priority patent/WO2019070246A1/fr
Priority to JP2020519030A priority patent/JP7246102B2/ja
Publication of WO2019070246A1 publication Critical patent/WO2019070246A1/fr
Priority to AU2022202534A priority patent/AU2022202534A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14Fungi; Culture media therefor
    • C12N1/16Yeasts; Culture media therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14Hydrolases (3)
    • C12N9/48Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/58Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from fungi
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00Preparation of peptides or proteins
    • C12P21/02Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12YENZYMES
    • C12Y304/00Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/23Aspartic endopeptidases (3.4.23)
    • C12Y304/23041Yapsin 1 (3.4.23.41)

Definitions

  • the present disclosure relates to methods of strain optimization to produce or enhance production of proteins or metabolites from cells.
  • the present disclosure also relates to compositions resulting from those methods.
  • the disclosure relates to yeast cells selected or genetically engineered to reduce degradation of recombinant proteins expressed by the yeast cells, and to methods of cultivating yeast cells for the production of useful compounds.
  • the methylotrophic yeast Pichia pastoris is widely used in the production of recombinant proteins.
  • P. pastoris grows to high cell density, provides tightly controlled methanol-inducible trans gene expression and efficiently secretes heterologous proteins in defined media.
  • a Pichia pastoris microorganism in which the activity of a YPS1-1 protease and a YPS1-2 protease has been attenuated or eliminated, wherein said microorganism expresses a recombinant polypeptide.
  • the YPS1-1 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 67. In some embodiments, the YPS1-1 protease comprises SEQ ID NO: 67. In some embodiments, the YPS1-1 protease is encoded by a YPS1-1 gene. In some embodiments, the YPS1-1 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 1. In some embodiments, the YPS1-1 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO: 1. In some embodiments, the YPS1-1 gene comprises SEQ ID NO: 1. In some embodiments, the YPS1- 1 gene is at locus PAS_chr4_0584 of said microorganism.
  • the YPS1-2 protease comprises a polypeptide sequence at least 95% identical to SEQ ID NO: 68. In some embodiments, the YPS1-2 protease comprises SEQ ID NO: 68. In some embodiments, the YPS1-2 protease is encoded by a YPS1-2 gene. In some embodiments, the YPS1-2 gene comprises a polynucleotide sequence at least 95% identical to SEQ ID NO: 2. In some embodiments, the YPS1-2 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO: 2. In some embodiments, the YPS1-2 gene comprises SEQ ID NO: 2. In some embodiments, the YPS1- 2 gene is at locus PAS_chr3_1157 of said microorganism.
  • the YPS1-1 gene or said YPS1-2 gene, or both has been mutated or knocked out.
  • the microorganism expresses a recombinant protein.
  • the recombinant protein comprises at least one block polypeptide sequence from a silk protein.
  • the recombinant protein comprises a silk-like polypeptide.
  • the silk-like polypeptide comprises comprises a polypeptide sequence encoded by SEQ ID NO: 462.
  • the activity of one or more additional proteases in the microorganism has been attenuated or eliminated.
  • the one or more additional proteases comprises YPS1-5, MCK7, or YPS1-3.
  • the YPS1-5 gene is at locus PAS_chr3_0688 of said microorganism.
  • the MCK7 protease is encoded by a MCK7 gene comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 7.
  • the MCK7 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO: 7.
  • the MCK7 gene comprises SEQ ID NO: 7.
  • the MCK7 gene is at locus PAS_chr1-1_0379 of said microorganism.
  • the YPS1-3 protease is encoded by a YPS1-3 gene comprising a polynucleotide sequence at least 95% identical to SEQ ID NO: 3.
  • the YPS1-3 gene comprises at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of SEQ ID NO: 3.
  • the YPS1-3 gene comprises SEQ ID NO: 3.
  • the YPS1-3 gene is at locus PAS_chr3_0299 of said microorganism.
  • the one or more additional proteases comprise a polypeptide sequence at least 95% identical to a polypeptide sequence selected from the group consisting of: SEQ ID NO: 68 - 130. In some embodiments, the one or more additional proteases comprise a polypeptide sequence selected from the group consisting of: SEQ ID NO: 68 - 130. In some embodiments, the one or more additional proteases are encoded by a polynucleotide sequence at least 95% identical to a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 3 - 66.
  • the one or more additional proteases are encoded by a polynucleotide sequence comprising at least 15, 20, 25, 30, 40, or 50 contiguous nucleotides of a polynucleotide sequence selected from the group consisting of: SEQ ID NO: 3 - 66.
  • the microorganism comprises a 3X, 4X or 5X protease knockout.
  • a Pichia pastoris engineered microorganism comprising YPS1-1 and YPS1-2 activity reduced by a mutation or deletion of the YPS1-1 gene comprising SEQ ID NO: 1 and the YPS1-2 gene comprising SEQ ID NO: 2, wherein said microorganism further comprises a recombinantly expressed protein comprising a polypeptide sequence encoded by SEQ ID NO: 462.
  • cell culture comprising a protease mitigated microorganism as described herein.
  • a cell culture comprising a microorganism whose YPS1-1 and YPS1-2 activity has been attenuated or eliminated as described herein, wherein the microorganism recombinantly expresses a protein, wherein said recombinantly expressed protein is less degraded than a cell culture comprising an otherwise identical Picha pastoris microorganism whose YPS1-1 and YPS1-2 activity has not been attenuated or eliminated.
  • a method of producing a recombinant protein with a reduced degradation comprising: culturing whose YPS1-1 and YPS1-2 activity has been attenuated or eliminated as described herein in a culture medium under conditions suitable for expression of the recombinantly expressed protein; and isolating the recombinant protein from the microorganism or the culture medium.
  • the recombinant protein is secreted from said
  • the recombinant protein has a decreased level of degradation as compared to said recombinant protein produced by an otherwise identical microorganism wherein said YPS1-1 and said YPS1-2 protease activity has not been attenuated or eliminated.
  • Also provided herein is a method of modifying Pichia pastoris to reduce the degradation of a recombinantly expressed protein, comprising knocking out or mutating a gene encoding a YPS1-1 protein and a YPS1-2 protein.
  • the method of modifying Pichia pastoris to reduce the degradation of a recombinantly expressed protein further comprises knocking out or mutating one or more additional genes encoding a YPS1-3 protein, a YPS1-5 protein, or an MCK7 protein.
  • the method of modifying Pichia pastoris to reduce the degradation of a recombinantly expressed protein further comprises knocking out one or more genes encoding a protein comprising a polypeptide selected from the group consisting of SEQ ID NO: 68-130.
  • the recombinantly expressed protein comprises a polyA sequence comprising at least at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous alanine residues.
  • the recombinantly expressed protein comprises a silk-like polypeptide.
  • the recombinantly expressed protein comprises a polypeptide sequence encoded by SEQ ID NO: 462. BRIEF DESCRIPTION OF THE DRAWINGS
  • Figure 1 is a plasmid map for KU 70 deletion with a zeocin resistance marker.
  • Figure 2 is a plasmid map of a plasmid comprising a nourseothricin marker used with homology arms for targeted protease gene deletion.
  • Figure 3A and Figure 3B are cassettes for protease knockout with homology arms targeting the desired protease gene flanking a nourseothricin resistance marker.
  • Figure 4 is a representative western blot of protein isolated from single KO strains to show protein degradation from these strains.
  • Figure 5 is a representative western blot of protein isolated from double KO strains to show protein degradation from these strains.
  • Figure 6 is a representative western blot of protein isolated from 2X, 3X, 4X, and 5X protease KO strains subcultured in BMGY or YPD to show protein degradation in these strains.
  • polynucleotide or“nucleic acid molecule” refers to a polymeric form of nucleotides of at least 10 bases in length.
  • the term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native
  • the nucleic acid can be in any topological conformation.
  • the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.
  • nucleic acid comprising SEQ ID NO:1 refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1.
  • the choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.
  • RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.
  • An“isolated” organic molecule e.g., a silk protein
  • a silk protein is one which is substantially separated from the cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it originated, or from the medium in which the host cell was cultured.
  • the term does not require that the biomolecule has been separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.
  • the term“recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or a portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature.
  • the term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and/or mRNAs encoded by such nucleic acids.
  • an endogenous nucleic acid sequence in the genome of an organism is deemed“recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered.
  • heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof).
  • a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it.
  • a nucleic acid is also considered“recombinant” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome.
  • an endogenous coding sequence is considered“recombinant” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention.
  • A“recombinant nucleic acid” also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.
  • the phrase“degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence.
  • the term“degenerate oligonucleotide” or“degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.
  • sequence identity refers to the residues in the two sequences which are the same when aligned for maximum correspondence.
  • the length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.
  • polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol.183:63-98 (1990) (hereby incorporated by reference in its entirety).
  • percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein
  • sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet.3:266-272 (1993); Madden et al., Meth. Enzymol.266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).
  • nucleic acid or fragment thereof indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 75%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.
  • nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions.
  • stringent hybridization conditions and“stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art.
  • One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of
  • “stringent hybridization” is performed at about 25°C below the thermal melting point (T m ) for the specific DNA hybrid under a particular set of conditions.
  • “Stringent washing” is performed at temperatures about 5°C lower than the T m for the specific DNA hybrid under a particular set of conditions.
  • the T m is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • “stringent conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65oC for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65°C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.
  • the nucleic acids (also referred to as polynucleotides) of this present invention may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g.,
  • phosphorothioates phosphorodithioates, etc.
  • pendent moieties e.g., polypeptides
  • intercalators e.g., acridine, psoralen, etc.
  • chelators e.g., alkylators
  • modified linkages e.g., alpha anomeric nucleic acids, etc.
  • synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions.
  • Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule.
  • Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in“locked” nucleic acids.
  • nucleic acid sequences when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence.
  • a nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as“error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic.2:28- 33 (1992)); and“oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).
  • mutagenesis techniques such as“error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained
  • Attenuate generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product non-functional.
  • a functional deletion is described as a knockout mutation.
  • Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art.
  • the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant is lessened such that the enzyme activity is not impacted by the presence of a compound.
  • an enzyme that has been altered to be less active can be referred to as attenuated.
  • deletion refers to the removal of one or more nucleotides from a nucleic acid molecule or one or more amino acids from a protein, the regions on either side being joined together.
  • knock-out is intended to refer to a gene whose level of expression or activity has been reduced to zero.
  • a gene is knocked-out via deletion of some or all of its coding sequence.
  • a gene is knocked-out via introduction of one or more nucleotides into its open reading frame, which results in translation of a non-sense or otherwise non-functional protein product.
  • vector as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • a“plasmid” which generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double-stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme.
  • Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC).
  • BAC bacterial artificial chromosome
  • YAC yeast artificial chromosome
  • Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below).
  • vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell).
  • Other vectors can be integrated into the genome of a host cell upon introduction into the host cell, and are thereby replicated along with the host genome.
  • certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as
  • “Operatively linked” or“operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.
  • expression control sequence refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post- transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion.
  • control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence.
  • control sequences is intended to include, at a minimum, all components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • regulatory element refers to any element which affects transcription or translation of a nucleic acid molecule. These include, by way of example but not limitation: regulatory proteins (e.g., transcription factors), chaperones, signaling proteins, RNAi molecules, antisense RNA molecules, microRNAs and RNA aptamers. Regulatory elements may be endogenous to the host organism. Regulatory elements may also be exogenous to the host organism. Regulatory elements may be synthetically generated regulatory elements.
  • promoter refers to a DNA sequence which when ligated to a nucleotide sequence of interest is capable of controlling the transcription of the nucleotide sequence of interest into mRNA.
  • a promoter is typically, though not necessarily, located 5 ⁇ (i.e., upstream) of a nucleotide sequence of interest whose transcription into mRNA it controls, and provides a site for specific binding by RNA polymerase and other transcription factors for initiation of transcription. Promoters may be endogenous to the host organism. Promoters may also be exogenous to the host organism. Promoters may be synthetically generated regulatory elements.
  • Promoters useful for expressing the recombinant genes described herein include both constitutive and inducible/repressible promoters. Where multiple recombinant genes are expressed in an engineered organism of the invention, the different genes can be controlled by different promoters or by identical promoters in separate operons, or the expression of two or more genes may be controlled by a single promoter as part of an operon.
  • recombinant host cell (or simply“host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term“host cell” as used herein.
  • a recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.
  • peptide refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long.
  • the term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.
  • polypeptide encompasses both naturally-occurring and non-naturally- occurring proteins, and fragments, mutants, derivatives and analogs thereof.
  • a polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.
  • isolated protein or“isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds).
  • polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be“isolated” from its naturally associated components.
  • a polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art.
  • “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.
  • polypeptide fragment refers to a polypeptide that has a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion compared to a full-length polypeptide.
  • the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.
  • a protein has“homology” or is“homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
  • a protein has homology to a second protein if the two proteins have "similar” amino acid sequences.
  • homology between two regions of amino acid sequence is interpreted as implying similarity in function.
  • A“conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative amino acid substitutions.
  • the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol.
  • Examples of unconventional amino acids include: 4-hydroxyproline, ⁇ -carboxyglutamate, ⁇ -N,N,N- trimethyllysine, ⁇ -N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3- methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand end corresponds to the amino terminal end and the right-hand end corresponds to the carboxy- terminal end, in accordance with standard usage and convention.
  • Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG),
  • GCG contains programs such as“Gap” and“Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.
  • a useful algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol.215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res.7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)).
  • Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max.
  • Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max.
  • polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues.
  • searching a database containing sequences from a large number of different organisms it is preferable to compare amino acid sequences.
  • Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
  • FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, herein incorporated by reference.
  • recombinant strains and methods of producing recombinant strains to increase production of a full-length desired product in a target cell e.g., by reducing protease degradation.
  • the genes encoding these enzymes are inactivated or mutated to reduce or eliminate activity. This can be done through mutations or insertions into the gene itself of through modification of a gene regulatory element. This can be achieved through standard yeast genetics techniques.
  • Examples of such techniques include gene replacement through double homologous recombination, in which homologous regions flanking the gene to be inactivated are cloned in a vector flanking a selectable maker gene (such as an antibiotic resistance gene or a gene complementing an auxotrophy of the yeast strain).
  • a selectable maker gene such as an antibiotic resistance gene or a gene complementing an auxotrophy of the yeast strain.
  • the homologous regions can be PCR-amplified and linked through overlapping PCR to the selectable marker gene. Subsequently, such DNA fragments are transformed into Pichia pastoris through methods known in the art, e.g., electroporation. Transformants that then grow under selective conditions are analyzed for the gene disruption event through standard techniques, e.g. PCR on genomic DNA or Southern blot.
  • gene inactivation can be achieved through single homologous recombination, in which case, e.g. the 5 ⁇ end of the gene's ORF is cloned on a promoterless vector also containing a selectable marker gene.
  • such vector Upon linearization of such vector through digestion with a restriction enzyme only cutting the vector in the target-gene homologous fragment, such vector is transformed into Pichia pastoris. Integration at the target gene site is confirmed through PCR on genomic DNA or Southern blot. In this way, a duplication of the gene fragment cloned on the vector is achieved in the genome, resulting in two copies of the target gene locus: a first copy in which the ORF is incomplete, thus resulting in the expression (if at all) of a shortened, inactive protein, and a second copy which has no promoter to drive transcription.
  • transposon mutagenesis is used to inactivate the target gene.
  • a library of such mutants can be screened through PCR for insertion events in the target gene.
  • the functional phenotype (i.e., deficiencies) of an engineered/knockout strain can be assessed using techniques known in the art.
  • a deficiency of an engineered strain in protease activity can be ascertained using any of a variety of methods known in the art, such as an assay of hydrolytic activity of chromogenic protease substrates, band shifts of substrate proteins for the selected protease, among others.
  • Attenuation of a protease activity described herein can be achieved through mechanisms other than a knockout mutation.
  • a desired protease can be attenuated via amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art.
  • the protease activity of proteases encoded at PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) is attenuated by any of the methods described above.
  • the invention is directed to methylotrophic yeast strains, especially Pichia pastoris strains, wherein a YPS1-1 and a YPS1-2 gene (e.g., as set forth in SEQ ID NO: 1 and SEQ ID NO: 2) have been inactivated.
  • additional protease encoding genes may also be knocked-out in accordance with the methods provided herein to further reduce protease activity of a desired protein product expressed by the strain.
  • vectors to deliver recombinant genes or to knock-out or otherwise attenuate endogenous genes as desired.
  • vectors can take the form of a vector backbone containing a replication origin and a selection marker (typically antibiotic resistance, although many other methods are possible), or a linear fragment that enables incorporation into the target cell’s chromosome.
  • the vectors should correspond to the organism and insertion method chosen.
  • construction of the vector can be performed in many different ways.
  • a DNA synthesis service or a method to individually make every vector may be used.
  • Overlap assembly provides a method to ensure all of the elements get assembled in the correct position and do not introduce any undesired sequences.
  • the vectors generated above can be inserted into target cells using standard molecular biology techniques, e.g., molecular cloning.
  • the target cells are already engineered or selected such that they already contain the genes required to make the desired product, although this may also be done during or after further vector insertion.
  • microorganisms able to take up and replicate DNA from the local environment
  • transformation by electroporation or chemical means, transduction with a virus or phage, mating of two or more cells, or conjugation from a different cell.
  • Non limiting examples of commercial kits and bacterial host cells for electroporation include ZappersTM electrocompetent cells (EMD Chemicals Inc., NJ, USA), XL1-Blue Electroporation-competent cells (Stratagene, CA, USA), ElectroMAXTM A. tumefaciens LBA4404 Cells (Invitrogen Corp., Carlsbad, Calif., USA).
  • Non-limiting examples of commercial kits and reagents for transfection of recombinant nucleic acid to eukaryotic cell include LipofectamineTM 2000, OptifectTM Reagent, Calcium Phosphate Transfection Kit (Invitrogen Corp., Carlsbad, Calif., USA), GeneJammer® Transfection Reagent, LipoTAXI® Transfection Reagent (Stratagene, CA, USA).
  • recombinant nucleic acid may be introduced into insect cells (e.g. sf9, sf21, High FiveTM) by using baculo viral vectors.
  • Transformed cells are isolated so that each clone can be tested separately. In an embodiment, this is done by spreading the culture on one or more plates of culture media containing a selective agent (or lack of one) that will ensure that only transformed cells survive and reproduce.
  • This specific agent may be an antibiotic (if the library contains an antibiotic resistance marker), a missing metabolite (for auxotroph complementation), or other means of selection.
  • the cells are grown into individual colonies, each of which contains a single clone.
  • Colonies are screened for desired production of a protein, metabolite, or other product, or for reduction in protease activity.
  • screening identifies recombinant cells having the highest (or high enough) product production titer or efficiency. This includes a decreased proportion of degradation products or an increased total amount of full-length desired polypeptides collected from a cell culture.
  • This assay can be performed by growing individual clones, one per well, in multi- well culture plates. Once the cells have reached an appropriate biomass density, they are induced with methanol. After a period of time, typically 24-72 hours of induction, the cultures are harvested by spinning in a centrifuge to pellet the cells and removing the supernatant. The supernatant from each culture can then be tested for protease activity and/or protein degradation.
  • the modified strains with reduced protease activity described herein recombinantly express a silk-like polypeptide sequence.
  • the silk-like polypeptide sequences are 1) block copolymer polypeptide compositions generated by mixing and matching repeat domains derived from silk polypeptide sequences and/or 2) recombinant expression of block copolymer polypeptides having sufficiently large size (approximately 40 kDa) to form useful fibers by secretion from an industrially scalable microorganism.
  • Small (approximately 40 kDa to approximately 100 kDa) block copolymer polypeptides engineered from silk repeat domain fragments, including sequences from almost all published amino acid sequences of spider silk polypeptides, can be expressed in the modified microorganisms described herein.
  • silk polypeptide sequences are matched and designed to produce highly expressed and secreted polypeptides capable of fiber formation.
  • knock-out of protease genes or reduction of protease activity in the host modified strain reduces degradation of the silk like polypeptides.
  • methods of secreting block copolymers in scalable organisms e.g., yeast, fungi, and gram positive bacteria
  • the block copolymer polypeptide comprises 0 or more N-terminal domains (NTD), 1 or more repeat domains (REP), and 0 or more C- terminal domains (CTD).
  • the block copolymer polypeptide is >100 amino acids of a single polypeptide chain.
  • the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a sequence of a block copolymer polypeptide as disclosed in International Publication No. WO/2015/042164,“Methods and Compositions for Synthesizing Improved Silk Fibers,” incorporated by reference in its entirety.
  • Aciniform (AcSp) silks tend to have high toughness, a result of moderately high strength coupled with moderately high extensibility.
  • AcSp silks are characterized by large block (“ensemble repeat”) sizes that often incorporate motifs of poly serine and GPX.
  • TuSp silks tend to have large diameters, with modest strength and high extensibility.
  • TuSp silks are characterized by their poly serine and poly threonine content, and short tracts of poly alanine.
  • Major Ampullate (MaSp) silks tend to have high strength and modest extensibility.
  • MaSp silks can be one of two subtypes: MaSp1 and MaSp2.
  • MaSp1 silks are generally less extensible than MaSp2 silks, and are characterized by poly alanine, GX, and GGX motifs.
  • MaSp2 silks are characterized by poly alanine, GGX, and GPX motifs.
  • MiSp silks tend to have modest strength and modest extensibility.
  • MiSp silks are characterized by GGX, GA, and poly A motifs, and often contain spacer elements of approximately 100 amino acids.
  • Flagelliform (Flag) silks tend to have very high extensibility and modest strength.
  • Flag silks are usually characterized by GPG, GGX, and short spacer motifs.
  • each silk type can vary from species to species, and spiders leading distinct lifestyles (e.g. sedentary web spinners vs. vagabond hunters) or that are
  • a list of putative silk sequences can be compiled by searching GenBank for relevant terms, e.g.“spidroin”“fibroin”“MaSp”, and those sequences can be pooled with additional sequences obtained through independent sequencing efforts. Sequences are then translated into amino acids, filtered for duplicate entries, and manually split into domains (NTD, REP, CTD). In some embodiments, candidate amino acid sequences are reverse translated into a DNA sequence optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are each cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, various silk domains demonstrating successful expression and secretion are subsequently assembled in combinatorial fashion to build silk molecules capable of fiber formation.
  • Silk polypeptides are characteristically composed of a repeat domain (REP) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains).
  • C-terminal and N-terminal domains are between 75-350 amino acids in length.
  • the repeat domain exhibits a hierarchical architecture, as depicted in Figure 1.
  • the repeat domain comprises a series of blocks (also called repeat units). The blocks are repeated, sometimes perfectly and sometimes imperfectly (making up a quasi-repeat domain), throughout the silk repeat domain.
  • the length and composition of blocks varies among different silk types and across different species. Table 1 lists examples of block sequences from selected species and silk types, with further examples presented in Rising, A.
  • blocks may be arranged in a regular pattern, forming larger macro-repeats that appear multiple times (usually 2-8) in the repeat domain of the silk sequence. Repeated blocks inside a repeat domain or macro-repeat, and repeated macro- repeats within the repeat domain, may be separated by spacing elements.
  • block sequences comprise a glycine rich region followed by a polyA region.
  • short ( ⁇ 1-10) amino acid motifs appear multiple times inside of blocks.
  • blocks from different natural silk polypeptides can be selected without reference to circular permutation (i.e., identified blocks that are otherwise similar between silk polypeptides may not align due to circular permutation).
  • a“block” of SGAGG is, for the purposes of the present invention, the same as GSGAG (SEQ ID NO: 495) and the same as GGSGA (SEQ ID NO: 496); they are all just circular permutations of each other.
  • Fiber-forming block copolymer polypeptides from the blocks and/or macro-repeat domains is described in International Publication No. WO/2015/042164, incorporated by reference.
  • Natural silk sequences obtained from a protein database such as GenBank or through de novo sequencing are broken up by domain (N-terminal domain, repeat domain, and C-terminal domain).
  • the N-terminal domain and C-terminal domain sequences selected for the purpose of synthesis and assembly into fibers include natural amino acid sequence information and other modifications described herein.
  • a properly formed block copolymer polypeptide comprises at least one repeat domain comprising at least 1 repeat sequence, and is optionally flanked by an N-terminal domain and/or a C-terminal domain.
  • a repeat domain comprises at least one repeat sequence.
  • the repeat sequence is 150-300 amino acid residues.
  • the repeat sequence comprises a plurality of blocks.
  • the repeat sequence comprises a plurality of macro-repeats.
  • a block or a macro-repeat is split across multiple repeat sequences.
  • the repeat sequence starts with a Glycine, and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to satisfy DNA assembly requirements.
  • some of the repeat sequences can be altered as compared to native sequences.
  • the repeat sequencess can be altered such as by addition of a serine to the C terminus of the polypeptide (to avoid terminating in F, Y, W, C, H, N, M, or D).
  • the repeat sequence can be modified by filling in an incomplete block with homologous sequence from another block.
  • the repeat sequence can be modified by rearranging the order of blocks or macrorepeats.
  • non-repetitive N- and C-terminal domains can be selected for synthesis.
  • N-terminal domains can be by removal of the leading signal sequence, e.g., as identified by SignalP (Peterson, T.N., et. Al., SignalP 4.0:
  • the N-terminal domain, repeat sequence, or C-terminal domain sequences can be derived from Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp.
  • the silk polypeptide nucleotide coding sequence can be operatively linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operatively linked to another endogenous or heterologous secretion signal coding sequence. In some
  • the silk polypeptide nucleotide coding sequence can be operatively linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operatively linked to other affinity tags such as 6-8 His residues.
  • the P. pastoris strains disclosed herein have been modified to express a silk-like polypeptide.
  • Methods of manufacturing preferred embodiments of silk- like polypeptides are provided in WO 2015/042164, especially at Paragraphs 114-134, incorporated herein by reference.
  • Disclosed therein are synthetic proteinaceous copolymers based on recombinant spider silk protein fragment sequences derived from MaSp2, such as from the species Argiope bruennichi.
  • Silk-like polypeptides are described that include two to twenty repeat units, in which a molecular weight of each repeat unit is greater than about 20 kDa.
  • each repeat unit of the copolymer are more than about 60 amino acid residues that are organized into a number of“quasi-repeat units.”
  • the repeat unit of a polypeptide described in this disclosure has at least 95% sequence identity to a MaSp2 dragline silk protein sequence.
  • each“repeat unit” of a silk-like polypeptide comprises from two to twenty“quasi-repeat” units (i.e., n3 is from 2 to 20). Quasi-repeats do not have to be exact repeats. Each repeat can be made up of concatenated quasi-repeats. Equation 1 shows the composition of a repeat unit according the present disclosure and that incorporated by reference from WO 2015/042164. Each silk-like polypeptide can have one or more repeat units as defined by Equation 1. [0090] The variable compositional element X1 (termed a“motif”) is according to any one of the following amino acid sequences shown in Equation 2 and X1 varies randomly within each quasi-repeat unit.
  • X 1 SGGQQ or GAGQQ or GQGPY or AGQQ or SQ (Equation 2)
  • the compositional element of a quasi-repeat unit represented by“GGY-[GPG-X 1 ] n1 -GPS” in Equation 1 is referred to a“first region.”
  • a quasi-repeat unit is formed, in part by repeating from 4 to 8 times the first region within the quasi-repeat unit. That is, the value of n 1 indicates the number of first region units that are repeated within a single quasi-repeat unit, the value of n1 being any one of 4, 5, 6, 7 or 8.
  • compositional element represented by“(A)n2” (i.e., a polyA sequence) is referred to as a “second region” and is formed by repeating within each quasi-repeat unit the amino acid sequence“A” n2 times. That is, the value of n2 indicates the number of second region units that are repeated within a single quasi-repeat unit, the value of n2 being any one of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
  • the repeat unit of a polypeptide of this disclosure has at least 95% sequence identity to a sequence containing quasi-repeats described by Equations 1 and 2.
  • the repeat unit of a polypeptide of this disclosure has at least 80%, or at least 90%, or at least 95%, or at least 99% sequence identity to a sequence containing quasi-repeats described by Equations 1 and 2.
  • 3“long” quasi repeats are followed by 3“short” quasi- repeat units.
  • all of the short quasi- repeats have the same X 1 motifs in the same positions within each quasi-repeat unit of a repeat unit.
  • no more than 3 quasi-repeat units out of 6 share the same X1 motifs.
  • a repeat unit is composed of quasi-repeat units that do not use the same X1 more than two occurrences in a row within a repeat unit.
  • a repeat unit is composed of quasi-repeat units where at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 of the quasi-repeats do not use the same X1 more than 2 times in a single quasi-repeat unit of the repeat unit.
  • strains of yeast that recombinantly express silk-like polypeptides with a reduced degradation to increase the amount of full- length polypeptides present in the isolated product from a cell culture.
  • the strain expressing a silk-like polypeptide is a P. pastoris strain comprises a PAS_chr4_0584 knock-out and a PAS_chr3_1157 knock-out.
  • articles such as“a,”“an,” and“the” may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include“or” between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context.
  • the invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process.
  • the invention includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.
  • Example 1 Production of recombinant yeast expressing 18B
  • Transformation was accomplished by electroporation as described in PMID 15679083, incorporated by reference herein.
  • Each vector includes an 18B expression cassette with the polynucleotide sequence encoding the silk-like protein in the recombinant vectors flanked by a promoter (pGCW14) and a terminator (tAOX1 pA signal).
  • the recombinant vectors further comprised dominant resistance markers for selection of bacterial and yeast transformants, and a bacterial origin of replication.
  • the first recombinant vector included targeting regions that directed integration of the 18B polynucleotide sequences immediately 3 ⁇ of the AOX2 loci in the Pichia pastoris genome.
  • the resistance marker in the first vector conferred resistance to G418 (aka geneticin).
  • the second recombinant vector included targeting regions that directed integration of the 18B polynucleotide sequences immediately 3 ⁇ of the TEF1 loci in the Pichia pastoris genome.
  • the resistance marker in the second vector conferred resistance to Hygromycin B.
  • Homology arms used for each target were amplified by the primers provided in Table 7, and inserted into the nourseothricin resistance plasmid. Homology arms were inserted into the nourseothricin plasmid to generate cassettes comprising a nourseothricin resistance marker flanded by 3 ⁇ and 5 ⁇ homology arms to the target protease as shown in Figure 3A and Figure 3B.
  • the resistance cassette Nour Resistance Cassette
  • HA1 and HA2 homology arms
  • nourseothricin marker are shown, including the promoter from ILV5 gene from
  • Streptomyces noursei (nat)
  • the polyA signal from CYC1 gene from Saccharomyces cerevisiae.
  • the homology arms in each vector targeted one of the 65 desired protease loci as provided in Table 2.
  • Transformants were plated on YPD agar plates supplemented with nourseothricin, and incubated for 48 hours at 30°C.
  • Example 3 Testing single protease knockout clones for reduced protein degradation.
  • Example 4 Generating a library of protease double knock-outs
  • proteases were knocked out. These proteases were selected, in part, because they were paralogs that may have compensatory function with respect to each other.
  • nourseothricin resistance was eliminated from the single protease knock-out strains produced in Example 2, and a second protease deleted by transformation with a second nourseothricin resistance cassette as provided in Example 2.
  • Transformants were plated on YPD agar plates supplemented with nourseothricin, and incubated for 48 hours at 30°C.
  • Double protease knock-outs tested are provided in Table 3.
  • Table 3 Protease double KO strains of P. Pastoris expressing silk-like polypeptide
  • Resulting clones were inoculated into 400 ⁇ L of Buffered Glycerol-complex Medium (BMGY) in 96-well blocks, and incubated for 48 hours at 30°C with agitation at 1,000 rpm. Following the 48-hour incubation, 4 ⁇ L of each culture was used to inoculate 400 ⁇ L of BMGY in 96-well blocks, which were then incubated for 48 hours at 30°C. Guanidine thiocyanate was added to a final concentration of 2.5M to the cell cultures to extract the recombinant protein. After a 5 min incubation, solutions were centrifuged and the supernatant was sampled and analyzed by western blot.
  • BMGY Buffered Glycerol-complex Medium
  • Figure 4 shows representative results from different protease double knockout strains. As shown, despite the presence of protein degradation in all single knockout strains tested, the combination of PAS_chr4_0584 + PAS_chr3_1157 protease knockout (Strain 3 from Table 3) resulted in the near-complete elimination of degradation products. None of the other combinations of proteases resulted in the elimination of degradation products.
  • PAS_chr4_0584 and PAS_chr3_1157 to mitigate degradation of the desired protein. We further knocked out one or more additional proteases to enhance the production of full-length products and minimize degradation.
  • an additional protease gene was deleted from a single protease KO (1X KO), double protease KO (2X KO), triple protease KO (3X KO), or quadruple protease KO (4X KO) by transformation with a nourseothricin resistance cassette with homology arms targeting the desired gene as provided in Example 2.
  • the protease genes knocked out in each strain are shown in Table 4:
  • Protein expressed by the cells was isolated and analyzed for degradation as follows: Guanidine thiocyanate was added to a final concentration of 2.5M to the cell cultures to extract the recombinant protein. After a 5 min incubation, solutions were centrifuged and the supernatant was sampled and analyzed by western blot.
  • Figure 5 shows the results of a Western Blot of purified protein from the 2X KO, 3X KO, 4X KO and 5X KO strains inoculated in BMGY or YPD. As shown, the deletion of additional protease genes from the strain having the PAS_chr4_0584 + PAS_chr3_1157 protease knockout (Strain 3 from Table 3) resulted in the further elimination of degradation products.

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Abstract

L'invention concerne des souches modifiées pour réduire la dégradation de produits exprimés par recombinaison sécrétés par un organisme hôte et des procédés d'utilisation des souches modifiées.
PCT/US2017/054997 2017-10-03 2017-10-03 Souches modifiées pour la production de soie recombinante WO2019070246A1 (fr)

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EP3794151A4 (fr) * 2018-05-17 2022-03-23 Bolt Threads, Inc. Souches modifiées sec pour sécrétion améliorée de protéines recombinantes
WO2023215221A3 (fr) * 2022-05-02 2023-12-28 North Carolina State University Micro-organismes modifiés présentant une expression et une sécrétion de protéines améliorées

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AU2022202534A1 (en) 2022-05-12
AU2017434920A1 (en) 2020-05-07
KR20200058482A (ko) 2020-05-27
KR102558303B1 (ko) 2023-07-21
JP7246102B2 (ja) 2023-03-27
EP3692054A4 (fr) 2021-06-09
EP3692054A1 (fr) 2020-08-12
CN111315763A (zh) 2020-06-19
JP2021503275A (ja) 2021-02-12
MX2020003362A (es) 2020-07-29

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