WO2020069142A1

WO2020069142A1 - Optimized expression systems for expressing berberine bridge enzyme and berberine bridge enzyme-like polypeptides

Info

Publication number: WO2020069142A1
Application number: PCT/US2019/053173
Authority: WO
Inventors: Andrew HORWITZ; Leo D'ESPAUX; Jeff Wong
Original assignee: Demetrix, Inc.
Priority date: 2018-09-26
Filing date: 2019-09-26
Publication date: 2020-04-02

Abstract

The present disclosure provides modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide. The modified host cells of the disclosure may be useful for expressing a BBE or BBE-like polypeptide. The present disclosure also provides methods, nucleic acids, and modified host cells for expressing a BBE or BBE-like polypeptide.

Description

OPTIMIZED EXPRESSION SYSTEMS FOR EXPRESSING BERBERINE BRIDGE ENZYME AND BERBERINE BRIDGE ENZYME-LIKE POLYPEPTIDES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of ET.S. Provisional Application No.

62/737,006, filed September 26, 2018, the contents of which are incorporated herein by reference in its entirety.

INTRODUCTION

[0002] Synthetic biology allows for the engineering of industrial host organisms— e.g., microbes— to convert simple sugar feedstocks into medicines. This approach includes identifying genes that produce the target molecules and optimizing their activities in the industrial host. Microbial production can be significantly cost-advantaged over agriculture and chemical synthesis, less variable, and allows tailoring of the target molecule. However, reconstituting or creating a pathway to produce a target molecule in an industrial host organism can require significant engineering of both the pathway genes and the host.

[0003] Medicines are attractive target molecules for microbial production because they are often high-value and produced by metabolic pathways found in or derived from nature. Astonishingly, more than 50% of known medicines originate from plants and many are currently manufactured via agriculture, at times followed by chemical modification.

[0004] Plants that naturally produce medicinal molecules can contain specialized genes, pathways, and anatomical features not present in industrial microbes. For example, in its native host Cannabis sativa, the enzyme tetrahydrocannabinolic acid (THCA) synthase is produced primarily in specialized epidermal cells within the glandular trichome and targeted via the secretory pathway to an oily extracellular cavity where the enzyme actively produces THCA. In Papaver somniferum , the opium poppy, Berberine Bridge Enzyme (BBE)— an enzyme evolutionarily related to THCA synthase— is primarily produced in specialized cells in the root, within subcellular vacuolar compartments, where BBE forms a carbon-carbon bond characteristic of the Berberine branch of opiates. Downstream opiate biosynthetic enzymes are produced and are active in different cell types, e.g., laticifer cells that secrete product opiates into the latex (Weid et al. 2004).

[0005] It is thought that targeting enzymes and the metabolites they produce via the secretory pathway can serve to spatially separate toxic products or by-products from central cellular metabolism and to store defensive molecules to disengage a would-be herbivore. The evolutionary relationships giving rise to such organization can lead to pinpoint accuracy in the interaction between a plant metabolite and a signaling network from another organism, creating a treasure trove of potential medicines.

[0006] Many other molecules of medicinal interest originate from bacterial or fungal cells and are often manufactured using complex and challenging chemical synthesis. The metabolic pathways for producing these molecules within bacterial or fungal cells sometimes involve BBE or BBE-like polypeptides, a group of polypeptides involved in the biosynthesis of diverse and medicinally important molecules (e.g., opiates, cannabinoids, and nicotine). BBE or BBE-like polypeptides often contain disulfide bonds, glycosylations, and are the only polypeptides known to contain a bi-covalently attached FAD cofactor. In bacteria and fungi, these polypeptides are typically processed through the secretory pathway or system.

[0007] One possible method to access these potential medicines is production via fermentation of engineered microbes, such as yeast. By engineering production of the relevant plant, fungal, or bacterial enzymes in microbes, it may be possible to achieve conversion of various feedstocks into a range of metabolites, potentially at much lower cost and with much higher purity than what is available from the plant, fungi, or bacteria, or through chemical synthesis. A key challenge to this effort is the difficulty of expressing BBE, BBE-like, or secreted enzymes, such as secreted plant enzymes, in the microbe, as these enzymes must successfully traverse the microbe’s secretory pathway to fold and function properly. Specific challenges for processing heterologous secretory polypeptides in microbes include 1) identification of plant enzyme secretory sequences which may require replacement with analogous microbe sequences, 2) optimization of codon usage to promote efficient translation, 3) support of protein folding and disulfide formation requirements, 4) support of co-factor requirements (e.g., covalently linked flavin adenine dinucleotide (FAD)), and 5) potential for microbe glycosylation patterns to interfere with protein folding or function. New methods and modified microbes are needed to address these challenges.

SUMMARY

[0008] The present disclosure provides modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide. The modified host cells of the disclosure may be useful for expressing a BBE or BBE-like polypeptide. The present disclosure also provides methods, nucleic acids, and modified host cells for the expression of BBE or BBE-like polypeptides. [0009] One aspect of the disclosure relates to a modified host cell for expressing a

Berberine bridge enzyme (BBE) or BBE-like polypeptide, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides or a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, or a combination of any of the foregoing, and wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide. In certain such embodiments, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In certain such embodiments, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), or one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon- optimized.

[0010] In some embodiments described herein, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, a JEM1 polypeptide, a LHS1 polypeptide, a SIS1 polypeptide, a SSB1 polypeptide, a CNE1 polypeptide, a CNS1 polypeptide, a PFD2s polypeptide, a PFDl polypeptide, a SSA1 polypeptide, a YDJ1 polypeptide, a SIL1 polypeptide, a SCJ1 polypeptide, a ROT1 polypeptide, a CPR5 polypeptide, or a FPR1 polypeptide, or a combination of any of the foregoing. In certain such embodiments, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. In certain such embodiments, the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[0011] In some embodiments described herein, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response. In certain such embodiments, the one or more polypeptides involved in unfolded protein response comprise an IRE1 polypeptide or a HACls polypeptide, or a combination of any of the foregoing. In some embodiments described herein, the one or more polypeptides involved in unfolded protein response comprise a transcription factor polypeptide or a lumenal sensor polypeptide that binds to an unfolded protein response element. In some embodiments described herein, the modified host cell comprises a synthetic polypeptide that binds to an unfolded protein response element.

[0012] In some embodiments described herein, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In certain such embodiments, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FADl polypeptide.

[0013] In some embodiments described herein, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides. In certain such embodiments, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide.

[0014] In some embodiments described herein, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. In certain such embodiments, the one or more thiol oxidase polypeptides comprise an EROl polypeptide or an ERV2 polypeptide, or a combination of any of the foregoing. In certain such embodiments, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide.

[0015] In some embodiments described herein, the modified host cell comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In certain such embodiments, the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, or one or more genes encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing.

[0016] In some embodiments described herein, the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene or a MNS1 gene, or a combination of any of the foregoing.

[0017] In some embodiments described herein, the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism. In certain such embodiments, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene.

[0018] In some embodiments described herein, the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides. In certain such embodiments, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, a PRB1 gene, or a PRC1 gene, or a combination of any of the foregoing.

[0019] In some embodiments described herein, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in EIPR, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, wherein the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and a SSB1 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in UPR comprise a HACls polypeptide, and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

[0020] In some embodiments described herein, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more

heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, and one or more genes encoding one or more glycosidase polypeptides, wherein the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene. [0021] In some embodiments described herein, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding the

Berberine bridge enzyme (BBE) or BBE-like polypeptide.

[0022] In some embodiments described herein, the nucleotide sequence encoding the

Berberine bridge enzyme (BBE) or BBE-like polypeptide is codon-optimized.

[0023] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue, a fungal cell, or a bacterial cell.

[0024] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a tetrahydrocannabinolic acid synthase polypeptide.

[0025] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a cannabichromenic acid synthase polypeptide.

[0026] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a cannabidiolic acid synthase polypeptide.

[0027] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE polypeptide from Eschscholzia californica.

[0028] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum.

[0029] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans .

[0030] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a daurichromenic acid synthase polypeptide from Rhododendron dauricum.

[0031] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[0032] In some embodiments described herein, the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprises a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide.

[0033] In some embodiments described herein, the tetrahydrocannabinolic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 92.

[0034] In some embodiments described herein, the cannabichromenic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:94.

[0035] In some embodiments described herein, the cannabidiolic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 108 or SEQ ID NO: 132.

[0036] In some embodiments described herein, the BBE polypeptide from

Eschscholzia californica comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 116.

[0037] In some embodiments described herein, the BBEdike nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 120.

[0038] In some embodiments described herein, the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 122.

[0039] In some embodiments described herein, the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 118.

[0040] In some embodiments described herein, the xylooligosaccharide oxidase polypeptide from Myceliophthor a thermophila comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 124.

[0041] In some embodiments described herein, the modified host cell is a eukaryotic cell. In certain such embodiments, the eukaryotic cell is a yeast cell. In certain such embodiments, the yeast cell is Saccharomyces cerevisiae. In certain such embodiments, the Saccharomyces cerevisiae is a protease-deficient strain of Saccharomyces cerevisiae.

[0042] In some embodiments described herein, at least one of the one or more heterologous nucleic acids are integrated into the chromosome of the modified host cell.

[0043] In some embodiments described herein, at least one of the one or more heterologous nucleic acids are maintained extrachromosomally (e.g., on a plasmid or artificial chromosome).

[0044] In some embodiments described herein, at least one of the one or more heterologous nucleic acids are operably-linked to an inducible promoter.

[0045] In some embodiments described herein, at least one of the one or more heterologous nucleic acids are operably-linked to a constitutive promoter.

[0046] In some embodiments described herein, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding an Internal Ribosome Entry Site (IRES) element.

[0047] In some embodiments described herein, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding a T2A element.

[0048] In some embodiments described herein, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding a green fluorescent protein (GFP) polypeptide.

[0049] One aspect of the disclosure relates to a method of making a modified host cell disclosed herein, the method comprising introducing into a host cell: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

[0050] Another aspect of the disclosure relates to a method of making a modified host cell disclosed herein, the method comprising introducing into a host cell: a) one or more vectors comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more vectors comprising one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides. [0051] In some embodiments of the method of making a modified host cell described herein, the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[0052] One aspect of the disclosure relates to a method for expressing a Berberine bridge enzyme (BBE) or BBE-like polypeptide, the method comprising culturing a modified host cell of any one of the foregoing or following embodiments in a culture medium. In some embodiments described herein, the culture medium comprises a fermentable sugar. In some embodiments described herein, the culture medium comprises a pretreated cellulosic feedstock. In some embodiments described herein, the culture medium comprises a non- fermentable carbon source. In some embodiments described herein, the non-fermentable carbon source comprises ethanol.

BRIEF DESCRIPTION OF THE DRAWINGS

[0053] FIG. 1 depicts a phylogenetic analysis of BBE and BBE-like proteins. See,

Vacuole-Localized Berberine Bridge Enzyme-Like Proteins Are Required for a Late Step of Nicotine Biosynthesis in Tobacco, Kajikawa et al ., Plant Physiology Apr 2011, 155 (4) 2010-2022; DOI: 10. H04/pp.110.170878.

[0054] FIG. 2 depicts that the daurichromenic acid synthase polypeptide from

Rhododendron dauricum catalyzes an oxidative ring closure. See, Daurichromenic acid synthase from Rhododendron dauricum , Iijima et al ., Plant Physiology Jul

2017, rr.00586.2017; DOI: 10.1104/rr.17.00586.

[0055] FIGS. 3A, 3B, and 3C depict expression constructs used in the production of the S29 strain. The expression constructs depicted in FIGS. 3A, 3B, and 3C were also used in the production of the following strains: S61, S122, S171, S181, S206, S220, S228, S241, S270, S280, S311, S312, S313, S314, S315, S316, S317, S318, S319, S320, S321, S322, S323, S324, S325, S326, and S369. Throughout the figures, in addition to the specified coding sequences from Table 1, construct maps depict regulatory, non-coding and genomic cassette sequences described in Table 4. Construct maps also depict genes denoted with a preceding“m” (e.g., mERGl3), which specify open reading frames from Table 1 with 200- 250 base pairs (bp) of downstream regulatory (terminator) sequence. Arrows in construct maps indicate the directionality of certain DNA parts. The“!” preceding a part name is an output of the DNA design software used, is redundant with the arrow directionality, and can be ignored. [0056] FIG. 4 depicts an expression construct used in the production of the S181 strain. The expression construct depicted in FIG. 4 was also used in the production of following strains: S220, S241, S270, and S369.

[0057] FIG. 5 depicts an expression construct used in the production of the S220 strain. The expression construct depicted in FIG. 5 was also used in the production of following strains: S241, S270, and S369.

[0058] FIG. 6 depicts expression constructs used in the production of the S241 strain.

The expression constructs depicted in FIG. 6 were also used in the production of following strains: S270 and S369.

[0059] FIG. 7 depicts an expression construct used in the production of the S61 strain. The expression construct depicted in FIG. 7 was also used in the production of the following strains: S122, S171, S181, S220, S241, S270, and S369.

[0060] FIG. 8 depicts expression constructs used in the production of the S122 strain.

The expression constructs depicted in FIG. 8 were also used in the production of the following strains: S171, S181, S220, S241, S270, and S369.

[0061] FIG. 9 depicts an expression construct used in the production of the S 171 strain. The expression construct depicted in FIG. 9 was also used in the production of the following strains: S181, S220, S241, S270, and S369.

[0062] FIGS. 10A and 10B depict expression constructs used in the production of the

S206 strain. The expression constructs depicted in FIGS. 10A and 10B were also used in the production of the following strains: S228, S280, S318, S319, S320, S321, S322, S323, S324, and S326.

[0063] FIG. 11 depicts expression constructs used in the production of the S270 strain. The expression constructs depicted in FIG. 11 were also used in the production of the S369 strain.

[0064] FIG. 12 depicts expression constructs used in the production of the S228 strain. The expression constructs depicted in FIG. 12 were also used in the production of the following strains: S280, S318, S319, S320, S321, S322, S323, S324, and S326.

[0065] FIG. 13 depicts an expression construct used in the production of the S311 strain.

[0066] FIG. 14 depicts an expression construct used in the production of the S312 strain.

[0067] FIG. 15 depicts an expression construct used in the production of the S313 strain. [0068] FIG. 16 depicts an expression construct used in the production of the S314 strain.

[0069] FIG. 17 depicts an expression construct used in the production of the S315 strain.

[0070] FIG. 18 depicts an expression construct used in the production of the S316 strain.

[0071] FIG. 19 depicts an expression construct used in the production of the S317 strain.

[0072] FIG. 20 depicts an expression construct used in the production of the S318 strain.

[0073] FIG. 21 depicts an expression construct used in the production of the S319 strain.

[0074] FIG. 22 depicts an expression construct used in the production of the S320 strain.

[0075] FIG. 23 depicts an expression construct used in the production of the S321 strain.

[0076] FIG. 24 depicts an expression construct used in the production of the S322 strain.

[0077] FIG. 25 depicts an expression construct used in the production of the S323 strain.

[0078] FIG. 26 depicts an expression construct used in the production of the S324 strain.

[0079] FIG. 27 depicts expression constructs used in the production of the S280 and

S369 strains.

[0080] FIG. 28 depicts microscopy images of the secretory engineering-naive strain

S29 expressing CBDASco5-GFP (left panel = S325) and the secretory engineered strain S228 expressing CBDASco5-GFP (right panel = S326). Green fluorescent protein (GFP) fluorescence appears in white against gray /black background. Small amounts of GFP in sharp foci are visible in the left panel, compared to diffuse and distributed GFP in the right panel.

[0081] FIG. 29 depicts a graphical representation of Table 2 data. Cannabidiolic acid

(CBDA) titer (y-axis) versus GFP polypeptide expression level (x-axis) of strains expressing various codon optimizations of cannabidiolic acid synthase (CBDAS) fused via T2A linker to GFP. Data in the left panel are from strains S311-S317; right panel from S318-S324. [0082] FIG. 30 depicts that GFP expression of CBDAS codon-opts fused to GFP via

T2A cleavable linker is similar in naive strains (left panel, S311 -S317) versus secretory engineered strains (right panel, S318-S324).

[0083] FIG. 31 depicts expression constructs used in the production of the S325 strain.

[0084] FIG. 32 depicts expression constructs used in the production of the S326 strain.

DETAILED DESCRIPTION

[0085] For many industrially useful polypeptides— including metabolic enzymes involved in the biosynthesis of cannabinoids, opiates, and other drugs, as well as therapeutic or otherwise useful proteins such as antibodies and secreted proteins— processing along the secretory pathway or system is crucial for their maturation. Functionally reconstituting the activity of BBE or BBE-like polypeptide or other polypeptides processed through the secretory pathway or system in a heterologous host organism can require extensive re- engineering of the host’s secretory pathway and fine tuning of the expression of the target polypeptide(s). The present disclosure provides modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide. The modified host cells of the disclosure may be useful for expressing a BBE or BBE-like polypeptide. The present disclosure also provides methods, nucleic acids, and modified host cells for expressing a BBE or BBE-like polypeptide.

[0086] The Berberine Bridge Enzyme (BBE) and BBE-like sub-family are a unique group of polypeptides involved in the biosynthesis of diverse and medicinally important molecules (e.g., opiates, cannabinoids, and nicotine). These polypeptides are often processed via the secretory pathway or system and may be found in many organisms, including plants, bacteria, and fungi. BBE enzymes include the namesake enzymes in P. somniferum and related plants; cannabinoid biosynthetic genes such as THCA synthase in C. sativa ; BBE- like polypeptides involved in nicotine biosynthesis in Nicotiana tabacum and related plants; and carbohydrate-degrading enzymes in Myceliophthora thermophila and other microbes (FIG. 1). BBE and BBE-like polypeptides often contain disulfide bonds, glycosylations, and are the only polypeptides known to contain a bi-covalently attached FAD cofactor— thought to increase the enzyme’s catalytic potential over the more common mono-covalent FAD.

[0087] Among BBE-like polypeptides, cannabinoid synthase polypeptides from C. sativa are attractive targets for biotechnological applications, yet have proven difficult to reconstitute in a new host for several reasons. The three known cannabinoid synthase polypeptides— tetrahydrocannabinolic acid (THCA)-, cannabidiolic acid (CBDA)- and cannabichromenic acid (CBCA)-synthase polypeptides— convert the central cannabinoid precursor cannabigerolic acid (CBGA) into THCA, CBDA, or CBCA, respectively. These acidic cannabinoids are the more common cannabinoid constituents in live Cannabis and can decarboxylate in the presence of heat— e.g., via smoking— into their respective neutral forms, e.g., THCA into tetrahydrocannabinol (THC). THC is known to be psychoactive— while THCA is not— and several cannabinoids including cannabidiol (CBD), THC, and others, are FDA-approved drugs. The three known cannabinoid synthase polypeptides— producing THCA, CBDA, or CBCA— are BBE-like enzymes and share more than 80% identity at the polypeptide level. Additionally, each contain one disulfide bond, a bi- covalently bound FAD, several glycosylation sites, and an N-terminal secretory signal sequence. The signal sequence is thought to mediate routing through the secretory pathway where the enzyme is matured; polypeptides not processed through the secretory pathway are inactive.

[0088] The namesake enzyme of the BBE sub-family is found in P. somniferum and catalyzes the formation of a carbon-carbon bond called the berberine bridge, found in the berberine branch of the opiate pathway. The Eschscholzia California BBE 1 polypeptide (ETniprot P30986) is predicted to contain a signal peptide, a disulfide bond, three

glycosylation sites, and a bi-covalently bound FAD.

[0089] In Nicotiana tabacum and related plants, BBE-like enzymes catalyze another carbon-carbon bond involved in the biosynthesis of nicotine. N. tabacum BBL 1 polypeptide (Uniprot F1T160) contains characteristic BBE features such as an FAD-binding domain and a predicted N-terminal signal sequence that is thought to mediate the polypeptide’s transport to the vacuole (Kajikawa et al. 2011).

[0090] Besides biosynthesis, BBE-like polypeptides can also degrade metabolites, as in the nicotine-degrading 6-hydroxy -D-nicotine oxidase (HDNO) polypeptide from the bacterium Paenarthrobacter nicotinovorans (ETniprot Q8GAG1). The structure of HDNO has been solved and contains multiple FAD-binding sites (Koetter and Schulz 2005).

[0091] In Rhododendron dauricum , a BBE-like enzyme named daurichromenic acid synthase polypeptide (DC AS, ETniprot A0A218PFV5) catalyzes the last step in the biosynthesis of daurichromenic acid, an anti-HIV meroterpenoid (Iijima et al. 2017). Like the THC AS polypeptide, the DCAS polypeptide contains a signal sequence and a bi- covalently bound FAD, and catalyzes an oxidative ring closure (FIG. 2). [0092] The BBE-like xylooligosaccharide oxidase polypeptide from Myceliophthora thermophile oxidizes oligosaccharides from cellulose and contains a bi-covalently bound FAD.

[0093] The disclosure provides for modification of the secretory pathway of a host cell modified with one or more nucleic acids (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide. In some embodiments, the nucleotide sequence encoding a BBE or BBE-like polypeptide is codon-optimized.

Modification of the secretory pathway in the host cell may improve expression and solubilization of BBE or BBE-like polypeptides, as these polypeptides are often processed through the secretory pathway or system. Reconstituting the activity of polypeptides typically processed through the secretory pathway or system, such as BBE or BBE-like polypeptides, in a modified host cell, such as a modified yeast cell, can be challenging and unreliable. Often the expressed polypeptides are misfolded or mislocalized, resulting in low expression, expressed polypeptides lacking activity, polypeptide aggregation, reduced host cell viability, and/or cell death. Additionally, a backlog of misfolded or mislocalized expressed polypeptides can induce metabolic stress within the modified host cell, harming the modified host cell. The expressed polypeptides may lack necessary posttranslational modifications for folding and activity, such as disulfide bonds, glycosylation and trimming, and cofactors, affording inactive polypeptides or polypeptides with reduced enzymatic activity. Moreover, the addition of signal sequence polypeptides to the BBE or BBE-like polypeptides, such as secretory signal sequence polypeptides, may facilitate proper transport of BBE or BBE-like polypeptides through the secretory pathway, also possibly improving host cell viability and expression, solubilization, folding, and enzymatic activity of the BBE or BBE-like polypeptides.

[0094] The modified host cell of the disclosure may be a modified yeast cell. Yeast cells may be reasonable host cells for protein expression because they are easily cultured using known conditions, grow rapidly, and are generally regarded as safe. Yeast cells contain the secretory pathway common to all eukaryotes. As disclosed herein, manipulation of that secretory pathway in yeast host cells modified with one or more nucleic acids (e.g., heterologous nucleic acids) comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide may improve expression, folding, and enzymatic activity of the BBE or BBE- like polypeptide as well as viability of the modified yeast host cell, such as modified Saccharomyces cerevisiae. Further, addition of signal sequence polypeptides to the BBE or BBE-like polypeptides, such as secretory signal sequence polypeptides, and/or use of codon- optimized nucleotide sequences encoding BBE or BBE-like polypeptides, may improve expression and activity of the BBE or BBE-like polypeptide and viability of modified yeast host cells, such as modified Saccharomyces cerevisiae.

[0095] The modified host cells of the disclosure may be useful for producing bioactive compounds such as naturally-occurring and non-naturally occurring cannabinoids; opiates; alkaloids, such as benzylisoquinoline alkaloids, nicotine, and anabasine;

polyketides; terpenes; isoprenoids; terpenoids; antibiotics; aldonic acids; or industrially or medicinally useful proteins such as antibodies or secreted proteins. Naturally-occurring cannabinoids, opiates, alkaloids, polyketides, terpenes, isoprenoids, terpenoids, antibiotics, and aldonic acids and non-naturally occurring cannabinoids, opiates, alkaloids, polyketides, terpenes, isoprenoids, terpenoids, antibiotics, and aldonic acids can be challenging to produce using chemical synthesis due to their complex structures. Bioproduction also enables synthesis of cannabinoids, opiates, alkaloids, polyketides, terpenes, isoprenoids, terpenoids, antibiotics, and aldonic acids with defined stereochemistries, which is challenging to do using chemical synthesis. ETsing the modified host cells of the disclosure may afford a more reliable and economical process than agriculture-based production of cannabinoids, opiates, alkaloids, polyketides, terpenes, isoprenoids, terpenoids, antibiotics, and aldonic acids. Microbial fermentations can be completed in days versus the months necessary for an agricultural crop, are not affected by climate variation or soil contamination (e.g., by heavy metals), and may produce pure products at high titer.

[0096] The modified host cells of the present disclosure may also provide a platform for the economical production of high-value naturally-occurring and non-naturally occurring cannabinoids, opiates, alkaloids, polyketides, terpenes, isoprenoids, terpenoids, antibiotics, and aldonic acids. The modified host cells also may provide for the production of different naturally-occurring and non-naturally occurring cannabinoids, opiates, alkaloids, polyketides, terpenes, isoprenoids, terpenoids, antibiotics, and aldonic acids for which no viable method of production exists.

Secretory Pathway

[0097] BBE and BBE-like polypeptides are often processed through the secretory pathway or system— a diverse set of transport and modification routes for cargo

polypeptides that can be different for different polypeptides and organisms. Secretory processing typically includes initial transport into the endoplasmic reticulum (ER), where a cargo polypeptide can undergo folding and refolding, disulfide-bond formation and isomerization, proline isomerization, flavin cofactor binding, various forms of glycosylation and trimming, and other modifications. From the ER, secretory cargo polypeptides can be targeted to various organelles or extracellular localizations where they can undergo additional processing. For many polypeptides, processing along the secretory pathway or system is crucial for their maturation. Functionally reconstituting the activity of BBE and BBE-like polypeptides in a heterologous host cell can require engineering the host cell’s secretory pathway and tuning the expression of the target polypeptide(s).

[0098] In eukaryotic cells, including yeast, polypeptides destined for the ER, the

Golgi, the vacuole and the extracellular space are processed and sorted through the secretory pathway (reviewed extensively in Barlowe and Miller, 2013; Delic et al. 2013). The process begins in the cytoplasm where translation of secretory polypeptide mRNAs is initiated. The polypeptide is then translocated to the ER, either co-translationally (via interaction between the ribosome-bound native polypeptide and the signal recognition particle (SRP) complex), or post-translationally (via the SEC63 complex with recruitment by cytosolic chaperones). In both cases, the polypeptide enters the ER through the SEC61 pore complex. As the polypeptide enters the ER, it interacts first with the chaperone KAR2, a luminal HSP70 ATPase that assists with the translocation and folding of the polypeptide. Signal sequence processing by the Signal Peptidase Complex (SPC) and the attachment of asparagine-linked (N-linked) glycosylations by the oligosaccharyltransferase (OST) enzymes also occur during translocation.

[0099] Once inside the ER, further maturation of the polypeptide occurs. Some polypeptides undergo O-linked glycosylation on serine and threonine residues and some are modified on their carboxy-terminus by attachment of a lipid-anchored

glycosylphosphatidylinositol (GPI) moiety. Proper disulfide bond formation is catalyzed by disulfide isomerases such as PDI1, which require oxidizing equivalents from polypeptides such as EROl or ERV2. KAR2 continues to play a chaperone role in maturation and peptidyl-prolyl isomerases such as CPR5 are another class of chaperone that assists with polypeptide folding at this stage. Processing of the initial l4-residue N-linked glycosylations by glucosidases such as ROT2 plays an important role in quality control, and the polypeptide may enter a calnexin (CNEl)-dependent folding cycle, where improperly folded

polypeptides are modified such that they remain bound to CNE1, while properly folded substrates acquire a specific glycosylation pattern that allows exit from the cycle. In the case of terminally misfolded polypeptides, the glycosylations are trimmed by the mannosidases MNS1 and HTM1, allowing interaction with the ER lectin YOS9 and targeting for ER- associated degradation (ERAD). ERAD comprises a series of pathways that remove misfolded polypeptides for ubquitination and degradation in the proteasome.

[00100] These complex ER functions and capabilities are regulated by a

transcriptional program known as the ETnfolded Protein Response (EIPR). Accumulation of unfolded polypeptides is initially sensed by association between the lumenal sensor polypeptides IRE1 and KAR2, formation of oligomers and activation of the kinase and ribonuclease activity of IRE1, which splices HAC1 mRNA into the mature form. The transcription factor HAC1 is a strong activator of genes containing EIPR elements in their regulatory regions, and in response to ER stress it upregulates functions that save as well as destroy misfolded polypeptides.

[00101] Following maturation in the ER, secretory forward traffic involves assembly (via interaction with SEC24) into COPII-protein complex coated transport vesicles that cross the cytoplasm to fuse with the Golgi via the activity of SNARE complexes. Inside the Golgi, further processing of the glycosylation chain and/or proteolytic processing may occur.

Polypeptides bearing an ER retention signal (HDEL or KDEL signal) may be returned to the ER in COPI-coated vesicles, a process known as retrograde transport. Polypeptides destined for the vacuole may be routed to the endosome by the sorting receptor PEP1. Finally, polypeptides destined for the extracellular space move to the trans-face of the Golgi and are tethered to the plasma membrane by the exocyst, where SNARE proteins are once again involved in membrane fusion and release of the secreted polypeptide.

[00102] Many BBE and BBE-like polypeptides contain one or more disulfide bridges and/or one or more glycosylation sites, features that pose a challenge for expression in yeast. In plants, for example, many BBE and BBE-like polypeptides are processed through the secretory pathway in specialized cells and tissues, e.g., glandular trichomes, to install these post-translational modifications. As disclosed herein, engineering of the yeast secretory pathway may achieve substantial BBE and BBE-like polypeptide expression, function, and solubilization in yeast.

General Information

[00103] In certain aspects, the practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature:“ Molecular Cloning: A Laboratory Manual ,” second edition (Sambrook et ak, 1989);“ Oligonucleotide Synthesis’’ (M. J. Gait, ed., 1984);“ Animal Cell Culture” (R. I. Freshney, ed., 1987);“Methods in Enzymology” (Academic Press, Inc.);“Current Protocols in Molecular Biology” (F. M. Ausubel et al., eds., 1987, and periodic updates);“ PCR : The Polymerase Chain Reaction,” (Mullis et al., eds., 1994). Singleton et al., Dictionary of Microbiology andMolecular Biology 2nd ed., J. Wiley & Sons (New York, N.Y. 1994), and March, Advanced Organic Chemistry Reactions, Mechanisms and Structure 4th ed., John Wiley & Sons (New York, N.Y. 1992), provide one skilled in the art with a general guide to many of the terms used in the present application.

[00104] The term“nucleic acid” or“nucleic acids” used herein, may refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides. Thus, this term may include, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, genes, synthetic DNA or RNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other naturally-occurring, chemically or biochemically modified, non- naturally-occurring, or derivatized nucleotide bases.

[00105] The terms“peptide,”“polypeptide,” and“protein” may be used

interchangeably herein, and may refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids and chemically or biochemically modified or derivatized amino acids. The polypeptides disclosed herein may include full- length polypeptides, fragments of polypeptides, truncated polypeptides, fusion polypeptides, or polypeptides having modified peptide backbones. The polypeptides disclosed herein may also be variants differing from a specifically recited“reference” polypeptide (e.g., a wild- type polypeptide) by amino acid insertions, deletions, mutations, and/or substitutions.

[00106] As used herein, the term“heterologous” may refer to what is not normally found in nature. The term“heterologous nucleotide sequence” or the term“heterologous nucleic acid” may refer to a nucleic acid or nucleotide sequence not normally found in a given cell in nature. A heterologous nucleotide sequence may be: (a) foreign to its host cell (i.e., is“exogenous” to the cell); (b) naturally found in the host cell (i.e.,“endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); (c) be naturally found in the host cell but positioned outside of its natural locus; or (d) be naturally found in the host cell, but with introns removed or added. A heterologous nucleic acid may be: (a) foreign to its host cell (i.e., is“exogenous” to the cell); (b) naturally found in the host cell (i.e.,“endogenous”) but present at an unnatural quantity in the cell (i.e., greater or lesser quantity than naturally found in the host cell); or (c) be naturally found in the host cell but positioned outside of its natural locus. In some embodiments, a heterologous nucleic acid may comprise a codon-optimized nucleotide sequence. A codon-optimized nucleotide sequence may be an example of a heterologous nucleotide sequence. In some embodiments, the heterologous nucleic acids disclosed herein may comprise nucleotide sequences that encode a polypeptide disclosed herein, such as a cannabinoid synthase polypeptide, but do not comprise nucleotide sequences that do not encode the polypeptide disclosed herein (e.g., vector sequences, promoters, enhancers, upstream or downstream elements). In some embodiments, the heterologous nucleic acids disclosed herein may comprise nucleotide sequences encoding a polypeptide disclosed herein, such as a cannabinoid synthase polypeptide, along with nucleotide sequences that do not encode the polypeptide disclosed herein (e.g., vector sequences, promoters, enhancers, upstream or downstream elements). In some embodiments, the nucleic acids disclosed herein are heterologous.

[00107] The term“heterologous enzyme” or“heterologous polypeptide” may refer to an enzyme or polypeptide that is not normally found in a given cell in nature. The term encompasses an enzyme or polypeptide that is: (a) exogenous to a given cell (i.e., encoded by a nucleic acid that is not naturally present in the host cell or not naturally present in a given context in the host cell); or (b) naturally found in the host cell (e.g., the enzyme or polypeptide is encoded by a nucleic acid that is endogenous to the cell) but that is produced in an unnatural amount (e.g., greater or lesser than that naturally found) in the host cell. For example, a heterologous polypeptide may include a mutated version of a polypeptide naturally occurring in a host cell.

[00108] As used herein, the term“one or more heterologous nucleic acids” or“one or more heterologous nucleotide sequences” may refer to heterologous nucleic acids comprising one or more nucleotide sequences encoding one or more polypeptides. In some embodiments, the one or more heterologous nucleic acids may comprise a nucleotide sequence encoding one polypeptide. In other embodiments, the one or more heterologous nucleic acids may comprise nucleotide sequences encoding more than one polypeptide. In certain such embodiments, the nucleotide sequences encoding the more than one polypeptide may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof. In some embodiments, the one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of the same polypeptide. In certain such embodiments, the nucleotide sequences encoding the multiple copies of the same polypeptide may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof. In some embodiments, the one or more heterologous nucleic acids may comprise nucleotide sequences encoding multiple copies of different polypeptides. In certain such embodiments, the nucleotide sequences encoding the multiple copies of the different polypeptides may be present on the same heterologous nucleic acid or on different heterologous nucleic acids, or combinations thereof.

[00109] “Operably linked” may refer to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, control sequences operably linked to a coding sequence are capable of effecting the expression of the coding sequence. The control sequences need not be contiguous with the coding sequence, so long as they function to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked” to the coding sequence.

[00110] “Isolated” may refer to polypeptides or nucleic acids that are substantially or essentially free from components that normally accompany them in their natural state.

An isolated polypeptide or nucleic acid may be other than in the form or setting in which it is found in nature. Isolated polypeptides and nucleic acids therefore may be distinguished from the polypeptides and nucleic acids as they exist in natural cells. An isolated nucleic acid or polypeptide may be purified from one or more other components in a mixture with the isolated nucleic acid or polypeptide, if such components are present.

[00111] A“modified host cell” (also may be referred to as a“recombinant host cell”) may refer to a host cell into which has been introduced a nucleic acid (e.g., a heterologous nucleic acid), e.g., an expression vector or construct. For example, a modified eukaryotic host cell may be produced through introduction into a suitable eukaryotic host cell of a nucleic acid (e.g., a heterologous nucleic acid).

[00112] “Plant secretory tissue” may refer to cells or multicellular structures that secrete a variety of chemical compounds. Multicellular secretory tissues are classified as laticiferous or glandular. Laticiferous tissues secrete latex. Glandular tissues, including the trichome, secrete and accumulate a variety of different metabolites.

[00113] In some embodiments, conservative substitutions may be made in the amino acid sequence of a polypeptide without disrupting the three-dimensional structure or function of the polypeptide. Conservative substitutions may be accomplished by the skilled artisan by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Additionally, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating amino acid residues that have been mutated between species without altering the basic functions of the encoded proteins. The term“conservative amino acid substitution” may refer to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains may consist of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains may consist of serine and threonine; a group of amino acids having amide containing side chains may consist of asparagine and glutamine; a group of amino acids having aromatic side chains may consist of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains may consist of lysine, arginine, and histidine; a group of amino acids having acidic side chains may consist of glutamate and aspartate; and a group of amino acids having sulfur containing side chains may consist of cysteine and methionine.

Exemplary conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

[00114] A polynucleotide or polypeptide has a certain percent“sequence identity” to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different manners. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/ebi.ac.uk/ Tools/msa/muscle/mafft.cbrc.jp/alignment/software/. See, e.g., Altschul et al. (1990), J. Mol. Biol. 215:403-10.

[00115] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00116] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also

encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. [00117] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[00118] It must be noted that as used herein and in the appended claims, the singular forms“a,”“an,” and“the” may include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to“the modified host cell” may include reference to one or more modified host cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element.

As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as“solely,”“only” and the like in connection with the recitation of claim elements, or use of a“negative” limitation.

[00119] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

Modified Host Cells for Expressing BBE and BBE-Like Polypeptides

[00120] The present disclosure provides modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, such as a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue; a BBE or BBE-like polypeptide produced in a fungal cell; a BBE or BBE-like polypeptide produced in a bacterial cell; cannabinoid synthase polypeptides, such as a tetrahydrocannabinolic acid synthase polypeptide, a cannabichromenic acid synthase polypeptide, or a cannabidiolic acid synthase polypeptide; a BBE polypeptide from Eschscholzia californica ; a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacunr, a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans a daurichromenic acid synthase polypeptide from Rhododendron dauricum or a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila. In certain such embodiments, the modified host cells of the disclosure are for expressing the BBE or BBE-like polypeptide. In some embodiments, the nucleotide sequence encoding a BBE or BBE-like polypeptide is codon-optimized.

Exemplary Berberine bridge enzyme (BBE) or BBE-like polypeptides produced in a plant cell within a plant secretory tissue include, but are not limited to, cannabinoid synthase polypeptides from Cannabis saliva, such as a tetrahydrocannabinolic acid synthase polypeptide, a cannabichromenic acid synthase polypeptide, or a cannabidiolic acid synthase polypeptide; a BBE polypeptide from Eschscholzia californica ; a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacunr, and a daurichromenic acid synthase polypeptide from Rhododendron dauricum. Exemplary Berberine bridge enzyme (BBE) or BBE-like polypeptides produced in a fungal cell include, but are not limited to, a

xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. Exemplary Berberine bridge enzyme (BBE) or BBE-like polypeptides produced in a bacterial cell include, but are not limited to, a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans .

[00121] The disclosure also provides nucleic acids (e.g., heterologous nucleic acids), which can be introduced into microorganisms (e.g., modified host cells), resulting in expression or overexpression of one or more polypeptides. In some embodiments, these nucleic acids comprise codon-optimized nucleotide sequences.

[00122] To express a BBE or BBE-like polypeptide, the modified host cells may express or overexpress heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide. In some embodiments, the nucleotide sequence encoding a BBE or BBE-like polypeptide is codon-optimized. In some embodiments, the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprise one or more modifications to modulate the expression of one or more secretory pathway polypeptides. The one or more modifications to modulate the expression of one or more secretory pathway polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure for expressing a BBE or BBE-like polypeptide comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, resulting in expression or overexpression of the one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized. In some embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, reducing or eliminating the expression of the one or more secretory pathway polypeptides. In certain such embodiments, the modified host cells comprise a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the modified host cells comprise a downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00123] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

Secretory Pathway Modifications

[00124] Secretory pathway polypeptides with modulated expression in the modified host cells of the disclosure may include, but are not limited to: chaperone or co-chaperone polypeptides, flavin adenine dinucleotide (FAD) synthetase polypeptides, glycosidase polypeptides, protein disulfide isomerase polypeptides, thiol oxidase polypeptides, polypeptides involved in unfolded protein response (UPR), polypeptides involved in regulation of lipid metabolism, and vacuolar proteinase polypeptides. Expression of secretory pathway polypeptides may be modulated by introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and/or deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides in a host cell. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.

[00125] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more of the following genes: a ROT2 gene, a MNS1 gene, an OPI1 gene, a PEP4 gene, a PRC1 gene, or a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of one or more of the following genes: a ROT2 gene, a MNS1 gene, an OPI1 gene, a PEP4 gene, a PRC1 gene, or a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a

downregulation of one or more of the following genes: a ROT2 gene, a MNS1 gene, an OPI1 gene, a PEP4 gene, a PRC1 gene, or a PRB1 gene.

[00126] The secretory pathway polypeptides and the nucleotide sequences the secretory pathway polypeptides may be derived from any suitable source, for example, bacteria, yeast, fungi, algae, human, plant, or mouse. In some embodiments, the secretory pathway polypeptides and the nucleotide sequences encoding the secretory pathway polypeptides may be derived from Pichia pastoris (now known as Komagataella phaffii ), Pichia fmlandica , Pichia trehalophila , Pichia koclamae , Pichia memhranaefaciens , Pichia opuntiae , Pichia thermotolerans , Pichia salictaria , Pichia guercuum , Pichia pijperi , Pichia stiptis , Pichia methanolica , Pichia sp., Saccharomyces cerevisiae, Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta ), Yarrowia lipolytica , Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pomhe ,

Scheffer somyces stipites, Dekkera hruxellensis , Blastobotrys adeninivorans (formerly Arxula adeninivorans ), Candida albicans , Aspergillus nidulans , Aspergillus niger , Aspergillus oryzae, I richoderma reese /, Chrysosporium lucknowense , Fusarium sp., Fusarium gramineum , Fusarium venenatum , Neurospora crassa, and the like. In some embodiments, the disclosure also encompasses orthologous genes encoding the secretory pathway polypeptides disclosed herein. Exemplary secretory pathway polypeptides disclosed herein may also include a full-length secretory pathway polypeptide, a fragment of a secretory pathway polypeptide, a variant of a secretory pathway polypeptide, a truncated secretory pathway polypeptide, or a fusion polypeptide that has at least one activity of a secretory pathway polypeptide. The disclosure also provides for nucleotide sequences encoding secretory pathway polypeptides, such as, a full-length secretory pathway polypeptide, a fragment of a secretory pathway polypeptide, a variant of a secretory pathway polypeptide, a truncated secretory pathway polypeptide, or a fusion polypeptide that has at least one activity of a secretory pathway polypeptide. In some embodiments, the nucleotide sequences encoding the secretory pathway polypeptides are codon-optimized.

Chaperone and Co-Chaperone Polypeptides

[00127] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more chaperone or co-chaperone polypeptides. The one or more modifications to modulate the expression of one or more chaperone or co-chaperone polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides and/or deletion or downregulation of one or more genes encoding one or more chaperone or co-chaperone polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, resulting in expression or overexpression of the one or more chaperone or co- chaperone polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more chaperone or co-chaperone polypeptides, reducing or eliminating the expression of the one or more chaperone or co-chaperone polypeptides.

[00128] Exemplary chaperone or co-chaperone polypeptides disclosed herein may also include a full-length chaperone or co-chaperone polypeptide, a fragment of a chaperone or co-chaperone polypeptide, a variant of a chaperone or co-chaperone polypeptide, a truncated chaperone or co-chaperone polypeptide, or a fusion polypeptide that has at least one activity of a chaperone or co-chaperone polypeptide.

[00129] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more chaperone or co-chaperone polypeptides including, but not limited to, cytoplasmic chaperone or co-chaperone polypeptides (e.g., a SSA1 polypeptide, a SSB1 polypeptide, a PFD2s polypeptide, or a CNS1 polypeptide), a peptidyl-prolyl isomerase polypeptide (e.g., a CPR5 polypeptide or a FPR1 polypeptide), and ER chaperone polypeptides (e.g., protein folding chaperone polypeptides, a KAR2 polypeptide, a LHS1 polypeptide, a ROT1 polypeptide, a JEM1 polypeptide, a CNS1 polypeptide, a CNE1 polypeptide, a SCJ1 polypeptide, or a SIL1 polypeptide).

[00130] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co- chaperone polypeptides are selected from the group consisting of: a KAR2 polypeptide, a JEM1 polypeptide, a LHSl polypeptide, a SIS1 polypeptide, a SSB1 polypeptide, a CNE1 polypeptide, a CNS1 polypeptide, a PFD2s polypeptide, a PFDl polypeptide, a SSA1 polypeptide, a YDJ1 polypeptide, a SIL1 polypeptide, a SCJ1 polypeptide, a ROTl polypeptide, a FPR1 polypeptide, and a CPR5 polypeptide.

[00131] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co- chaperone polypeptides are selected from the group consisting of chaperone or co-chaperone polypeptides comprising the amino acid sequences set forth in: SEQ ID NO: 12 (a KAR2 polypeptide), SEQ ID NO: 14 (a JEM1 polypeptide), SEQ ID NO: 16 (a LHS1 polypeptide), SEQ ID NO: 18 (a SIS1 polypeptide), SEQ ID NO:4 (a SSB1 polypeptide), SEQ ID NO:20 (a CNE1 polypeptide), SEQ ID NO:8 (a CNS1 polypeptide), SEQ ID NO: 10 (a PFD2s polypeptide), SEQ ID NO:64 (a PFD1 polypeptide), SEQ ID NO:2 (a SSA1 polypeptide), SEQ ID NO:6 (a YDJ1 polypeptide), SEQ ID NO:22 (a SIL1 polypeptide), SEQ ID NO:24 (a SCJ1 polypeptide), SEQ ID NO:28 (a ROT1 polypeptide), SEQ ID NO:32 (a FPR1 polypeptide), and SEQ ID NO:30 (a CPR5 polypeptide).

[00132] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co- chaperone polypeptides are selected from the group consisting of chaperone or co-chaperone polypeptides comprising the amino acid sequences set forth in: SEQ ID NO: 12 (a KAR2 polypeptide), SEQ ID NO: 14 (a JEM1 polypeptide), SEQ ID NO: 16 (a LHS1 polypeptide), SEQ ID NO: 18 (a SIS1 polypeptide), SEQ ID NO:4 (a SSB1 polypeptide), SEQ ID NO:20 (a CNE1 polypeptide), SEQ ID NO:8 (a CNS1 polypeptide), SEQ ID NO: 10 (a PFD2s polypeptide), SEQ ID NO:64 (a PFD1 polypeptide), SEQ ID NO:2 (a SSA1 polypeptide), SEQ ID NO:6 (a YDJ1 polypeptide), SEQ ID NO:22 (a SIL1 polypeptide), SEQ ID NO:24 (a SCJ1 polypeptide), SEQ ID NO:28 (a ROT1 polypeptide), SEQ ID NO:32 (a FPR1 polypeptide), and SEQ ID NO:30 (a CPR5 polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.

[00133] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co- chaperone polypeptides are selected from the group consisting of chaperone or co-chaperone polypeptides comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: SEQ ID NO: 12 (a KAR2 polypeptide), SEQ ID NO: 14 (a JEM1 polypeptide), SEQ ID NO: 16 (a LHS1 polypeptide), SEQ ID NO: 18 (a SIS1 polypeptide), SEQ ID NO:4 (a SSB1 polypeptide), SEQ ID NO:20 (a CNE1 polypeptide), SEQ ID NO:8 (a CNS1 polypeptide), SEQ ID NO: 10 (a PFD2s polypeptide), SEQ ID NO:64 (a PFD1 polypeptide), SEQ ID NO:2 (a SSA1 polypeptide), SEQ ID NO:6 (a YDJ1 polypeptide), SEQ ID NO:22 (a SIL1 polypeptide), SEQ ID NO:24 (a SCJ1 polypeptide), SEQ ID NO:28 (a ROT1 polypeptide), SEQ ID NO:32 (a FPR1 polypeptide), and SEQ ID NO:30 (a CPR5 polypeptide).

[00134] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co-chaperone polypeptides are selected from the group consisting of a KAR2 polypeptide, a JEM1 polypeptide, a LHSl polypeptide, a SIS1 polypeptide, a SSB1 polypeptide, a CNE1 polypeptide, a CNS1 polypeptide, a PFD2s polypeptide, a PFDl polypeptide, a SSA1 polypeptide, a YDJ1 polypeptide, a SIL1 polypeptide, a SCJ1 polypeptide, a ROTl polypeptide, a FPR1 polypeptide, and a CPR5 polypeptide.

[00135] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co-chaperone polypeptides are selected from the group consisting of chaperone or co-chaperone polypeptides comprising the amino acid sequences set forth in: SEQ ID NO: 12 (a KAR2 polypeptide), SEQ ID NO: 14 (a JEM1 polypeptide), SEQ ID NO: 16 (a LHS1 polypeptide), SEQ ID NO: 18 (a SIS1 polypeptide), SEQ ID NO:4 (a SSB1 polypeptide), SEQ ID NO:20 (a CNE1 polypeptide), SEQ ID NO:8 (a CNS1 polypeptide), SEQ ID NO: 10 (a PFD2s polypeptide), SEQ ID NO:64 (a PFD1 polypeptide), SEQ ID NO:2 (a SSA1 polypeptide), SEQ ID NO:6 (a YDJ1 polypeptide), SEQ ID NO:22 (a SIL1 polypeptide), SEQ ID NO:24 (a SCJ1 polypeptide), SEQ ID NO:28 (a ROT1 polypeptide), SEQ ID NO:32 (a FPR1 polypeptide), and SEQ ID NO:30 (a CPR5 polypeptide).

[00136] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more chaperone or co-chaperone polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more chaperone or co-chaperone polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more chaperone or co-chaperone polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more chaperone or co-chaperone polypeptides.

[00137] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more chaperone or co- chaperone polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more chaperone or co-chaperone polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more chaperone or co-chaperone polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more chaperone or co-chaperone polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more chaperone or co-chaperone polypeptides.

[00138] In some embodiments, the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides are codon-optimized. In some embodiments, the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides are a spliced form with the intron removed of the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides. For example, the nucleotide sequence encoding the PFD2s polypeptide may be a spliced form that requires no additional splicing prior to translation.

[00139] In some embodiments, one or more chaperone or co-chaperone polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a chaperone or co-chaperone polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00140] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a chaperone or co-chaperone polypeptide, such as, a full-length chaperone or co-chaperone polypeptide, a fragment of a chaperone or co-chaperone polypeptide, a variant of a chaperone or co-chaperone polypeptide, a truncated chaperone or co-chaperone polypeptide, or a fusion polypeptide that has at least one activity of a chaperone or co-chaperone polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[00141] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:l 1 (encodes a KAR2 polypeptide), SEQ ID NO: l3 (encodes a JEM1 polypeptide), SEQ ID NO:l5 (encodes a LHSl polypeptide), SEQ ID NO: 17 (encodes a SIS1 polypeptide), SEQ ID NO:3 (encodes a SSB1 polypeptide), SEQ ID NO: 19 (encodes a CNE1 polypeptide), SEQ ID NO:7 (encodes a CNS1 polypeptide), SEQ ID NO: 9 (encodes a PFD2s polypeptide), SEQ ID NO: 63 (encodes a PFD1

polypeptide), SEQ ID NO: l (encodes a SSA1 polypeptide), SEQ ID NO:5 (encodes a YDJ1 polypeptide), SEQ ID NO:2l (encodes a SIL1 polypeptide), SEQ ID NO:23 (encodes a SCJ1 polypeptide), SEQ ID NO:27 (encodes a ROT1 polypeptide), SEQ ID NO:3 l (encodes a FPR1 polypeptide), and SEQ ID NO:29 (encodes a CPR5 polypeptide).

[00142] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:l 1 (encodes a KAR2 polypeptide), SEQ ID NO: l3 (encodes a JEM1 polypeptide), SEQ ID NO:l5 (encodes a LHSl polypeptide), SEQ ID NO: 17 (encodes a SIS1 polypeptide), SEQ ID NO:3 (encodes a SSB1 polypeptide), SEQ ID NO: 19 (encodes a CNE1 polypeptide), SEQ ID NO:7 (encodes a CNS1 polypeptide), SEQ ID NO: 9 (encodes a PFD2s polypeptide), SEQ ID NO: 63 (encodes a PFD1

polypeptide), SEQ ID NO: l (encodes a SSA1 polypeptide), SEQ ID NO:5 (encodes a YDJ1 polypeptide), SEQ ID NO:2l (encodes a SIL1 polypeptide), SEQ ID NO:23 (encodes a SCJ1 polypeptide), SEQ ID NO:27 (encodes a ROT1 polypeptide), SEQ ID NO:3 l (encodes a FPR1 polypeptide), and SEQ ID NO:29 (encodes a CPR5 polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.

[00143] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, the nucleotide sequences encoding the one or more chaperone or co-chaperone polypeptides are selected from the group consisting of nucleotide sequences having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to: SEQ ID NO: 11 (encodes a KAR2 polypeptide), SEQ ID NO: l3 (encodes a JEM1 polypeptide), SEQ ID NO:l5 (encodes a LHS1

polypeptide), SEQ ID NO: 17 (encodes a SIS1 polypeptide), SEQ ID NO: 3 (encodes a SSB1 polypeptide), SEQ ID NO: 19 (encodes a CNE1 polypeptide), SEQ ID NO:7 (encodes a CNS1 polypeptide), SEQ ID NO:9 (encodes a PFD2s polypeptide), SEQ ID NO:63 (encodes a PFD1 polypeptide), SEQ ID NO: l (encodes a SSA1 polypeptide), SEQ ID NO:5 (encodes a YDJ1 polypeptide), SEQ ID NO:2l (encodes a SIL1 polypeptide), SEQ ID NO:23 (encodes a SCJ1 polypeptide), SEQ ID NO:27 (encodes a ROT1 polypeptide), SEQ ID NO:3 l (encodes a FPRl polypeptide), and SEQ ID NO:29 (encodes a CPR5 polypeptide).

[00144] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more chaperone or co-chaperone polypeptides, the one or more chaperone or co-chaperone polypeptides are encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO: 11 (encodes a KAR2 polypeptide), SEQ ID NO: l3 (encodes a JEM1 polypeptide), SEQ ID NO:l5 (encodes a LHSl polypeptide), SEQ ID NO: 17 (encodes a SIS1 polypeptide), SEQ ID NO:3 (encodes a SSB1 polypeptide), SEQ ID NO: 19 (encodes a CNE1 polypeptide), SEQ ID NO:7 (encodes a CNS1 polypeptide),

SEQ ID NO: 9 (encodes a PFD2s polypeptide), SEQ ID NO: 63 (encodes a PFD1

Flavin Adenine Dinucleotide (FAD) Synthetase Polypeptides

[00145] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. The one or more modifications to modulate the expression of one or more flavin adenine dinucleotide (FAD) synthetase polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides and/or deletion or downregulation of one or more genes encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, resulting in expression or overexpression of the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, reducing or eliminating the expression of the one or more flavin adenine dinucleotide (FAD) synthetase

polypeptides. Flavin adenine dinucleotide (FAD) synthetase polypeptides catalyze adenylation of flavin mononucleotide (FMN) to form flavin adenine dinucleotide (FAD) coenzyme.

[00146] Exemplary flavin adenine dinucleotide (FAD) synthetase polypeptides disclosed herein may also include a full-length flavin adenine dinucleotide (FAD) synthetase polypeptide, a fragment of a flavin adenine dinucleotide (FAD) synthetase polypeptide, a variant of a flavin adenine dinucleotide (FAD) synthetase polypeptide, a truncated flavin adenine dinucleotide (FAD) synthetase polypeptide, or a fusion polypeptide that has at least one activity of a flavin adenine dinucleotide (FAD) synthetase polypeptide.

[00147] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a FAD1 polypeptide.

[00148] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a FAD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:26.

[00149] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a FAD1 polypeptide comprising the amino acid sequence set forth in: SEQ ID NO:26, or a conservatively substituted amino acid sequence thereof.

[00150] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a FAD1 polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:26. [00151] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a FADl polypeptide.

[00152] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a FAD1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:26.

[00153] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. In some

embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more flavin adenine dinucleotide (FAD) synthetase polypeptides.

[00154] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more flavin adenine dinucleotide (FAD) synthetase

polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more flavin adenine dinucleotide (FAD) synthetase polypeptides.

[00155] In some embodiments, the nucleotide sequences encoding the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides are codon-optimized.

[00156] In some embodiments, one or more flavin adenine dinucleotide (FAD) synthetase polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a flavin adenine dinucleotide (FAD) synthetase polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00157] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a full-length flavin adenine

dinucleotide (FAD) synthetase polypeptide, such as, a full-length flavin adenine dinucleotide (FAD) synthetase polypeptide, a fragment of a flavin adenine dinucleotide (FAD) synthetase polypeptide, a variant of a flavin adenine dinucleotide (FAD) synthetase polypeptide, a truncated flavin adenine dinucleotide (FAD) synthetase polypeptide, or a fusion polypeptide that has at least one activity of a flavin adenine dinucleotide (FAD) synthetase polypeptide.

In some embodiments, the nucleotide sequence is codon-optimized.

[00158] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the nucleotide sequence encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a nucleotide sequence encoding a FAD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:25.

[00159] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the nucleotide sequence encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a nucleotide sequence encoding a FAD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:25, or a codon degenerate nucleotide sequence thereof.

[00160] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the nucleotide sequence encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides is a nucleotide sequence encoding a FAD1 polypeptide, wherein the nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:25.

[00161] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides are encoded by a nucleotide sequence encoding a FAD1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:25. Glycosidase Polypeptides

[00162] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more glycosidase polypeptides. The one or more modifications to modulate the expression of one or more glycosidase polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides and/or deletion or downregulation of one or more genes encoding one or more glycosidase polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, resulting in expression or overexpression of the one or more glycosidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more glycosidase polypeptides, reducing or eliminating the expression of the one or more glycosidase polypeptides. Glycosidase polypeptides (also may be referred to as glycoside hydrolase polypeptides or glycosyl hydrolase polypeptides) catalyze the hydrolysis of glycosidic bonds in carbohydrates.

[00163] Exemplary glycosidase polypeptides disclosed herein may also include a full- length glycosidase polypeptide, a fragment of a glycosidase polypeptide, a variant of a glycosidase polypeptide, a truncated glycosidase polypeptide, or a fusion polypeptide that has at least one activity of a glycosidase polypeptide.

[00164] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more glycosidase polypeptides including, but not limited to, glucosidase polypeptides (e.g., a ROT2 polypeptide) and mannosidase

polypeptides (e.g., a MNSl polypeptide).

[00165] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are selected from the group consisting of: a ROT2 polypeptide and a MNS1 polypeptide.

[00166] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are selected from the group consisting of glycosidase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:66 (a ROT2 polypeptide) and SEQ ID NO:68 (a MNS1

polypeptide). [00167] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are selected from the group consisting of glycosidase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:66 (a ROT2 polypeptide) and SEQ ID NO:68 (a MNS1

polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.

[00168] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are selected from the group consisting of glycosidase polypeptides comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: SEQ ID NO:66 (a ROT2 polypeptide) and SEQ ID NO:68 (a MNS1 polypeptide).

[00169] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are selected from the group consisting of a ROT2 polypeptide and a MNS1 polypeptide.

[00170] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are selected from the group consisting of glycosidase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:66 (a ROT2 polypeptide) and SEQ ID NO:68 (a MNS1 polypeptide).

[00171] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a glycosidase polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more glycosidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more glycosidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more glycosidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more glycosidase polypeptides.

[00172] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more glycosidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more glycosidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more glycosidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more glycosidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more glycosidase polypeptides.

[00173] In some embodiments, the nucleotide sequences encoding the one or more glycosidase polypeptides are codon-optimized.

[00174] In some embodiments, one or more glycosidase polypeptides are

overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more glycosidase polypeptides to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a glycosidase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a glycosidase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a glycosidase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a glycosidase polypeptide. In some

embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a glycosidase polypeptide. In some

embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a glycosidase polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00175] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a glycosidase polypeptide, such as, a full-length glycosidase polypeptide, a fragment of a glycosidase polypeptide, a variant of a glycosidase polypeptide, a truncated glycosidase polypeptide, or a fusion polypeptide that has at least one activity of a glycosidase polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[00176] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the nucleotide sequences encoding the one or more glycosidase polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:65 (encodes a ROT2 polypeptide) and SEQ ID NO:67 (encodes a MNS1 polypeptide).

[00177] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the nucleotide sequences encoding the one or more glycosidase polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:65 (encodes a ROT2 polypeptide) and SEQ ID NO:67 (encodes a MNS1 polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.

[00178] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, the nucleotide sequences encoding the one or more glycosidase polypeptides are selected from the group consisting of nucleotide sequences having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to: SEQ ID NO:65 (encodes a ROT2 polypeptide) and SEQ ID NO:67 (encodes a MNSl polypeptide). [00179] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more glycosidase polypeptides, the one or more glycosidase polypeptides are encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO: 65 (encodes a ROT2 polypeptide) and SEQ ID NO: 67 (encodes a MNS1 polypeptide).

[00180] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more of the following genes: a ROT2 gene or a MNS1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of one or more of the following genes: a ROT2 gene or a MNS1 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of one or more of the following genes: a ROT2 gene or a MNS1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a ROT2 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a MNS1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a MNS1 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a MNS1 gene.

Protein Disulfide Isomerase Polypeptides

[00181] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more protein disulfide isomerase polypeptides. The one or more modifications to modulate the expression of one or more protein disulfide isomerase polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides and/or deletion or downregulation of one or more genes encoding one or more protein disulfide isomerase polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, resulting in expression or overexpression of the one or more protein disulfide isomerase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more protein disulfide isomerase polypeptides, reducing or eliminating the expression of the one or more protein disulfide isomerase polypeptides. Protein disulfide isomerase polypeptides may catalyze the formation and breakage of disulfide bonds between cysteine residues within polypeptides as they fold.

[00182] Exemplary protein disulfide isomerase polypeptides disclosed herein may also include a full-length protein disulfide isomerase polypeptide, a fragment of a protein disulfide isomerase polypeptide, a variant of a protein disulfide isomerase polypeptide, a truncated protein disulfide isomerase polypeptide, or a fusion polypeptide that has at least one activity of a protein disulfide isomerase polypeptide.

[00183] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides is a PDI1 polypeptide.

[00184] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides is a PDI1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:38.

[00185] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides is a PDI1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:38, or a conservatively substituted amino acid sequence thereof.

[00186] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides is a PDI1 polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:38.

[00187] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides is a PDI1 polypeptide.

[00188] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides is a PDI1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:38.

[00189] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more protein disulfide isomerase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more protein disulfide isomerase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more protein disulfide isomerase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more protein disulfide isomerase polypeptides.

[00190] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more protein disulfide isomerase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more protein disulfide isomerase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more protein disulfide isomerase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more protein disulfide isomerase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more protein disulfide isomerase polypeptides.

[00191] In some embodiments, the nucleotide sequences encoding the one or more protein disulfide isomerase polypeptides are codon-optimized. [00192] In some embodiments, one or more protein disulfide isomerase polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more protein disulfide isomerase polypeptides to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a protein disulfide isomerase polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00193] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a protein disulfide isomerase polypeptide, such as, a full-length protein disulfide isomerase polypeptide, a fragment of a protein disulfide isomerase polypeptide, a variant of a protein disulfide isomerase polypeptide, a truncated protein disulfide isomerase polypeptide, or a fusion polypeptide that has at least one activity of a protein disulfide isomerase polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[00194] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more protein disulfide isomerase polypeptides, the nucleotide sequence encoding one or more protein disulfide isomerase polypeptides is a nucleotide sequence encoding a PDI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:37. [00195] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more protein disulfide isomerase polypeptides, the nucleotide sequence encoding one or more protein disulfide isomerase polypeptides is a nucleotide sequence encoding a PDI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:37, or a codon degenerate nucleotide sequence thereof.

[00196] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more protein disulfide isomerase polypeptides, the nucleotide sequence encoding one or more protein disulfide isomerase polypeptides is a nucleotide sequence encoding a PDI1 polypeptide, wherein the nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:37.

[00197] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more protein disulfide isomerase polypeptides, the one or more protein disulfide isomerase polypeptides are encoded by a nucleotide sequence encoding a PDI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:37.

Thiol Oxidase Polypeptides

[00198] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more thiol oxidase polypeptides. The one or more modifications to modulate the expression of one or more thiol oxidase polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides and/or deletion or downregulation of one or more genes encoding one or more thiol oxidase polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, resulting in expression or overexpression of the one or more thiol oxidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more thiol oxidase polypeptides, reducing or eliminating the expression of the one or more thiol oxidase polypeptides. Thiol oxidase polypeptides transfer electrons from reduced protein disulfide isomerase polypeptides to a terminal acceptor such as oxygen.

[00199] Exemplary thiol oxidase polypeptides disclosed herein may also include a full-length thiol oxidase polypeptide, a fragment of a thiol oxidase polypeptide, a variant of a thiol oxidase polypeptide, a truncated thiol oxidase polypeptide, or a fusion polypeptide that has at least one activity of a thiol oxidase polypeptide.

[00200] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are selected from the group consisting of: an EROl polypeptide and an ERV2 polypeptide. The EROl and ERV2 polypeptides may serve as partners to the PDI1 polypeptide, a protein disulfide isomerase polypeptide.

[00201] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are selected from the group consisting of thiol oxidase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:34 (an EROl polypeptide) and SEQ ID NO:36 (an ERV2 polypeptide).

[00202] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are selected from the group consisting of thiol oxidase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:34 (an EROl polypeptide) and SEQ ID NO:36 (an ERV2 polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.

[00203] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are selected from the group consisting of thiol oxidase polypeptides comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: SEQ ID NO:34 (an EROl polypeptide) and SEQ ID NO:36 (an ERV2 polypeptide).

[00204] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are selected from the group consisting of an EROl polypeptide and an ERV2 polypeptide.

[00205] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are selected from the group consisting of thiol oxidase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:34 (an EROl polypeptide) and SEQ ID NO:36 (an ERV2 polypeptide).

[00206] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a thiol oxidase polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more thiol oxidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more thiol oxidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more thiol oxidase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more thiol oxidase polypeptides.

[00207] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more thiol oxidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more thiol oxidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more thiol oxidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more thiol oxidase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more thiol oxidase polypeptides.

[00208] In some embodiments, the nucleotide sequences encoding the one or more thiol oxidase polypeptides are codon-optimized.

[00209] In some embodiments, one or more thiol oxidase polypeptides are

overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more thiol oxidase polypeptides to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a thiol oxidase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a thiol oxidase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a thiol oxidase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a thiol oxidase polypeptide. In some

embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a thiol oxidase polypeptide. In some

embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a thiol oxidase polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00210] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a thiol oxidase polypeptide, such as, a full-length thiol oxidase polypeptide, a fragment of a thiol oxidase polypeptide, a variant of a thiol oxidase polypeptide, a truncated thiol oxidase polypeptide, or a fusion polypeptide that has at least one activity of a thiol oxidase polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[00211] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the nucleotide sequences encoding the one or more thiol oxidase polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:33 (encodes an EROl polypeptide) and SEQ ID NO:35 (encodes an ERV2 polypeptide).

[00212] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the nucleotide sequences encoding the one or more thiol oxidase polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:33 (encodes an EROl polypeptide) and SEQ ID NO:35 (encodes an ERV2 polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.

[00213] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, the nucleotide sequences encoding the one or more thiol oxidase polypeptides are selected from the group consisting of nucleotide sequences having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to: SEQ ID NO:33 (encodes an EROl polypeptide) and SEQ ID NO:35 (encodes an ERV2 polypeptide).

[00214] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more thiol oxidase polypeptides, the one or more thiol oxidase polypeptides are encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:33 (encodes an EROl polypeptide) and SEQ ID NO:35 (encodes an ERV2 polypeptide).

Polypeptides Involved in Unfolded Protein Response (UPR)

[00215] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more polypeptides involved in unfolded protein response. The one or more modifications to modulate the expression of one or more polypeptides involved in unfolded protein response may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response and/or deletion or downregulation of one or more genes encoding one or more polypeptides involved in unfolded protein response in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, resulting in expression or overexpression of the one or more polypeptides involved in unfolded protein response. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in unfolded protein response, reducing or eliminating the expression of the one or more polypeptides involved in unfolded protein response.

[00216] Exemplary polypeptides involved in unfolded protein response disclosed herein may also include a full-length polypeptide involved in unfolded protein response, a fragment of a polypeptide involved in unfolded protein response, a variant of a polypeptide involved in unfolded protein response, a truncated polypeptide involved in unfolded protein response, or a fusion polypeptide that has at least one activity of a polypeptide involved in unfolded protein response.

[00217] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more polypeptides involved in unfolded protein response including, but not limited to, lumenal sensor polypeptides (e.g., an IRE1 polypeptide) and transcription factor polypeptides (e.g., a HACls polypeptide). In some embodiments, manipulation of the unfolded protein response might also be achieved in a synthetic fashion, e.g., by fusion of a transcriptional regulatory domain polypeptide, e.g., the VP 16 activator or the Gal4 activation domain to a DNA binding domain polypeptide that interacts with genomic unfolded protein response element (UPRE) nucleotide sequences to regulate gene expression. Such a DNA binding domain polypeptide could either be naturally occurring (e.g., found in the HACls polypeptide) or synthetic (e.g., a Zinc finger, TALEN or Cas9 system). Modulating the expression of one or more polypeptides involved in EIPR may prevent degradation of expressed Berberine Bridge Enzyme and Berberine Bridge Enzyme- like polypeptides.

[00218] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the one or more

polypeptides involved in unfolded protein response are selected from the group consisting of: an IRE1 polypeptide and a HACls polypeptide. In some embodiments, the IRE1 polypeptide is a fragment of an IRE1 polypeptide (e.g., missing the first 7 amino acids).

[00219] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the one or more polypeptides involved in unfolded protein response are selected from the group consisting of polypeptides involved in unfolded protein response comprising the amino acid sequences set forth in: SEQ ID NO:42 (an IRE1 polypeptide), SEQ ID NO:206 (a fragment IRE1 polypeptide) and SEQ ID NO:40 (a HACls polypeptide).

[00220] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the one or more

polypeptides involved in unfolded protein response are selected from the group consisting of polypeptides involved in unfolded protein response comprising the amino acid sequences set forth in: SEQ ID NO:42 (an IRE1 polypeptide), SEQ ID NO:206 (a fragment IRE1 polypeptide) and SEQ ID NO:40 (a HACls polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.

[00221] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the one or more

polypeptides involved in unfolded protein response are selected from the group consisting of polypeptides involved in unfolded protein response comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: SEQ ID NO:42 (an IRE1 polypeptide), SEQ ID NO:206 (a fragment IRE1 polypeptide) and SEQ ID NO:40 (a HACls polypeptide).

[00222] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in unfolded protein response, the one or more polypeptides involved in unfolded protein response are selected from the group consisting of an IRE1 polypeptide, and a HACls polypeptide.

[00223] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in unfolded protein response, the one or more polypeptides involved in unfolded protein response are selected from the group consisting of polypeptides involved in unfolded protein response comprising the amino acid sequences set forth in: SEQ ID NO:42 (an IRE1 polypeptide), SEQ ID NO:206 (a fragment IRE1 polypeptide) and SEQ ID NO:40 (a HACls polypeptide).

[00224] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more polypeptides involved in unfolded protein response. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more polypeptides involved in unfolded protein response. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more polypeptides involved in unfolded protein response. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more polypeptides involved in unfolded protein response.

[00225] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in unfolded protein response. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more polypeptides involved in unfolded protein response. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more polypeptides involved in unfolded protein response. In some embodiments, the modified host cells of the disclosure comprise a deletion or

downregulation of four or more genes encoding four or more polypeptides involved in unfolded protein response. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more polypeptides involved in unfolded protein response.

[00226] In some embodiments, the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response are codon-optimized. In some embodiments, the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response are a spliced form with the intron removed of the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response (e.g., the nucleotide sequence encoding the HACls polypeptide). In some embodiments, the intron from the native HAC1 locus is excised.

[00227] In some embodiments, one or more polypeptides involved in unfolded protein response are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response to a strong promoter. In some embodiments, the modified host cell has one copy of a

heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in unfolded protein response. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00228] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a polypeptide involved in unfolded protein response, such as a full-length polypeptide involved in unfolded protein response, a fragment of a polypeptide involved in unfolded protein response, a variant of a polypeptide involved in unfolded protein response, a truncated polypeptide involved in unfolded protein response, or a fusion polypeptide that has at least one activity of a polypeptide involved in unfolded protein response. In some embodiments, the nucleotide sequence is codon- optimized. [00229] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:4l (encodes an IRE1 polypeptide), SEQ ID NO:205 (encodes a fragment IRE1 polypeptide), and SEQ ID NO:39 (encodes a HACls polypeptide).

[00230] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:4l (encodes an IRE1 polypeptide), SEQ ID NO:205 (encodes a fragment IRE1 polypeptide), and SEQ ID NO:39 (encodes a HACls polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.

[00231] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response, the nucleotide sequences encoding the one or more polypeptides involved in unfolded protein response are selected from the group consisting of nucleotide sequences having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to: SEQ ID NO:4l

(encodes an IRE1 polypeptide), SEQ ID NO:205 (encodes a fragment IRE1 polypeptide), and SEQ ID NO:39 (encodes a HACls polypeptide).

[00232] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in unfolded protein response, the one or more polypeptides involved in unfolded protein response are encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:4l (encodes an IRE1

polypeptide), SEQ ID NO:205 (encodes a fragment IRE1 polypeptide), and SEQ ID NO:39 (encodes a HACls polypeptide). Vacuolar Proteinase Polypeptides

[00233] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more vacuolar proteinase polypeptides. The one or more modifications to modulate the expression of one or more vacuolar proteinase polypeptides may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides and/or deletion or downregulation of one or more genes encoding one or more vacuolar proteinase polypeptides in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, resulting in expression or overexpression of the one or more vacuolar proteinase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more vacuolar proteinase polypeptides, reducing or eliminating the expression of the one or more vacuolar proteinase polypeptides.

[00234] Exemplary vacuolar proteinase polypeptides disclosed herein may also include a full-length vacuolar proteinase polypeptide, a fragment of a vacuolar proteinase polypeptide, a variant of a vacuolar proteinase polypeptide, a truncated vacuolar proteinase polypeptide, or a fusion polypeptide that has at least one activity of a vacuolar proteinase polypeptide.

[00235] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are selected from the group consisting of: a PEP4 polypeptide, a PRC1 polypeptide, and a PRBl polypeptide.

[00236] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are selected from the group consisting of vacuolar proteinase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:72 (a PEP4 polypeptide),

SEQ ID NO:82 (a PRC1 polypeptide), and SEQ ID NO:84 (a PRB1 polypeptide).

[00237] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are selected from the group consisting of vacuolar proteinase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:72 (a PEP4 polypeptide),

SEQ ID NO: 82 (a PRC1 polypeptide), and SEQ ID NO: 84 (a PRB1 polypeptide), or a conservatively substituted amino acid sequence of any of the foregoing.

[00238] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are selected from the group consisting of vacuolar proteinase polypeptides comprising amino acid sequences having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to: SEQ ID NO:72 (a PEP4 polypeptide), SEQ ID NO: 82 (a PRC1 polypeptide), and SEQ ID NO: 84 (a PRB1 polypeptide).

[00239] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are selected from the group consisting of a PEP4 polypeptide, a PRC1 polypeptide, and a PRB1 polypeptide.

[00240] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are selected from the group consisting of vacuolar proteinase polypeptides comprising the amino acid sequences set forth in: SEQ ID NO:72 (a PEP4 polypeptide), SEQ ID NO:82 (a PRC1 polypeptide), and SEQ ID NO:84 (a PRB1 polypeptide).

[00241] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more vacuolar proteinase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more vacuolar proteinase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more vacuolar proteinase polypeptides. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more vacuolar proteinase polypeptides.

[00242] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more vacuolar proteinase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more vacuolar proteinase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more vacuolar proteinase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more vacuolar proteinase polypeptides. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more vacuolar proteinase polypeptides.

[00243] In some embodiments, the nucleotide sequences encoding the one or more vacuolar proteinase polypeptides are codon-optimized.

[00244] In some embodiments, one or more vacuolar proteinase polypeptides are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more vacuolar proteinase polypeptides to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide.

In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a vacuolar proteinase polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00245] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a vacuolar proteinase polypeptide, such as a full-length vacuolar proteinase polypeptide, a fragment of a vacuolar proteinase polypeptide, a variant of a vacuolar proteinase polypeptide, a truncated vacuolar proteinase polypeptide, or a fusion polypeptide that has at least one activity of a vacuolar proteinase polypeptide. In some embodiments, the nucleotide sequence is codon-optimized.

[00246] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the nucleotide sequences encoding the one or more vacuolar proteinase polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:7l (encodes a PEP4 polypeptide), SEQ ID NO:8l (encodes a PRC1 polypeptide), and SEQ ID NO: 83 (encodes a PRB1 polypeptide).

[00247] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the nucleotide sequences encoding the one or more vacuolar proteinase polypeptides are selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:7l (encodes a PEP4 polypeptide), SEQ ID NO:8l (encodes a PRC1 polypeptide), and SEQ ID NO: 83 (encodes a PRB1 polypeptide), or a codon degenerate nucleotide sequence of any of the foregoing.

[00248] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, the nucleotide sequences encoding the one or more vacuolar proteinase polypeptides are selected from the group consisting of nucleotide sequences having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to: SEQ ID NO:7l (encodes a PEP4 polypeptide), SEQ ID NO:8l (encodes a PRC1 polypeptide), and SEQ ID NO: 83 (encodes a PRB1 polypeptide).

[00249] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more vacuolar proteinase polypeptides, the one or more vacuolar proteinase polypeptides are encoded by nucleotide sequences selected from the group consisting of nucleotide sequences set forth in: SEQ ID NO:7l (encodes a PEP4 polypeptide), SEQ ID NO: 81 (encodes a PRC1

polypeptide), and SEQ ID NO:83 (encodes a PRB1 polypeptide).

[00250] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more of the following genes: a PEP4 gene, a PRC1 gene, or a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of one or more of the following genes: a PEP4 gene, a PRC1 gene, or a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of one or more of the following genes: a PEP4 gene, a PRC1 gene, or a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a PEP4 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a PRC1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PRC1 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a PRC1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of a PRB1 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of a PRB1 gene.

Polypeptides Involved in Regulation of Lipid Metabolism

[00251] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more polypeptides involved in regulation of lipid metabolism. The one or more modifications to modulate the expression of one or more polypeptides involved in regulation of lipid metabolism may include introducing into a host cell one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism and/or deletion or downregulation of one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism in a host cell. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism, resulting in expression or overexpression of the one or more polypeptides involved in regulation of lipid metabolism. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, reducing or eliminating the expression of the one or more polypeptides involved in regulation of lipid metabolism.

[00252] Exemplary polypeptides involved in regulation of lipid metabolism disclosed herein may also include a full-length polypeptide involved in regulation of lipid metabolism, a fragment of a polypeptide involved in regulation of lipid metabolism, a variant of a polypeptide involved in regulation of lipid metabolism, a truncated polypeptide involved in regulation of lipid metabolism, or a fusion polypeptide that has at least one activity of a polypeptide involved in regulation of lipid metabolism.

[00253] Modified host cells of the disclosure may comprise one or more modifications to modulate the expression of one or more polypeptides involved in the regulation of lipid metabolism including, but not limited to, transcriptional regulator polypeptides (e.g., an OPI1 polypeptide).

[00254] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism is an OPI1 polypeptide.

[00255] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism is an OPI1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:70.

[00256] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism is an OPI1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:70, or a conservatively substituted amino acid sequence thereof. [00257] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism is an OPI1 polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:70.

[00258] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism is an OPI1 polypeptide.

[00259] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism is an OPI1 polypeptide comprising the amino acid sequence set forth in SEQ ID NO:70.

[00260] In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a polypeptide involved in regulation of lipid metabolism. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding two or more polypeptides involved in regulation of lipid metabolism. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding three or more polypeptides involved in regulation of lipid metabolism. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding four or more polypeptides involved in regulation of lipid metabolism. In some embodiments, a modified host cell of the present disclosure comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding five or more polypeptides involved in regulation of lipid metabolism.

[00261] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of two or more genes encoding two or more polypeptides involved in regulation of lipid metabolism. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of three or more genes encoding three or more polypeptides involved in regulation of lipid metabolism. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of four or more genes encoding four or more polypeptides involved in regulation of lipid metabolism. In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of five or more genes encoding five or more polypeptides involved in regulation of lipid metabolism.

[00262] In some embodiments, the nucleotide sequences encoding the one or more polypeptides involved in regulation of lipid metabolism are codon-optimized.

[00263] In some embodiments, one or more polypeptides involved in regulation of lipid metabolism are overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in regulation of lipid metabolism, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequences encoding the one or more polypeptides involved in regulation of lipid metabolism to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in regulation of lipid metabolism. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in regulation of lipid metabolism. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding polypeptide involved in regulation of lipid metabolism. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in regulation of lipid metabolism. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in regulation of lipid metabolism. In some embodiments, the modified host cell has five or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide involved in regulation of lipid metabolism. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide activity in the modified host cell.

[00264] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a polypeptide involved in regulation of lipid metabolism, such as, a full-length polypeptide involved in regulation of lipid metabolism, a fragment of a polypeptide involved in regulation of lipid metabolism, a variant of a polypeptide involved in regulation of lipid metabolism, a truncated polypeptide involved in regulation of lipid metabolism, or a fusion polypeptide that has at least one activity of a polypeptide involved in regulation of lipid metabolism. In some embodiments, the nucleotide sequence is codon-optimized.

[00265] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more polypeptides involved in regulation of lipid metabolism, the nucleotide sequence encoding one or more polypeptides involved in regulation of lipid metabolism is a nucleotide sequence encoding an OPI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:69.

[00266] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more polypeptides involved in regulation of lipid metabolism, the nucleotide sequence encoding one or more polypeptides involved in regulation of lipid metabolism is a nucleotide sequence encoding an OPI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO: 69, or a codon degenerate nucleotide sequence thereof.

[00267] In some embodiments, wherein the modified host cells of the disclosure comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more polypeptides involved in regulation of lipid metabolism, the nucleotide sequence encoding one or more polypeptides involved in regulation of lipid metabolism is a nucleotide sequence encoding an OPI1 polypeptide, wherein the nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:69.

[00268] In some embodiments, wherein the modified host cells of the disclosure comprise a deletion or downregulation of one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, the one or more polypeptides involved in regulation of lipid metabolism are encoded by a nucleotide sequence encoding an OPI1 polypeptide, wherein the nucleotide sequence is that set forth in SEQ ID NO:69.

[00269] In some embodiments, the modified host cells of the disclosure comprise a deletion or downregulation of an OPI1 gene. In some embodiments, the modified host cells of the disclosure comprise a deletion of an OPI1 gene. In some embodiments, the modified host cells of the disclosure comprise a downregulation of an OPI1 gene.

BBE and BBE-Like Polypeptides

[00270] The present disclosure provides modified host cells comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, such as a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue; a BBE or BBE-like polypeptide produced in a fungal cell; a BBE or BBE-like polypeptide produced in a bacterial cell; cannabinoid synthase polypeptides, such as a tetrahydrocannabinolic acid synthase polypeptide, a cannabichromenic acid synthase polypeptide, or a cannabidiolic acid synthase polypeptide; a BBE polypeptide from Eschscholzia californica ; a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacunr, a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans a daurichromenic acid synthase polypeptide from Rhododendron dauricum or a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila.

[00271] In plants, many BBE and BBE-like polypeptides are processed through the secretory pathway in specialized cells and tissues, e.g., glandular trichomes. In fungi and bacteria, these peptides are often processed through the secretory pathway or system. As secreted polypeptides, BBE or BBE-like polypeptides have structural features that may hinder expression in modified host cells, such as modified yeast cells. BBE or BBE-like polypeptides may comprise disulfide bonds, numerous glycosylation sites, including N- glycosylation sites, and a bicovalently attached flavin adenine dinucleotide (FAD) cofactor moiety. Accordingly, reconstituting the activity and expression of BBE or BBE-like polypeptides in a modified host cell, such as a modified yeast cell, can be challenging and unreliable. Often these secreted polypeptides are misfolded or mislocalized, resulting in low expression, polypeptides lacking activity, reduced host cell viability, and/or cell death. As disclosed herein, manipulation of secretory pathway in host cells modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide may improve expression, folding, and enzymatic activity of the BBE or BBE- like polypeptide as well as viability of the modified host cell. In certain such embodiments, the the nucleotide sequence encoding a BBE or BBE-like polypeptide is codon-optimized.

[00272] The BBE or BBE-like polypeptides and the nucleotide sequences encoding the BBE or BBE-like polypeptides may be derived from any suitable source, for example, bacteria, yeast, fungi, algae, human, plant, or mouse. In some embodiments, the disclosure also encompasses orthologous genes encoding the BBE or BBE-like polypeptides disclosed herein. Exemplary BBE or BBE-like polypeptides disclosed herein may also include a full- length BBE or a full-length BBE-like polypeptide, a fragment of a BBE or a fragment of a BBE-like polypeptide, a variant of a BBE or a variant of a BBE-like polypeptide, a truncated BBE or a truncated BBE-like polypeptide, or a fusion polypeptide that has at least one activity of a BBE or a fusion polypeptide that has at least one activity of a BBE-like polypeptide. The disclosure also provides for nucleotide sequences encoding BBE or BBE- like polypeptides, such as, a full-length BBE or a full-length BBE-like polypeptide, a fragment of a BBE or a fragment of a BBE-like polypeptide, a variant of a BBE or a variant of a BBE-like polypeptide, a truncated BBE or a truncated BBE-like polypeptide, or a fusion polypeptide that has at least one activity of a BBE or a fusion polypeptide that has at least one activity of a BBE-like polypeptide. In some embodiments, the nucleotide sequences encoding the BBE or BBE-like polypeptide are codon-optimized.

Cannabinoid Synthase Polypeptides

[00273] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a cannabinoid synthase polypeptide. Different cannabinoid synthase polypeptides can convert CBGA into other cannabinoids, for example, THCA, cannabichromenic acid (CBCA), and CBDA. In some embodiments, the cannabinoid synthase polypeptide is a tetrahydrocannabinolic acid synthase polypeptide. In some embodiments, the cannabinoid synthase polypeptide is a cannabichromenic acid synthase polypeptide. In some embodiments, the cannabinoid synthase polypeptide is a cannabidiolic acid synthase polypeptide.

[00274] Exemplary cannabinoid synthase polypeptides disclosed herein may include a fragment of a cannabinoid synthase polypeptide, a full-length cannabinoid synthase polypeptide, a variant of a cannabinoid synthase polypeptide, a truncated cannabinoid synthase polypeptide, or a fusion polypeptide that has at least one activity of a cannabinoid synthase polypeptide.

[00275] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a cannabinoid synthase polypeptide, such as, a fragment of a cannabinoid synthase polypeptide, a variant of a cannabinoid synthase polypeptide, a full-length cannabinoid synthase polypeptide, a truncated

cannabinoid synthase polypeptide, or a fusion polypeptide that has at least one activity of a cannabinoid synthase polypeptide. In some embodiments, the nucleotide sequences are codon-optimized.

[00276] In some embodiments, a cannabinoid synthase polypeptide is a

tetrahydrocannabinolic acid synthase (THCAS) polypeptide. THCAS polypeptides can catalyze the conversion of cannabigerolic acid to THCA. Exemplary THCAS polypeptides disclosed herein may include a fragment of a THCAS polypeptide, a full-length THCAS polypeptide, a variant of a THCAS polypeptide, a truncated THCAS polypeptide, or a fusion polypeptide that has at least one activity of a THCAS polypeptide.

[00277] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide, wherein the THCAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:92. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide, wherein the THCAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:92, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a THCAS

polypeptide, wherein the THCAS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:92. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide, wherein the THCAS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:92. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide, wherein the THCAS polypeptide comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:92.

[00278] The THCAS polypeptide may include a modified THCAS polypeptide with an N-terminal truncation to remove the native signal sequence polypeptide. For example, in some embodiments, the THCAS polypeptide lacks N-terminal amino acids 1-28 of the amino acid sequence set forth in SEQ ID NO:92. In certain such embodiments, the THCAS polypeptide may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00279] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a THCAS polypeptide, such as, a fragment of a THCAS polypeptide, a variant of a THCAS polypeptide, a full-length THCAS polypeptide, a truncated THCAS polypeptide, or a fusion polypeptide that has at least one activity of a THCAS polypeptide. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a THCAS polypeptide lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding THCAS polypeptides lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated THCAS polypeptide also comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting THCAS polypeptide is modified with the signal sequence polypeptide. In some

embodiments, the nucleotide sequences are codon-optimized.

[00280] In some embodiments, the THCAS polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the THCAS polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the THCAS polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the THCAS polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00281] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequences encoding a THCAS polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:9l. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequences encoding a THCAS polypeptide, wherein the codon- optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:9l, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a THCAS polypeptide, wherein the codon- optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:9l. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a THCAS polypeptide, wherein the codon- optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:9l.

[00282] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a THCAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:9l. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a THCAS polypeptide, wherein the codon- optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:9l. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a THCAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO:9l. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a THCAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO:9l.

[00283] In some embodiments, a cannabinoid synthase polypeptide is a

cannabichromenic acid synthase (CBCAS) polypeptide. CBCAS polypeptides can produce cannabichromenic acid. Exemplary CBCAS polypeptides disclosed herein may include a fragment of a CBCAS polypeptide, a full-length CBCAS polypeptide, a variant of a CBCAS polypeptide, a truncated CBCAS polypeptide, or a fusion polypeptide that has at least one activity of a CBCAS polypeptide.

[00284] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBCAS polypeptide, wherein the CBCAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:94. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBCAS polypeptide, wherein the CBCAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO:94, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a CBCAS

polypeptide, wherein the CBCAS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:94. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBCAS polypeptide, wherein the CBCAS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:94. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBCAS polypeptide, wherein the CBCAS polypeptide comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:94.

[00285] The CBCAS polypeptide may include a modified CBCAS polypeptide with an N-terminal truncation to remove the native signal sequence polypeptide. For example, in some embodiments, the CBCAS polypeptide lacks N-terminal amino acids 1-28 of the amino acid sequence set forth in SEQ ID NO:94. In certain such embodiments, the CBCAS polypeptide may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00286] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a CBCAS polypeptide, such as, a fragment of a CBCAS polypeptide, a variant of a CBCAS polypeptide, a full-length CBCAS polypeptide, a truncated CBCAS polypeptide, or a fusion polypeptide that has at least one activity of a CBCAS polypeptide. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a CBCAS polypeptide lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding CBCAS polypeptides lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated CBCAS polypeptide also comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting CBCAS polypeptide is modified with the signal sequence polypeptide. In some

embodiments, the nucleotide sequences are codon-optimized.

[00287] In some embodiments, the CBCAS polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the CBCAS polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the CBCAS polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBCAS polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00288] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:93. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon- optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 93, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon- optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:93. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon- optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:93.

[00289] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:93. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon- optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:93. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 93. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBCAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO:93.

[00290] In some embodiments, a cannabinoid synthase polypeptide is cannabidiolic acid synthase (CBDAS) polypeptide. CBDAS polypeptides can catalyze the conversion of cannabigerolic acid to cannabidiolic acid (CBDA). Exemplary CBDAS polypeptides disclosed herein may include a full-length CBDAS polypeptide, a fragment of a CBDAS polypeptide, a variant of a CBDAS polypeptide, a truncated CBDAS polypeptide, or a fusion polypeptide that has at least one activity of a CBDAS polypeptide.

[00291] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: l08 or SEQ ID NO:l32. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 108 or SEQ ID NO: 132, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 108 or SEQ ID NO: 132. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS

polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 108 or SEQ ID NO: 132. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 108 or SEQ ID NO: 132.

[00292] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 108. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 108, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS

polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 108. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 108. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 108.

[00293] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 132. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 132, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS

polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 132. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 132. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 132.

[00294] The CBDAS polypeptide may include a modified CBDAS polypeptide with an N-terminal truncation to remove the native signal sequence polypeptide. For example, in some embodiments, the CBDAS polypeptide lacks N-terminal amino acids 1-28 of the amino acid sequence set forth in SEQ ID NO: 132. In certain such embodiments, the CBDAS polypeptide may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising the amino acid sequence set forth in SEQ ID NO:96 or SEQ ID NO: 134. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising the amino acid sequence set forth in SEQ ID NO:96 or SEQ ID NO: 134, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:96 or SEQ ID NO: 134. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:96 or SEQ ID NO: 134. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:96 or SEQ ID NO: 134.

[00295] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising the amino acid sequence set forth in SEQ ID NO:96. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising the amino acid sequence set forth in SEQ ID NO:96, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO:96. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO:96. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO:96.

[00296] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide

comprising the amino acid sequence set forth in SEQ ID NO: 134. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 134, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 134. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 134. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated CBDAS polypeptide comprising an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 134.

[00297] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a CBDAS polypeptide, such as, a full- length CBDAS polypeptide, a fragment of a CBDAS polypeptide, a variant of a CBDAS polypeptide, a truncated CBDAS polypeptide, or a fusion polypeptide that has at least one activity of a CBDAS polypeptide. In some embodiments, the nucleotide sequences are codon-optimized.

[00298] In some embodiments, the CBDAS polypeptide is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the CBDAS polypeptide, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the CBDAS polypeptide to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid

comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the CBDAS polypeptide. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00299] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129,

SEQ ID NO: 130, or SEQ ID NO: 131, or a codon degenerate nucleotide sequence of any of the foregoing. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126,

SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131.

[00300] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131.

[00301] The disclosure provides nucleic acids comprising a nucleotide sequence encoding a CBDAS polypeptide lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding CBDAS polypeptides lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some

embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 107 (corresponds to SEQ ID NO: 125 lacking the stop codon), SEQ ID NO: 109 (corresponds to SEQ ID NO: 126 lacking the stop codon), SEQ ID NO: 110 (corresponds to SEQ ID NO: 127 lacking the stop codon), SEQ ID NO: 111 (corresponds to SEQ ID NO: 128 lacking the stop codon), SEQ ID NO: 112 (corresponds to SEQ ID NO: 129 lacking the stop codon), SEQ ID NO: 113 (corresponds to SEQ ID NO: 130 lacking the stop codon), or SEQ ID NO: 114 (corresponds to SEQ ID NO: 131 lacking the stop codon). In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114, or a codon degenerate nucleotide sequence of any of the foregoing. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 107, SEQ ID NO: l09, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114.

[00302] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114.

[00303] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide with an N-terminal truncation to remove the native signal sequence polypeptide. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 95 or SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:95 or SEQ ID NO: 133, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO:95 or SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated

polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 95 or SEQ ID NO: 133. In some embodiments, the one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide also comprise nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting CBDAS polypeptide is modified with the signal sequence polypeptide.

[00304] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:95 or SEQ ID NO:l33. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 95 or SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO:95 or SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 95 or SEQ ID

NO: l33.

[00305] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated

polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:95. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:95, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon- optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 95. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:95. In some embodiments, the one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide also comprise nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting CBDAS polypeptide is modified with the signal sequence polypeptide.

[00306] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:95. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:95. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 95. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO:95.

[00307] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated

polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 133, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon- optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a CBDAS polypeptide, wherein the CBDAS polypeptide is a truncated polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 133. In some embodiments, the one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide also comprise nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting CBDAS polypeptide is modified with the signal sequence polypeptide.

[00308] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 133. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated CBDAS polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 133.

[00309] In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a cannabinoid synthase polypeptide is operably linked to an inducible promoter. In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a cannabinoid synthase polypeptide is operably linked to a constitutive promoter.

[00310] In some embodiments of the modified host cells of the disclosure, the cannabinoid synthase polypeptide comprises signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a cannabinoid synthase polypeptide comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the cannabinoid synthase polypeptide.

[00311] In some embodiments of the modified host cells of the disclosure, the cannabinoid synthase polypeptide is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a cannabinoid synthase polypeptide comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell of the disclosure is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1 polypeptide. In certain such embodiments, the cannabinoid synthase-AGA2t fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N- terminus of the cannabinoid synthase polypeptide.

[00312] In some embodiments of the modified host cells of the disclosure, the cannabinoid synthase polypeptide is a fusion polypeptide with a GFP polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a cannabinoid synthase polypeptide comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N- terminus of the cannabinoid synthase polypeptide. BBE polypeptide from Eschscholzia californica

[00313] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica. The BBE polypeptide from Eschscholzia californica is involved in the conversion of (S)-reticuline to (S)-scoulerine in opiate biosynthesis.

[00314] Exemplary BBE from Eschscholzia californica polypeptides disclosed herein may include a fragment of a BBE polypeptide from Eschscholzia californica , a full-length BBE polypeptide from Eschscholzia californica , a variant of a BBE polypeptide from Eschscholzia californica , a truncated BBE polypeptide from Eschscholzia californica , or a fusion polypeptide that has at least one activity of a BBE polypeptide from Eschscholzia californica.

[00315] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the BBE polypeptide from Eschscholzia californica comprises the amino acid sequence set forth in SEQ ID NO: 116. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from

Eschscholzia californica , wherein the BBE polypeptide from Eschscholzia californica comprises the amino acid sequence set forth in SEQ ID NO: 116, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the BBE polypeptide from Eschscholzia californica comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 116. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the BBE polypeptide from Eschscholzia californica comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 116. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the BBE polypeptide from Eschscholzia californica comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 116.

[00316] In some embodiments, the BBE polypeptide from Eschscholzia californica may include a modified BBE polypeptide from Eschscholzia californica with an N-terminal truncation to remove the native signal sequence polypeptide. In certain such embodiments, the BBE polypeptide from Eschscholzia californica may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00317] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a BBE polypeptide from Eschscholzia californica , such as, a fragment of a BBE polypeptide from Eschscholzia californica , a variant of a BBE polypeptide from Eschscholzia californica , a full-length BBE polypeptide from Eschscholzia californica , a truncated BBE polypeptide from Eschscholzia californica , or a fusion polypeptide that has at least one activity of a BBE polypeptide from Eschscholzia californica. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding BBE polypeptides from Eschscholzia californica lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated BBE polypeptide from Eschscholzia californica also comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting BBE polypeptide from Eschscholzia californica is modified with the signal sequence polypeptide. In some embodiments, the nucleotide sequences are codon-optimized.

[00318] In some embodiments, the BBE polypeptide from Eschscholzia californica is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica , e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some

embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from

Eschscholzia californica. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. In some

embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE polypeptide from Eschscholzia californica. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00319] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 115. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 115, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon- optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 115. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 115.

[00320] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 115. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE

polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 115. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 115. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: l l5.

[00321] In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica is operably linked to an inducible promoter. In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica is operably linked to a constitutive promoter.

[00322] In some embodiments of the modified host cells of the disclosure, the BBE polypeptide from Eschscholzia californica comprises a signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some

embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the BBE polypeptide from Eschscholzia californica.

[00323] In some embodiments of the modified host cells of the disclosure, the BBE polypeptide from Eschscholzia californica is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell of the disclosure is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1

polypeptide. In certain such embodiments, the BBE polypeptide from Eschscholzia

fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N-terminus of the BBE polypeptide from Eschscholzia californica.

[00324] In some embodiments of the modified host cells of the disclosure, the BBE polypeptide from Eschscholzia californica is a fusion polypeptide with a GFP polypeptide.

In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N-terminus of the BBE polypeptide from Eschscholzia californica.

BBE-like Nicotine Bridge Enzyme Polypeptide from Nicotiana tabacum

[00325] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. The BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum is involved in a BBE-like reaction in the biosynthesis of nicotine.

[00326] Exemplary BBE-like nicotine bridge enzyme from Nicotiana tabacum polypeptides disclosed herein may include a fragment of a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , a full-length BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , a variant of a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , a truncated BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , or a fusion polypeptide that has at least one activity of a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum.

[00327] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises the amino acid sequence set forth in SEQ ID NO: 120. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises the amino acid sequence set forth in SEQ ID NO: 120, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE- like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 120. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 120. In some embodiments, a modified host cell of the disclosure comprises one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 120.

[00328] In some embodiments, the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum may include a modified BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum with an N-terminal truncation to remove the native signal sequence polypeptide. In certain such embodiments, the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00329] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , such as, a fragment of a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , a variant of a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , a full-length BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , a truncated BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , or a fusion polypeptide that has at least one activity of a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding BBE-like nicotine bridge enzyme polypeptides from Nicotiana tabacum lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum also comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum is modified with the signal sequence polypeptide. In some embodiments, the nucleotide sequences are codon-optimized.

[00330] In some embodiments, the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE- like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell. [00331] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 119. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 119, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 119. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 119.

[00332] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 119. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 119. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 119. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: l l9.

[00333] In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum is operably linked to an inducible promoter. In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from

Nicotiana tabacum is operably linked to a constitutive promoter.

[00334] In some embodiments of the modified host cells of the disclosure, the BBE- like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises a signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum.

[00335] In some embodiments of the modified host cells of the disclosure, the BBE- like nicotine bridge enzyme polypeptide from Nicotiana tabacum is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell of the disclosure is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1 polypeptide. In certain such embodiments, the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum- AGA2t fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N-terminus of the BBE-like nicotine bridge enzyme polypeptide from

Nicotiana tabacum.

[00336] In some embodiments of the modified host cells of the disclosure, the BBE- like nicotine bridge enzyme polypeptide from Nicotiana tabacum is a fusion polypeptide with a GFP polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N-terminus of the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum.

BBE-Like 6-Hydroxy-D-Nicotine Oxidase Polypeptide from Paenarthrobacter

nicotinovorans

[00337] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. The BBE- like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is involved in nicotine degradation.

[00338] Exemplary BBE-like 6-hydroxy-D-nicotine oxidase from Paenarthrobacter nicotinovorans polypeptides disclosed herein may include a fragment of a BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , a full- length BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , a variant of a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans, a truncated BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , or a fusion polypeptide that has at least one activity of a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans .

[00339] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6- hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises the amino acid sequence set forth in SEQ ID NO: 122. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the BBE-like 6-hydroxy-D- nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises the amino acid sequence set forth in SEQ ID NO: 122, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE- like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 122. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6- hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 122. In some

embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 122.

[00340] In some embodiments, the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans may include a modified BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans with an N- terminal truncation to remove the native signal sequence polypeptide. In certain such embodiments, the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00341] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , such as, a fragment of a BBE- like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , a variant of a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , a full-length BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , a truncated BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , or a fusion polypeptide that has at least one activity of a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding BBE-like 6-hydroxy-D-nicotine oxidase polypeptides from Paenarthrobacter nicotinovorans lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans also comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is modified with the signal sequence polypeptide. In some embodiments, the nucleotide sequences are codon-optimized.

[00342] In some embodiments, the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans, e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE- like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6- hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6- hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00343] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans, wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 121. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 121, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 121. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6- hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: l2l.

[00344] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 121. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 121. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 121. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy-D- nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans, wherein the codon- optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 121.

[00345] In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is operably linked to an inducible promoter. In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is operably linked to a constitutive promoter.

[00346] In some embodiments of the modified host cells of the disclosure, the BBE- like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises a signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some

embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans .

[00347] In some embodiments of the modified host cells of the disclosure, the BBE- like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell of the disclosure is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1 polypeptide. In certain such embodiments, the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans- AGA2t fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N-terminus of the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from

Paenarthrobacter nicotinovorans .

[00348] In some embodiments of the modified host cells of the disclosure, the BBE- like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans is a fusion polypeptide with a GFP polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE-like 6- hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N-terminus of the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans.

Daurichromenic Acid Synthase Polypeptide from Rhododendron dauricum

[00349] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum. The daurichromenic acid synthase polypeptide from Rhododendron dauricum is involved in an oxidative cyclization similar to that of the THCA synthase polypeptide.

[00350] Exemplary daurichromenic acid synthase from Rhododendron dauricum polypeptides disclosed herein may include a fragment of a daurichromenic acid synthase polypeptide from Rhododendron dauricum , a full-length daurichromenic acid synthase polypeptide from Rhododendron dauricum , a variant of a daurichromenic acid synthase polypeptide from Rhododendron dauricum , a truncated daurichromenic acid synthase polypeptide from Rhododendron dauricum , or a fusion polypeptide that has at least one activity of a daurichromenic acid synthase polypeptide from Rhododendron dauricum.

[00351] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a

daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises the amino acid sequence set forth in SEQ ID NO: 118. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from

Rhododendron dauricum , wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises the amino acid sequence set forth in SEQ ID NO: 118, or a conservatively substituted amino acid sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from

Rhododendron dauricum , wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 118. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 118. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from

Rhododendron dauricum , wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 118.

[00352] In some embodiments, the daurichromenic acid synthase polypeptide from Rhododendron dauricum may include a modified daurichromenic acid synthase polypeptide from Rhododendron dauricum with an N-terminal truncation to remove the native signal sequence polypeptide. In certain such embodiments, the daurichromenic acid synthase polypeptide from Rhododendron dauricum may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00353] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a daurichromenic acid synthase polypeptide from Rhododendron dauricum , such as, a fragment of a daurichromenic acid synthase polypeptide from Rhododendron dauricum , a variant of a daurichromenic acid synthase polypeptide from Rhododendron dauricum , a full-length daurichromenic acid synthase polypeptide from Rhododendron dauricum , a truncated daurichromenic acid synthase polypeptide from Rhododendron dauricum , or a fusion polypeptide that has at least one activity of a daurichromenic acid synthase polypeptide from Rhododendron dauricum. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding daurichromenic acid synthase polypeptides from Rhododendron dauricum lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated daurichromenic acid synthase polypeptide from Rhododendron dauricum also comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting daurichromenic acid synthase polypeptide from Rhododendron dauricum is modified with the signal sequence polypeptide. In some embodiments, the nucleotide sequences are codon-optimized.

[00354] In some embodiments, the daurichromenic acid synthase polypeptide from Rhododendron dauricum is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum , e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the

daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid

comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the daurichromenic acid synthase polypeptide from Rhododendron dauricum. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00355] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:l 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 117, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 117. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 117.

[00356] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:l 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:l 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO:l 17. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 117.

[00357] In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum is operably linked to an inducible promoter. In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from

Rhododendron dauricum is operably linked to a constitutive promoter.

[00358] In some embodiments of the modified host cells of the disclosure, the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises a signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase

polypeptide from Rhododendron dauricum comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the daurichromenic acid synthase polypeptide from Rhododendron dauricum.

[00359] In some embodiments of the modified host cells of the disclosure, the daurichromenic acid synthase polypeptide from Rhododendron dauricum is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell of the disclosure is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1 polypeptide. In certain such embodiments, the daurichromenic acid synthase polypeptide from Rhododendron dauricum- AG A2t fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N-terminus of the daurichromenic acid synthase polypeptide from Rhododendron dauricum.

[00360] In some embodiments of the modified host cells of the disclosure, the daurichromenic acid synthase polypeptide from Rhododendron dauricum is a fusion polypeptide with a GFP polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N- terminus of the daurichromenic acid synthase polypeptide from Rhododendron dauricum.

Xylooligosaccharide Oxidase Polypeptide from Myceliophthora thermophila

[00361] A modified host cell of the present disclosure may comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. The xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila oxidizes oligosaccharides that originate from the breakdown of cellulose.

[00362] Exemplary xylooligosaccharide oxidase from Myceliophthora thermophila polypeptides disclosed herein may include a fragment of a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , a full-length xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , a variant of a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , a truncated xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , or a fusion polypeptide that has at least one activity of a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[00363] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a

xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprises the amino acid sequence set forth in SEQ ID NO: 124. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila , wherein the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprises the amino acid sequence set forth in SEQ ID

NO: 124, or a conservatively substituted amino acid sequence thereof. In some

embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprises an amino acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, or at least 75% amino acid sequence identity to SEQ ID NO: 124. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% amino acid sequence identity to SEQ ID NO: 124. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprises an amino acid sequence having 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% amino acid sequence identity to SEQ ID NO: 124.

[00364] In some embodiments, the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila may include a modified xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila with an N-terminal truncation to remove the native signal sequence polypeptide. In certain such embodiments, the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila may comprise a signal sequence polypeptide different than that of the native signal sequence polypeptide.

[00365] Exemplary heterologous nucleic acids disclosed herein may include nucleic acids comprising a nucleotide sequence that encodes a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , such as, a fragment of a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , a variant of a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , a full-length xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , a truncated xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , or a fusion polypeptide that has at least one activity of a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. The disclosure also provides nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding xylooligosaccharide oxidase polypeptides from Myceliophthora thermophila lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. In some embodiments, the nucleic acid comprising a nucleotide sequence encoding a truncated xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila also comprises nucleotide sequences encoding a signal sequence polypeptide.

In certain such embodiments, after transcription and translation, the resulting

xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila is modified with the signal sequence polypeptide. In some embodiments, the nucleotide sequences are codon- optimized.

[00366] In some embodiments, the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila is overexpressed in the modified host cell. Overexpression may be achieved by increasing the copy number of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , e.g., through use of a high copy number expression vector (e.g., a plasmid that exists at 10-40 copies or about 100 copies per cell) and/or by operably linking the nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila to a strong promoter. In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the

xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. In some embodiments, the modified host cell has eight or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila. Increased copy number of the heterologous nucleic acid and/or codon optimization of the nucleotide sequence may result in an increase in the desired enzyme catalytic activity in the modified host cell.

[00367] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO:l23. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has the nucleotide sequence set forth in SEQ ID NO: 123, or a codon degenerate nucleotide sequence thereof. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% sequence identity to SEQ ID NO: 123. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 123.

[00368] In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 123. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 123. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 123. In some embodiments, a modified host cell of the disclosure comprises one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 123.

[00369] In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila is operably linked to an inducible promoter. In some embodiments, at least one of the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila is operably linked to a constitutive promoter.

[00370] In some embodiments of the modified host cells of the disclosure, the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprises a signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[00371] In some embodiments of the modified host cells of the disclosure, the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell of the disclosure is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1 polypeptide. In certain such embodiments, the

xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila- AG A2t fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N-terminus of the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[00372] In some embodiments of the modified host cells of the disclosure, the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila is a fusion polypeptide with a GFP polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N- terminus of the xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

Modified Host Cells to Express BBE or BBE-Like Polypeptides

[00373] The disclosure provides modified host cells modified to express or overexpress one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cells comprise one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more heterologous nucleic acids disclosed herein comprising nucleotide sequences encoding secretory pathway polypeptides. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In certain such embodiments, the modified host cell may comprise a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the modified host cell may comprise a downregulation of one or more genes encoding one or more secretory pathway

polypeptides. In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a codon-optimized nucleotide sequence. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide. In the modified host cells for expressing a BBE or BBE-like polypeptide disclosed herein, the BBE or BBE-like polypeptide may include a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue; a BBE or BBE-like polypeptide produced in a fungal cell; a BBE or BBE-like polypeptide produced in a bacterial cell; cannabinoid synthase polypeptides, such as a tetrahydrocannabinolic acid synthase polypeptide, a cannabichromenic acid synthase polypeptide, or a cannabidiolic acid synthase polypeptide; a BBE polypeptide from Eschscholzia californica ; a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacunr, a BBE-like 6-hydroxy -D- nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans a daurichromenic acid synthase polypeptide from Rhododendron dauricum or a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[00374] In some embodiments, to express or overexpress BBE or BBE-like polypeptides, expression or overexpression of one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide in a modified host cell may be done in combination with expression or overexpression by the modified host cell of one or more other heterologous nucleic acids disclosed herein (e.g., one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides) and/or with deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments, the nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a codon-optimized nucleotide sequence. In some embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments, the BBE or BBE-like polypeptide is a fusion

polypeptide with a GFP polypeptide. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.

[00375] In some embodiments, the growth and/or viability of modified host cells of the disclosure for expressing a BBE or BBE-like polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure for expressing a BBE or BBE-like polypeptide has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions.

[00376] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a codon- optimized nucleotide sequence encoding a BBE or BBE-like polypeptide is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some

embodiments of the modified host cells of the disclosure comprising one or more

heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

[00377] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more modifications to modulate the expression of one or more secretory pathway polypeptides is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more modifications to modulate the expression of one or more secretory pathway polypeptides has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In certain such embodiments, the nucleotide sequence encoding the BBE or BBE-like polypeptide is codon-optimized. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more modifications to modulate the expression of one or more secretory pathway polypeptides, the BBE or BBE- like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more modifications to modulate the expression of one or more secretory pathway polypeptides, the BBE or BBE-like

polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more modifications to modulate the expression of one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

[00378] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In certain such embodiments, the nucleotide sequence encoding the BBE or BBE-like polypeptide are codon-optimized. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

[00379] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In certain such embodiments, the nucleotide sequence encoding the BBE or BBE-like polypeptide is codon-optimized. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

[00380] In some embodiments, the growth and/or viability of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides is not significantly decreased compared to the growth and/or viability of an unmodified host cell. In some embodiments, a culture of modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides has a cell density that is at least 25% or greater, at least 30% or greater, at least 35% or greater, at least 40% or greater, at least 45% or greater, at least 50% or greater, at least 55% or greater, at least 60% or greater, at least 65% or greater, at least 70% or greater, at least 75% or greater, at least 80% or greater, at least 85% or greater at least 90% or greater, at least 95% or greater, at least 100% or greater, at least 110% or greater, at least 120% or greater, at least 130% or greater, at least 140% or greater, or at least 150% or greater than the cell density of a culture of unmodified control host cells grown for the same period, in the same culture medium, and under the same culture conditions. In certain such embodiments, the nucleotide sequence encoding the BBE or BBE-like polypeptide is codon- optimized. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some

embodiments of the modified host cells of the disclosure comprising one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, the BBE or BBE- like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cells of the disclosure comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

Exemplary Modified Host Cells for Expressing BBE and BBE-Like Polypeptides

[00381] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide.

In certain such embodiments, the nucleotide sequence encoding a BBE or BBE-like polypeptide is codon-optimized. In the modified host cells for expressing a BBE or BBE- like polypeptide disclosed herein, the BBE or BBE-like polypeptide may include a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue; a BBE or BBE-like polypeptide produced in a fungal cell; a BBE or BBE- like polypeptide produced in a bacterial cell; cannabinoid synthase polypeptides, such as a tetrahydrocannabinolic acid synthase polypeptide, a cannabichromenic acid synthase polypeptide, or a cannabidiolic acid synthase polypeptide; a BBE polypeptide from

Eschscholzia californica ; a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicolinovorans a daurichromenic acid synthase polypeptide from Rhododendron dauricum or a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[00382] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In certain such embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00383] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and b) a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In certain such embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides.

[00384] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide; b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides; and c) a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00385] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In certain such embodiments, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), or one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing.

[00386] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides. In certain such embodiments, the one or more chaperone or co- chaperone polypeptides comprise a JEM1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a LHS1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a SIL1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CNE1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a SIS1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a SSB1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a ROT1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a DED1 polypeptide and a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a SSB1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a LHS1 polypeptide. In some embodiments, the one or more chaperone or co- chaperone polypeptides comprise a SSA1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CNS1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a DED1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone

polypeptides comprise a PFD2s polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a JEM1 polypeptide. In some

embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CNE1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a SCJ1 polypeptide. In some embodiments, the one or more chaperone or co- chaperone polypeptides comprise a SIL1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a FPR1 polypeptide. In some embodiments, the one or more chaperone or co-chaperone polypeptides comprise a ROT1 polypeptide.

[00387] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (EIPR). In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise KAR2 and the one or more polypeptides involved in EIPR comprise a HACls polypeptide.

[00388] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (EIPR). In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and the one or more polypeptides involved in EIPR comprise an IRE1 polypeptide.

[00389] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more polypeptides involved in UPR comprise an IRE1 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, and the one or more thiol oxidase polypeptides comprise an EROl polypeptide.

[00390] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR). In certain such embodiments, the one or more polypeptides involved in UPR comprise a HACls polypeptide and/or an IRE1 polypeptide.

In some embodiments, the one or more polypeptides involved in UPR comprise a HACls polypeptide. In some embodiments, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide.

[00391] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. In certain such embodiments, the one or more chaperone or co- chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, and the one or more thiol oxidase polypeptides comprise an EROl polypeptide.

[00392] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. In certain such embodiments, the one or more chaperone or co- chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, and the one or more thiol oxidase polypeptides comprise an ERV2 polypeptide.

[00393] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

[00394] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more polypeptides involved in UPR, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and a SSB1 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in UPR comprise a HACls polypeptide, and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide. In certain such embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, wherein the signal sequence polypeptide is a vacuolar localization signal sequence polypeptide, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide.

[00395] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. In certain such embodiments, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide and the one or more thiol oxidase polypeptides comprise an EROl polypeptide and an ERV2 polypeptide.

[00396] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR). In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and a SSB1 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, and the one or more polypeptides involved in UPR comprise a HACls polypeptide.

[00397] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In certain such embodiments, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

[00398] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

[00399] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide.

[00400] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In certain such embodiments, the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, or one or more genes encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises a downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00401] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 and/or MNS1 gene. In some embodiments, the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene. In some

embodiments, the one or more genes encoding one or more glycosidase polypeptides comprise a MNSl gene.

[00402] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides. In certain such embodiments, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

[00403] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In certain such embodiments, the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), or one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing, and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, or one or more genes encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing. In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion of one or more genes encoding one or more secretory pathway polypeptides. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00404] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, and the one or more genes encoding one or more secretory pathway

polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

[00405] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

[00406] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and the one or more genes encoding one or more secretory pathway

polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides and one or more genes encoding one or more vacuolar proteinase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene, and the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

[00407] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides and one or more genes encoding one or more vacuolar proteinase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene, and the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

[00408] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides and one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism. In certain such

embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, and the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene. In certain such embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, wherein the signal sequence polypeptide is a vacuolar localization signal sequence polypeptide, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

[00409] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene. In certain such embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, wherein the signal sequence polypeptide is a vacuolar localization signal sequence polypeptide, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

[00410] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, and one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene. In certain such embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, wherein the signal sequence polypeptide is a vacuolar localization signal sequence polypeptide, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

[00411] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, and one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in unfolded protein response (EIPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene. In certain such embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, wherein the signal sequence polypeptide is a vacuolar localization signal sequence polypeptide, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

[00412] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides and one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

[00413] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides and one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an ERV2 polypeptide, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

[00414] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, and the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

[00415] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, and the one or more genes encoding one or more secretory pathway

polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, and the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

[00416] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (ETPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, and one or more genes encoding one or more glycosidase polypeptides. In certain such embodiments, the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide and an ERV2 polypeptide, the one or more polypeptides involved in unfolded protein response (ETPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene. In certain such embodiments, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, wherein the signal sequence polypeptide is a vacuolar localization signal sequence polypeptide, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

[00417] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide; b) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate olivetolic acid geranyltransferase (GOT, CsPT4) polypeptide; c) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMG-CoA synthase (HMGS, ERG13) polypeptide; d) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a truncated 3 -hydroxy-3 -methyl- glutaryl-CoA reductase (tHMGR) polypeptide; e) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate kinase (MK, ERG12) polypeptide; f) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a phosphomevalonate kinase (PMK, ERG8) polypeptide; g) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate pyrophosphate

decarboxylase (MVD1) polypeptide; h) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a isopentenyl diphosphate isomerase (IDI1) polypeptide; i) one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase (ERG10) polypeptide; j) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide; and k) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS, ERG20mut) polypeptide.

[00418] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide; b) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate olivetolic acid geranyltransferase (GOT, CsPT4) polypeptide; c) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a HMG-CoA synthase (HMGS, ERG13) polypeptide; d) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a truncated 3 -hydroxy-3 -methyl- glutaryl-CoA reductase (tHMGR) polypeptide; e) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate kinase (MK, ERG12) polypeptide; f) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a phosphomevalonate kinase (PMK, ERG8) polypeptide; g) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a mevalonate pyrophosphate decarboxylase (MVD1) polypeptide; h) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a isopentenyl diphosphate isomerase (IDI1) polypeptide; i) one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase (ERG10) polypeptide; j) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide; k) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS, ERG20mut) polypeptide; and 1) one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more secretory pathway polypeptides. In certain such embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00419] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more

decarboxylase (MVD1) polypeptide; h) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a isopentenyl diphosphate isomerase (IDI1) polypeptide; i) one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase (ERG10) polypeptide; j) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide; k) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS, ERG20mut) polypeptide; and 1) a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. In certain such embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

[00420] The present disclosure provides a modified host cell for expressing a BBE or BBE-like polypeptide, wherein the modified host cell comprises: a) one or more

decarboxylase (MVD1) polypeptide; h) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a isopentenyl diphosphate isomerase (IDI1) polypeptide; i) one or more heterologous nucleic acids comprising a nucleotide sequence encoding an acetoacetyl-CoA thiolase (ERG10) polypeptide; j) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a pyruvate decarboxylase (PDC) polypeptide; k) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a geranyl pyrophosphate synthetase (GPPS, ERG20mut) polypeptide; 1) one or more heterologous nucleic acids comprising a nucleotide sequence encoding one or more secretory pathway polypeptides; and m) a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00421] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide.

[00422] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a tHMGR polypeptide.

[00423] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises four or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a PDIl polypeptide.

[00424] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, a nucleotide sequence encoding a BBE or BBE-like polypeptide is codon- optimized. In some embodiments, the nucleotide sequences encoding the one or more secretory pathway polypeptides are codon-optimized.

[00425] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprise nucleotide sequences encoding a signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such

embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide.

In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)). In some embodiments, the signal sequence polypeptide is linked to the N-terminus of the BBE or BBE-like polypeptide.

[00426] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprise a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide is modified with one or more heterologous nucleic acids comprising a nucleotide sequence encoding an AGA1 polypeptide. In certain such embodiments, the BBE or BBE-like- AGA2t fusion polypeptide may be displayed on the cell surface of the modified host cell. In some embodiments, the AGA2t polypeptide is linked to the N-terminus of the BBE or BBE-like polypeptide.

[00427] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide. In certain such embodiments, the one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprise a nucleotide sequence encoding a GFP polypeptide. In some embodiments, the GFP polypeptide is linked to the N-terminus of the BBE or BBE-like polypeptide.

Suitable Host Cells

[00428] Parent host cells that are suitable for use in generating a modified host cell of the present disclosure may include eukaryotic cells. In some embodiments, the eukaryotic cells are yeast cells.

[00429] Host cells (including parent host cells and modified host cells) are in some embodiments unicellular organisms, or are grown in culture as single cells. In some embodiments, the host cell is a eukaryotic cell. Suitable eukaryotic host cells may include, but are not limited to, yeast cells and fungal cells. Suitable eukaryotic host cells may include, but are not limited to, Pichia pastoris (now known as Komagataella phaffii ), Pichia fmlandica , Pichia trehalophila , Pichia koclamae , Pichia membranaefaciens , Pichia opuntiae , Pichia thermotolerans , Pichia salictaria , Pichia guercuum , Pichia pijperi , Pichia stiptis , Pichia methanolica , Pichia sp., Saccharomyces cerevisiae , Saccharomyces sp., Hansenula polymorpha (now known as Pichia angusta), Yarrowia lipolytica , Kluyveromyces sp., Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pomhe ,

Scheffer somyces stipites, Dekkera hruxellensis , Blastobotrys adeninivorans (formerly Arxula adeninivorans ), Candida albicans , Aspergillus nidulans , Aspergillus niger , Aspergillus oryzae, I richoderma reese /, Chrysosporium lucknowense , Fusarium sp., Fusarium gramineum , Fusarium venenatum , Neurospora crassa, and the like. In some embodiments, the modified host cell disclosed herein is cultured in vitro.

[00430] In some embodiments, the host cell of the disclosure is a yeast cell. In some embodiments, the host cell is a protease-deficient strain of Saccharomyces cerevisiae. In some embodiments, the host cell is Saccharomyces cerevisiae. In some embodiments, the host cell for use in generating a modified host cell of the present disclosure may be selected because of ease of culture; rapid growth; availability of tools for modification, such as promoters and vectors; and the host cell’s safety profile. In some embodiments, the host cell for use in generating a modified host cell of the present disclosure may be selected because of its ability or inability to introduce certain posttranslational modifications onto expressed polypeptides, such as BBE or BBE-like polypeptides. For instance, modified Komagataella phaffli host cells may hyperglycosylate BBE or BBE-like polypeptides and

hyperglycosylation may alter the activity of the resultant expressed polypeptide.

Genetic Modification of Host Cells

[00431] The present disclosure provides for a method of making a modified host cell for expressing a BBE or BBE-like polypeptide, the method comprising introducing into a host cell one or more heterologous nucleic acids disclosed herein. In certain such embodiments, the method comprises introducing into a host cell: a) one or more

heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more heterologous nucleic acids comprising nucleotide sequence encoding one or more secretory pathway polypeptides. In some embodiments, the modified host cell for expressing a BBE or BBE-like polypeptide comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides.

[00432] In some embodiments, the modified host cell for expressing a BBE or BBE- like polypeptide comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. The disclosure provides a method of making a modified host cell for expressing a BBE or BBE-like polypeptide, the method comprising introducing into a host cell: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide and b) a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00433] In some embodiments, the modified host cell for expressing a BBE or BBE- like polypeptide comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptide, and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. The disclosure provides a method of making a modified host cell for expressing a BBE or BBE-like polypeptide, the method comprising introducing into a host cell: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a BBE or BBE-like polypeptide, b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, and c) a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00434] In some embodiments of the modified host cell for expressing a BBE or BBE- like polypeptide, the BBE or BBE-like polypeptide comprises a signal sequence polypeptide, such as a secretory signal sequence polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with an AGA2t polypeptide. In some embodiments of the modified host cell for expressing a BBE or BBE-like polypeptide, the BBE or BBE-like polypeptide is a fusion polypeptide with a GFP polypeptide.

[00435] To modify a parent host cell to produce a modified host cell of the present disclosure, one or more heterologous nucleic acids disclosed herein may be introduced stably or transiently into a host cell, using established techniques. Such techniques may include, but are not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-mediated transfection, the lithium acetate method, and the like. See Gietz, R.D. and R.A. Woods. (2002) TRANSFORMATION OF YEAST BY THE Liac/SS CARRIER DNA/PEG METHOD. For stable transformation, a plasmid, vector, expression construct, etc. comprising one or more nucleic acids (e.g., heterologous) disclosed herein will generally include a selectable marker, e.g., any of several well-known selectable markers such as neomycin resistance, ampicillin resistance, tetracycline resistance, chloramphenicol resistance, kanamycin resistance, and the like. In some embodiments, the selectable marker gene to provide a phenotypic trait for selection of transformed host cells is dihydrofolate reductase. In some embodiments, a parent host cell is modified to produce a modified host cell of the present disclosure using a CRISPR/Cas9 system to modify a parent host cell with one or more heterologous nucleic acids disclosed herein.

[00436] In some embodiments, varying BBE or BBE-like expression level in a modified host cell may be done by changing the gene copy number, promoter strength, and/or promoter regulation and/or by codon-optimization.

[00437] One or more heterologous nucleic acids disclosed herein can be present in an expression vector or construct. Suitable expression vectors may include, but are not limited to, plasmids, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as yeast). Such vectors may include chromosomal, non- chromosomal, and synthetic DNA sequences.

[00438] The present disclosure provides for a method of making a modified host cell for expressing a BBE or BBE-like polypeptide, the method comprising introducing into a host cell one or more vectors disclosed herein. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE- like polypeptide. In certain such embodiments, the one or more vectors comprise one or more vectors comprising one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides. In some embodiments, the method comprises introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00439] Numerous additional suitable expression vectors are known to those of skill in the art, and many are commercially available. The following vectors are provided by way of example; for yeast, the low copy CEN ARS and high copy 2 micron plasmids. However, any other plasmid or other vector may be used so long as it is compatible with the host cell.

[00440] In some embodiments, one or more of the heterologous nucleic acids disclosed herein are present in a single expression vector. In some embodiments, two or more of the heterologous nucleic acids disclosed herein are present in a single expression vector. In some embodiments, three or more of the heterologous nucleic acids disclosed herein are present in a single expression vector. In some embodiments, four or more of the heterologous nucleic acids disclosed herein are present in a single expression vector. In some embodiments, five or more of the heterologous nucleic acids disclosed herein are present in a single expression vector. In some embodiments, six or more of the heterologous nucleic acids disclosed herein are present in a single expression vector. In some

embodiments, seven or more of the heterologous nucleic acids disclosed herein are present in a single expression vector.

[00441] In some embodiments, two or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, three or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, four or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, five or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, six or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, seven or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, eight or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, nine or more heterologous nucleic acids disclosed herein are in separate expression vectors. In some embodiments, ten or more heterologous nucleic acids disclosed herein are in separate expression vectors.

[00442] In some embodiments, one or more of the heterologous nucleic acids disclosed herein are present in a single expression construct. In some embodiments, two or more of the heterologous nucleic acids disclosed herein are present in a single expression construct. In some embodiments, three or more of the heterologous nucleic acids disclosed herein are present in a single expression construct. In some embodiments, four or more of the heterologous nucleic acids disclosed herein are present in a single expression construct. In some embodiments, five or more of the heterologous nucleic acids disclosed herein are present in a single expression construct. In some embodiments, six or more of the heterologous nucleic acids disclosed herein are present in a single expression construct. In some embodiments, seven or more of the heterologous nucleic acids disclosed herein are present in a single expression construct.

[00443] In some embodiments, two or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, three or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, four or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, five or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, six or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, seven or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, eight or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, nine or more heterologous nucleic acids disclosed herein are in separate expression constructs. In some embodiments, ten or more heterologous nucleic acids disclosed herein are in separate expression constructs.

[00444] In some embodiments, one or more of the heterologous nucleic acids disclosed herein is present in a high copy number plasmid, e.g., a plasmid that exists in about 10-50 copies per cell, or more than 50 copies per cell. In some embodiments, one or more of the heterologous nucleic acids disclosed herein is present in a low copy number plasmid. In some embodiments, one or more of the heterologous nucleic acids disclosed herein is present in a medium copy number plasmid. The copy number of the plasmid may be selected to reduce expression of one or more polypeptides disclosed herein, such as a BBE or BBE-like polypeptide. Reducing expression by limiting the copy number of the plasmid may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability.

[00445] In some embodiments, the modified host cell has one copy of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has two copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has three copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has four copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has five copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has six copies of a heterologous nucleic acid

comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has seven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has eight copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has nine copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has ten copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has eleven copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has twelve copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein. In some embodiments, the modified host cell has twelve or more copies of a heterologous nucleic acid comprising a nucleotide sequence encoding a polypeptide disclosed herein.

[00446] Depending on the host/vector or host/construct system utilized, any of a number of suitable transcription and translation control elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector or construct (see e.g., Bitter et al. ( 1987) Methods in

Emymology , 153:516-544).

[00447] In some embodiments, the heterologous nucleic acids disclosed herein are operably linked to a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some

embodiments, the promoter is functional in a eukaryotic cell. In some embodiments, the promoter can be a strong driver of expression. In some embodiments, the promoter can be a weak driver of expression. In some embodiments, the promoter can be a medium driver of expression. The promoter may be selected to reduce expression of one or more polypeptides disclosed herein, such as a BBE or BBE-like polypeptide. Reducing expression through promoter selection may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. Examples of strong constitutive promoters include, but are not limited to: pTDEB and pFBAl. Examples of medium constitutive promoters include, but are not limited to: pACTl and pCYCl. An example of a weak constitutive promoter includes, but is not limited to: pSLNl. Examples of strong inducible promoters include, but are not limited to: pGALl and pGALlO. An example of a medium inducible promoter includes, but is not limited to:

pGAL7. An example of a weak inducible promoter includes, but is not limited to: pGAL3.

[00448] Non-limiting examples of suitable eukaryotic promoters may include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector, construct, and promoter is well within the level of ordinary skill in the art. The expression vector or construct may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector or construct may also include appropriate sequences for amplifying expression.

[00449] Inducible promoters are well known in the art. Suitable inducible promoters may include, but are not limited to, a tetracycline-inducible promoter; an estradiol inducible promoter, a sugar inducible promoter, e.g, pGall or pSUC2, an amino acid inducible promoter, e.g., pMet25; a metal inducible promoter, e.g., pCupl, a methanol-inducible promoter, e.g., pAOXl, and the like.

[00450] In yeast, a number of vectors or constructs containing constitutive or inducible promoters may be used. For a review see, Current Protocols in Molecular Biology, Vol. 2, 1988, Ed. Ausubel, et ak, Greene Publish. Assoc. & Wiley Interscience, Ch. 13; Grant, et ak, 1987, Expression and Secretion Vectors for Yeast, in Methods in Enzymology, Eds. Wu & Grossman, 31987, Acad. Press, N.Y., Vol. 153, pp.516-544; Glover, 1986, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3; and Bitter, 1987, Heterologous Gene Expression in Yeast, Methods in Enzymology, Eds. Berger & Kimmel, Acad. Press, N.Y., Vol. 152, pp. 673-684; and The Molecular Biology of the Yeast Saccharomyces, 1982, Eds. Strathem et ak, Cold Spring Harbor Press, Vols. I and II. A constitutive yeast promoter such as pADH, pTDH3, pFBAl, pACTl, pCYCl, and pSLNl or an inducible promoter such as pGALl, pGALlO, pGAL7, and pGAL3 may be used (Cloning in Yeast, Ch. 3, R. Rothstein In: DNA Cloning Vol. 11, A Practical Approach, Ed. DM Glover, 1986, IRL Press, Wash., D.C.). Alternatively, vectors may be used which promote integration of foreign DNA sequences into the yeast chromosome. Generally, recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the S. cerevisiae TRP1 gene or a gene cassette encoding resistance to an antibiotic, etc.; and a promoter derived from a highly-expressed gene to direct transcription of the coding sequence. Such promoters can be derived from genetic sequences encoding glycolytic enzymes such as 3 -phosphogly cerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins, among others.

[00451] In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into the genome of the modified host cell disclosed herein. In some embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein is integrated into a chromosome of the modified host cell disclosed herein. In some

embodiments, one or more nucleic acids (e.g., heterologous) disclosed herein remains episomal (i.e., is not integrated into the genome or a chromosome of the modified host cell). In some embodiments, at least one of the one or more heterologous nucleic acids disclosed herein is maintained extrachromosomally (e.g., on a plasmid or artificial chromosome). The gene copy number of one or more genes encoding one or more polypeptides disclosed herein, such as a BBE or BBE-like polypeptide, may be selected to reduce expression of the one or more polypeptides disclosed herein, such as a BBE or BBE-like polypeptide.

Reducing expression by limiting the gene copy number may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability.

[00452] As will be appreciated by the skilled artisan, slight changes in nucleotide sequence do not necessarily alter the amino acid sequence of the encoded polypeptide. It will be appreciated by persons skilled in the art that changes in the identities of nucleotides in a specific gene sequence that change the amino acid sequence of the encoded polypeptide may result in reduced or enhanced effectiveness of the genes and that, in some applications (e.g., anti-sense, co-suppression, or RNAi), partial sequences often work as effectively as full length versions. The ways in which the nucleotide sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. In certain embodiments, effectiveness may easily be tested by, for example, conventional gas chromatography. All such variations of the genes are therefore included as part of the present disclosure.

[00453] Genomic deletion of the open reading frame encoding the protein may abolish all expression of a gene. Downregulation of a gene can be accomplished in several ways at the DNA, RNA, or protein level, with the result being a reduction in the amount of active protein in the cell. Truncations of the open reading frame or the introduction of mutations that destabilize the protein or reduce catalytic activity achieve a similar goal, as does fusing a“degron” polypeptide that destabilizes the protein. Engineering of the regulatory regions of the gene can also be used to change gene expression. Alteration of the promoter sequence or replacement with a different promoter is one method. Truncation of the terminator, known as decreased abundance of mRNA perturbation (DAmP), is also known to reduce gene expression. Other methods that reduce the stability of the mRNA include the use of cis- or trans-acting ribozymes, e.g., self-cleaving ribozymes, or RNA elements that recruit an exonuclease, or antisense DNA. RNAi may be used to silence genes in budding yeast strains via import of the required protein factors from other species, e.g., Drosha or Dice

(Drinnenberg et al 2009). Gene expression may also be silenced in S. cerevisiae via recruitment of native or heterologous silencing factors or repressors, which may be accomplished at arbitrary loci using the D-Cas9 CRISPR system (Qi et al 2013). Protein level can also be reduced by engineering the amino acid sequence of the target protein. A variety of degron sequences may be used to target the protein for rapid degradation, including, but not limited to, ubiquitin fusions and N-end rule residues at the amino terminus. These methods may be implemented in a constitutive or conditional fashion.

[00454] In some embodiments, the modified host cell comprises two or more nucleotide sequences linked via a T2A or an IRES element, allowing for co-expression of the sequences under the control of a single promoter and terminator sequence. In some embodiments, a T2A element is introduced between a nucleotide sequence encoding a BBE or BBE-like polypeptide upstream and a nucleotide sequence encoding a reporter polypeptide, such as a GFP polypeptide, downstream, resulting in two polypeptides separated at the GP (glycine-proline) junction at the end of the T2A element. Use of a T2A element in combination with a reporter polypeptide, such as a GFP polypeptide, may allow for screening or selection of modified host cells that are expressing a polypeptide of interest, as any modified host cells positive for the reporter polypeptide should also express the polypeptide of interest because they are both derived from the same transcript. Such screening may allow for rapid assessment of the solubilization and expression of BBE or BBE-like polypeptides in modified host cells comprising one or more modifications to modulate the expression of one or more secretory pathway polypeptides and/or one or more heterologous nucleic acids comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide. In certain such embodiments, the BBE or BBE-like polypeptide may comprise a signal sequence polypeptide.

[00455] The disclosure provides an assay for screening for expression of a BBE or BBE-like polypeptide in a modified host cell disclosed herein, wherein the modified host cell comprises a nucleotide sequence encoding a BBE or BBE-like polypeptide and a nucleotide sequence encoding a reporter polypeptide, wherein a T2A or an IRES element is introduced between the nucleotide sequence encoding the BBE or BBE-like polypeptide upstream and the nucleotide sequence encoding a reporter polypeptide downstream, said assay comprising: a) culturing the modified host cell in a culture medium and b) detecting the reporter polypeptide. In certain such embodiments, the reporter polypeptide is a GFP polypeptide. In certain such embodiments, the GFP polypeptide is detected by fluorescence detection. In certain such embodiments, a T2A element is used. Induction Systems

[00456] To adapt to a constantly changing environment, microbes such as yeast have evolved a wide range of natural inducible promoter systems. Any promoter that is regulated by a small molecule or change in environment (temperature, pH, oxygen level, osmolarity, oxidative damage) can in principle be converted into an inducible system for the expression of heterologous genes. The best known system in S. cerevisiae is the galactose regulon, which is strongly repressed by glucose and activated by galactose. Heterologous genetic pathways under the control of galactose-inducible promoters are regulated in the same way, and thus an engineered strain can be grown in glucose media to build biomass, and then switched to galactose to induce pathway expression. A range of expression levels can be achieved, from very strong pGALl to relatively weak pGAL3. However, galactose may be expensive and a poor carbon source for S. cerevisiae. Therefore, for industrial applications, it may be advantageous to re-engineer the regulon such that the cells can be induced in a non galactose media. The galactose regulon can be modified for this purpose in many ways, including:

• Overexpressing the negative regulator of GAL80, GAL3, from an inducible promoter, e.g., pSUC2-GAL3, such that switching from glucose to sucrose relieves GAL80 expression and activates the pathway.

• Deleting the repressor GAL80 and replacing the native GAL4 cassette with a version under the control of a sucrose inducible promoter, e.g., pSUC2- GAL4, such that expression is induced by a switch from glucose to sucrose.

• Replacing the native GAL80 gene with an inducible version, e.g., pSUC2- GAL80, such that expression is induced by a switch from sucrose to glucose.

[00457] These strategies often require fine-tuning of the activator and repressor levels to achieve the proper dynamics (very low or no expression in the off state, and desired expression level in the on state). There are a variety of ways to fine tune protein expression, including use of protein stabilization or degradation tags (e.g., degrons) or use of

temperature sensitive mutants of the activators or regulators. In the examples above, the pSUC2 promoter is used to induce the galactose regulon in sucrose media. However, any inducible promoter can be used for this purpose, or for control of individual genes outside of the context of the galactose regulon. The list below provides some examples:

• Phosphate regulated promoters, e.g., pPH05

• Carbon source regulated promoters, e.g., pADH2

• Amino acid regulated promoters, e.g., pMET25 • Metal ion induced promoters, e.g., pCUPl

• Temperature regulated promoters, e.g., pHSPl2, pHSP26

• pH regulated promoters, e.g., pHSPl2, pHSP26

• Oxygen level regulated promoters, e.g., pDANl

• Oxidative stress regulated promoters, e.g., AHP1, TRR1, TRX2, TSA1, GPX2, GSH1, GSH2, GLR1, SOD1, or SOD2 genes.

• ER stress regulated promoters, e.g., unfolded protein response element

promoters.

[00458] In addition to these natural examples, there are a variety of synthetic inducible promoter systems. These are generally based on re-arrangement of native or foreign transcriptional elements into a basal promoter scaffold and/or fusions of activator domains and DNA binding domains to create novel transcription factors. Two examples are provided below:

• Estradiol-inducible systems involving fusion of the estradiol receptor to

DNA-binding and transcriptional activation domain, paired with synthetic or native promoters with binding sites.

• tet Trans Activator (tTA) or reverse tet Trans Activator (rtTA) systems paired with tetO-containing promoters.

[00459] In some embodiments, one of the above inducible promoter systems is used in a modified host cell of the disclosure. In some embodiments, the inducible promoter system is a natural inducible promoter system. In some embodiments, the inducible promoter system is a synthetic inducible promoter system. In some embodiments, a suitable media for culturing modified host cells of the disclosure comprises one or more of the inducers disclosed herein. Possible inducers include:

• Phosphate regulated promoters, e.g., pPH05

o KH2PO4

• Carbon source regulated promoters, e.g., pADH2

o Galactose (e.g., pGALl)

o Glucose (e.g., pADH2)

o Sucrose (e.g., pSETC2, pGPHl, pMALl2)

o Maltose (e.g., pMALl2, pMAL32)

• Amino acid regulated promoters, e.g., pMET25

o Methionine (e.g., pMET25)

o Lysine (e.g., pLYS9) o Other amino acids

• Metal ion induced promoters, e.g., pCUPl

o CuS0₄

• Temperature regulated promoters, e.g., pHSPl2, pHSP26

o Change in temperature, e.g., 30 °C to 37 °C

• pH regulated promoters, e.g., pHSPl2, pHSP26

o Change in pH, e.g., pH 6 to pH 4

• Oxygen level regulated promoters, e.g., pDANl

o Change in oxygen level, e.g., 20% to 1% dissolved oxygen levels

• Oxidative stress regulated promoters, e.g., pSODl

o Addition of hydrogen peroxide or superoxide-generating drug menadione

• ER stress regulated promoters, e.g., unfolded protein response element promoters. o Tunicamycin, or expression of proteins prone to misfolding (e.g., cannabinoid synthases)

• Estradiol-inducible systems involving fusion of the estradiol receptor to DNA- binding and transcriptional activation domain, paired with synthetic or native promoters with binding sites.

o Estradiol

o Doxycyclin

Codon Usage and Nucleic Acids

[00460] As is well known to those of skill in the art, it is possible to improve the expression of a heterologous nucleic acid in a host organism by replacing the nucleotide sequences coding for a particular amino acid (i.e., a codon) with another codon which is better expressed in the host organism (i.e., codon-optimization). One reason that this effect arises is due to the fact that different organisms show preferences for different codons. In some embodiments, a nucleic acid disclosed herein is modified or optimized such that the nucleotide sequence reflects the codon preference for the particular host cell. For example, the nucleotide sequence will in some embodiments be modified or optimized for yeast codon preference. In some embodiments, a nucleotide sequence disclosed herein is codon- optimized. See, e.g., Bennetzen and Hall (1982) J Biol. Chem. 257(6): 3026-3031. [00461] Statistical methods have been generated to analyze codon usage bias in various organisms and many computer algorithms have been developed to implement these statistical analyses in the design of codon optimized gene sequences (Lithwick G, Margalit H (2003) Hierarchy of sequence-dependent features associated with prokaryotic translation. Genome Research 13: 2665-73). Other modifications in codon usage to increase protein expression that are not dependent on codon bias have also been described (Welch et al. (2009). In some embodiments, codon optimization of the nucleotide sequence may result in an increase in the desired polypeptide or enzyme catalytic activity in the modified host cell.

[00462] In some embodiments, the codon usage of a nucleotide sequence is modified or optimized such that the level of translation of the encoded mRNA is decreased. In some embodiments, a codon-optimized nucleotide sequence may be optimized such that the level of translation of the encoded mRNA is decreased. Reducing the level of translation of an mRNA by modifying codon usage may be achieved by modifying the nucleotide sequence to include codons that are rare or not commonly used by the host cell. Codon usage tables for many organisms are available that summarize the percentage of time a specific organism uses a specific codon to encode for an amino acid. Certain codons are used more often than other,“rare” codons. The use of“rare” codons in a nucleotide sequence generally decreases its rate of translation. Thus, e.g., the nucleotide sequence is modified by introducing one or more rare codons, which affect the rate of translation, but not the amino acid sequence of the polypeptide translated. For example, there are six codons that encode for arginine: CGT, CGC, CGA, CGG, AGA, and AGG. In E. coli the codons CGT and CGC are used far more often (encoding approximately 40% of the arginines in E. coli each) than the codon AGG (encoding approximately 2% of the arginines in E. coli). Modifying a CGT codon within the sequence of a gene to an AGG codon would not change the sequence of the polypeptide, but would likely decrease the gene’s rate of translation. In some embodiments, a codon- optimized nucleotide sequence may be optimized such that the rate of translation of the encoded mRNA is decreased. Slowing translation of the mRNA encoded by nucleotide sequences encoding a BBE or BBE-like polypeptide through use of codon-optimized nucleotide sequences encoding a BBE or BBE-like polypeptide may prevent saturation of the secretory pathway leading to possible protein degradation and/or modified host cell death or a loss of modified host cell viability. Without wishing to be bound by theory, slowing translation of the mRNA encoded by nucleotide sequences encoding a BBE or BBE-like polypeptide may improve translocation of the nascent BBE or BBE-like polypeptide chain to the endoplasmic reticulum (ER), assisting in delivery of the nascent BBE or BBE-like polypeptide chain to the secretory pathway.

[00463] In some embodiments, a codon-optimized nucleotide sequence may be optimized for expression in a yeast cell. In certain such embodiments, the yeast cell is Saccharomyces cerevisiae.

[00464] Further, it will be appreciated that this disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art and illustrated in the following table.

Codon Degeneracies

[00465] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide, wherein the BBE or BBE- like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE- like polypeptide, wherein the BBE or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a fungal cell. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide, wherein the BBE or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a bacterial cell. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide. Some

embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from

Nicotiana tabacum. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans . Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum. Some

embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila.

[00466] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO:9l. Some embodiments of the disclosure relate to a nucleic acid comprising a codon- optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO:9l, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:9l.

[00467] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:9l. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:9l . Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO:9l. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a

tetrahydrocannabinolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO:9l.

[00468] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO:93. Some embodiments of the disclosure relate to a nucleic acid comprising a codon- optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO:93, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a

cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 93. [00469] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:93. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:93. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO:93. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabichromenic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO:93.

[00470] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. Some embodiments of the disclosure relate to a nucleic acid comprising a codon- optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131, or a codon degenerate nucleotide sequence of any of the foregoing. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131.

[00471] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a

cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131. Some

embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon- optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 125, SEQ ID NO: 126, SEQ ID NO: 127, SEQ ID NO: 128, SEQ ID NO: 129, SEQ ID NO: 130, or SEQ ID NO: 131.

[00472] The disclosure provides nucleic acids comprising a nucleotide sequence encoding a cannabidiolic acid synthase polypeptide lacking a stop codon. Nucleic acids comprising a nucleotide sequence encoding cannabidiolic acid synthase polypeptides lacking the stop codon may be useful for expressing said polypeptides in a construct comprising T2A elements. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 107

(corresponds to SEQ ID NO: 125 lacking the stop codon), SEQ ID NO: 109 (corresponds to SEQ ID NO: 126 lacking the stop codon), SEQ ID NO: 110 (corresponds to SEQ ID NO: 127 lacking the stop codon), SEQ ID NO: 111 (corresponds to SEQ ID NO: 128 lacking the stop codon), SEQ ID NO: 112 (corresponds to SEQ ID NO: 129 lacking the stop codon), SEQ ID NO: 113 (corresponds to SEQ ID NO: 130 lacking the stop codon), or SEQ ID NO: 114 (corresponds to SEQ ID NO: 131 lacking the stop codon). Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114, or a codon degenerate nucleotide sequence of any of the foregoing. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: l07, SEQ ID NO: l09, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114.

[00473] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a

cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114. Some

embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a cannabidiolic acid synthase polypeptide, wherein the codon- optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, or SEQ ID NO: 114.

[00474] In some embodiments, the nucleic acid comprises a codon-optimized nucleotide sequence encoding cannabidiolic acid synthase polypeptide, wherein the cannabidiolic acid synthase polypeptide is a truncated cannabidiolic acid synthase polypeptide with an N-terminal truncation to remove the native signal sequence polypeptide. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO:95 or SEQ ID NO: 133. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO:95 or SEQ ID NO: 133, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO:95 or SEQ ID NO: 133. In some embodiments, the nucleic acids comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide also comprise nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting cannabidiolic acid synthase polypeptide is modified with the signal sequence polypeptide.

[00475] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:95 or SEQ ID NO: 133. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO:95 or SEQ ID NO: 133. Some

embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO:95 or SEQ ID NO: 133. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a truncated cannabidiolic acid synthase polypeptide, wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 95 or SEQ ID NO: 133.

[00476] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID

NO: 115. Some embodiments of the disclosure relate to a nucleic acid comprising a codon- optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 115, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE

polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 115.

[00477] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 115. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 115. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE

polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 115. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE polypeptide from Eschscholzia californica , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 115.

[00478] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 119. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 119, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from

Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 119.

[00479] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 119. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE- like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon- optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 119. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from

Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 119. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 119.

[00480] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 121. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6- hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 121, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy-D- nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon- optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 121.

[00481] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 121. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 121. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy-D- nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon- optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 121. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 121.

[00482] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 117. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase

polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 117, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from

Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 117.

[00483] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO:l 17. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a

daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 117. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from

Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 117. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a daurichromenic acid synthase polypeptide from Rhododendron dauricum , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 117.

[00484] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 123. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence is set forth in SEQ ID NO: 123, or a codon degenerate nucleotide sequence thereof. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has 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%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to SEQ ID NO: 123.

[00485] Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 80% sequence identity to SEQ ID NO: 123. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a

xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 85% sequence identity to SEQ ID NO: 123. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from

Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 90% sequence identity to SEQ ID NO: 123. Some embodiments of the disclosure relate to a nucleic acid comprising a codon-optimized nucleotide sequence encoding a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila , wherein the codon-optimized nucleotide sequence has at least 95% sequence identity to SEQ ID NO: 123.

[00486] In some embodiments, the nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide comprises nucleotide sequences encoding a signal sequence polypeptide. In certain such embodiments, after transcription and translation, the resulting BBE or BBE-like polypeptide is modified with the signal sequence polypeptide. In some embodiments, the signal sequence polypeptide is a secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

In some embodiments, the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide. In certain such embodiments, the endoplasmic reticulum retention signal sequence polypeptide is a HDEL polypeptide or a KDEL polypeptide. In some embodiments, the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide. In certain such embodiments, the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide. In some embodiments, the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mitochondrial targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a Golgi targeting signal sequence polypeptide. In some embodiments, the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide. In some embodiments, the peroxisome targeting signal sequence polypeptide is a PEX8 polypeptide. In some embodiments, the secretory signal sequence polypeptide is a mating factor secretory signal sequence polypeptide (e.g., a MF polypeptide or an evolved MF polypeptide (MFev)).

[00487] In some embodiments, the nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a nucleotide sequence encoding an AGA2t polypeptide. In certain such embodiments, after transcription and translation, the resulting BBE or BBE-like polypeptide is fused to the AGA2t polypeptide (e.g., a fusion polypeptide).

[00488] In some embodiments, the nucleic acid comprising a codon-optimized nucleotide sequence encoding a BBE or BBE-like polypeptide comprises a nucleotide sequence encoding a GFP polypeptide. In certain such embodiments, after transcription and translation, the resulting BBE or BBE-like polypeptide is fused to the GFP polypeptide (e.g., a fusion polypeptide).

[00489] In some embodiments, the nucleic acid disclosed herein is isolated. In some embodiments, the nucleic acid disclosed herein is purified. In some embodiments, the nucleic acid disclosed herein is isolated or purified.

[00490] Further included are nucleic acids that hybridize to the nucleic acids disclosed herein. Hybridization conditions may be stringent in that hybridization will occur if there is at least a 90%, 95%, or 97% sequence identity with the nucleotide sequence present in the nucleic acid encoding the polypeptides disclosed herein. The stringent conditions may include those used for known Southern hybridizations such as, for example, incubation overnight at 42 °C in a solution having 50% formamide, 5><SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5xDenhardt’s solution, 10% dextran sulfate, and 20 micrograms/milliliter denatured, sheared salmon sperm DNA, following by washing the hybridization support in O.l xSSC at about 65 °C. Other known hybridization conditions are well known and are described in Sambrook et ak, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor, N.Y. (2001).

[00491] The length of the nucleic acids disclosed herein may depend on the intended use. For example, if the intended use is as a primer or probe, for example for PCR

amplification or for screening a library, the length of the nucleic acid will be less than the full length sequence, for example, 15-50 nucleotides. In certain such embodiments, the primers or probes may be substantially identical to a highly conserved region of the nucleotide sequence or may be substantially identical to either the 5’ or 3’ end of the nucleotide sequence. In some cases, these primers or probes may use universal bases in some positions so as to be“substantially identical” but still provide flexibility in sequence recognition. It is of note that suitable primer and probe hybridization conditions are well known in the art.

[00492] Some embodiments of the disclosure relate to a vector comprising one or more nucleic acids disclosed herein. Some embodiments of the disclosure relate to an expression construct comprising one or more nucleic acids disclosed herein.

Exemplary Cell Culture Conditions

[00493] The disclosure provides methods for expressing a Berberine bridge enzyme (BBE) or BBE-like polypeptide, the methods comprising culturing a modified host cell of the disclosure in a culture medium. [00494] Suitable media for culturing modified host cells of the disclosure may include standard culture media (e.g., Luria-Bertani broth, optionally supplemented with one or more additional agents, such as an inducer (e.g., where nucleic acids disclosed herein are under the control of an inducible promoter, etc.); standard yeast culture media; and the like). In some embodiments, the culture medium can be supplemented with a fermentable sugar (e.g., a hexose sugar or a pentose sugar, e.g., glucose, xylose, galactose, and the like). In some embodiments, the culture medium can be supplemented with pretreated cellulosic feedstock (e.g., wheat grass, wheat straw, barley straw, sorghum, rice grass, sugarcane straw, bagasse, switchgrass, com stover, corn fiber, grains, or any combination thereof). In some

embodiments, the culture medium can be supplemented with oleic acid. In some

embodiments, the culture medium comprises a non-fermentable carbon source. In certain such embodiments, the non-fermentable carbon source comprises ethanol. In some embodiments, the suitable media comprises an inducer. In certain such embodiments, the inducer comprises galactose. In some embodiments, the inducer comprises KH2PO4, galactose, glucose, sucrose, maltose, an amino acid (e.g., methionine, lysine), CuSCri, a change in temperature (e.g., 30 °C to 37 °C), a change in pH (e.g., pH 6 to pH 4), a change in oxygen level (e.g., 20% to 1% dissolved oxygen levels), addition of hydrogen peroxide or superoxide-generating drug menadione, tunicamycin, expression of proteins prone to misfolding (e.g., cannabinoid synthases), estradiol, or doxycycline. Additional induction systems are detailed herein.

[00495] The carbon source in the suitable media can vary significantly, from simple sugars like glucose to more complex hydrolysates of other biomass, such as yeast extract.

The addition of salts generally provide essential elements such as magnesium, nitrogen, phosphorus, and sulfur to allow the cells to synthesize polypeptides and nucleic acids. The suitable media can also be supplemented with selective agents, such as antibiotics, to select for the maintenance of certain plasmids and the like. For example, if a microorganism is resistant to a certain antibiotic, such as ampicillin or tetracycline, then that antibiotic can be added to the medium in order to prevent cells lacking the resistance from growing. The suitable media can be supplemented with other compounds as necessary to select for desired physiological or biochemical characteristics, such as particular amino acids and the like.

[00496] In some embodiments, modified host cells disclosed herein are grown in minimal medium or minimal media. As used herein, the terms“minimal medium” or “minimal media” may refer to media comprising a defined composition of nutrients, generally chosen for minimal cost, while still allowing for robust growth and production. As used herein, the terms“minimal medium” or“minimal media” may refer to media containing: (1) one or more carbon sources for cellular (e.g., bacterial or yeast) growth; (2) various salts, which can vary among cellular (e.g., bacterial or yeast) species and growing conditions; (3) vitamins and trace elements; and (4) water. Generally, but not always, minimal media lacks one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids). Minimal media may also comprise growth factors, inducers, and repressors. In some embodiments, minimal media or minimal medium affords higher biomass formation in a fermentation tank compared to rich medium or rich media.

[00497] In some embodiments, modified host cells disclosed herein are grown in rich medium or rich media. In certain such embodiments, the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, yeast extract,

Bacto peptone, and dextrose (glucose). In certain such embodiments, the rich medium or rich media comprises yeast extract peptone dextrose (YPD) media comprising water, 10 g/L yeast extract, 20 g/L Bacto peptone, and 20 g/L dextrose (glucose). In some embodiments, the rich medium or rich media comprises YP + galactose and glucose. In some

embodiments, the rich medium or rich media comprises YP + 20 g/L galactose or YP + 40 g/L galactose and 1 g/L glucose. In some embodiments, rich medium or rich media affords greater cell density in fermentation compared to minimal media or minimal medium.

[00498] Materials and methods suitable for the maintenance and growth of the recombinant cells of the disclosure are described herein, e.g., in the Examples section. Other materials and methods suitable for the maintenance and growth of cell (e.g., bacterial or yeast) cultures are well known in the art. Exemplary techniques can be found in International Publication No. W02009/076676, U.S. Patent Application No. 12/335,071 (U.S. Publ. No. 2009/0203102), WO 2010/003007, US Publ. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/0003716, Manual of Methods for General Bacteriology Gerhardt et al, eds), American Society for Microbiology, Washington, D.C. (1994) or Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989) Sinauer Associates, Inc., Sunderland, MA.

[00499] Standard cell culture conditions can be used to culture the modified host cells disclosed herein (see, for example, WO 2004/033646 and references cited therein). In some embodiments, cells are grown and maintained at an appropriate temperature, gas mixture, and pH (such as at about 20 °C to about 37 °C, at about 0.04% to about 84% CO2, at about 0% to about 100% dissolved oxygen, and at a pH between about 2 to about 9). In some embodiments, modified host cells disclosed herein are grown at about 34 °C in a suitable cell culture medium. In some embodiments, modified host cells disclosed herein are grown at about 20 °C to about 37 °C in a suitable cell culture medium. While the growth optimum for S. cerevisiae is about 30 °C, culturing cells at a higher temperature, e.g., 34 °C may be advantageous by reducing the costs to cool industrial fermentation tanks. In some embodiments, modified host cells disclosed herein are grown at 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, or about 37 °C in a suitable cell culture medium. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 9.0 (such as about pH 3.0, about pH 3.5, about pH 4.0, about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 6.0 to about pH 8.0 or about pH 6.5 to about pH 7.0). In some embodiments, the pH ranges for fermentation are between about pH 4.5 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 4.0 to about pH 6.0. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 6.0. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.5. In some embodiments, the pH ranges for fermentation are between about pH 3.0 to about pH 5.0. In some embodiments, the dissolved oxygen is between about 0% to about 10%, about 0% to about 20%, about 0% to about 30%, about 0% to about 40%, about 0% to about 50%, about 0% to about 60%, about 0% to about 70%, about 0% to about 80%, about 0% to about 90%, about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40% or about 10% to about 50%. In some embodiments, the CO2 level is between about 0.04% to about 0.1% CO2, about 0.04% to about 1% CO2, about 0.04% to about 5% CO2, about 0.04% to about 10% CO2, about 0.04% to about 20% CO2, about 0.04% to about 30% CO2, about 0.04% to about 40% CO2, about 0.04% to about 50% CO2, about 0.04% to about 60% CO2, about 0.04% to about 70% CO2, about 0.1% to about 5% CO2, about 0.1% to about 10% CO2, about 0.1% to about 20% CO2, about 0.1% to about 30% CO2, about 0.1% to about 40% CO2, about 0.1% to about 50% CO2, about 1% to about 5% CO2, about 1% to about 10% CO2, about 1% to about 20% CO2, about 1% to about 30% CO2, about 1% to about 40% CO2, about 1% to about 50% CO2, about 5% to about 10% CO2, about 10% to about 20% CO2, about 10% to about 30% CO2, about 10% to about 40% CO2, about 10% to about 50% CO2, about 10% to about 60% CO2, about 10% to about 70% CO2, about 10% to about 80% CO2, about 50% to about 60% CO2, about 50% to about 70% CO2, or about 50% to about 80% CO2. Modified host cells disclosed herein disclosed herein can be grown under aerobic, anoxic, microaerobic, or anaerobic conditions based on the requirements of the cells.

[00500] Standard culture conditions and modes of fermentation, such as batch, fed- batch, or continuous fermentation that can be used are described in International Publication No. WO 2009/076676, U.S. Patent Application No. 12/335,071 (U.S. Publ. No.

2009/0203102), WO 2010/003007, US Publ. No. 2010/0048964, WO 2009/132220, US Publ. No. 2010/0003716, the contents of each of which are incorporated by reference herein in their entireties. Batch and Fed- Batch fermentations are common and well known in the art and examples can be found in Brock, Biotechnology: A Textbook of Industrial

Microbiology, Second Edition (1989) Sinauer Associates, Inc.

Methods of Preparing Berberine Bridge Enzyme (BBE) or BBE-like Polypeptides

[00501] In an aspect, the present disclosure provides methods for preparing Berberine Bridge Enzyme (BBE) or BBE-like polypeptides. In certain such embodiments, the methods may comprise culturing a modified host cell of the disclosure in a culture medium. In some embodiments, the modified host cell of the disclosure is a Pichia sp. The method can comprise isolating and/or purifying the BBE or BBE-like polypeptides, as described herein.

[00502] In some embodiments, the method for preparing BBE or BBE-like

polypeptides comprises the step of isolating or purifying the BBE or BBE-like polypeptides. The BBE or BBE-like polypeptides of the disclosure can be expressed in modified host cells, as described herein, and isolated from the modified host cells and/or culture medium using any one or more of the well known techniques used for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography. Chromatographic techniques for isolation of the BBE or BBE-like polypeptides of the disclosure may include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel

electrophoresis, and affinity chromatography. In some embodiments, affinity

chromatography is used.

[00503] In some embodiments, the BBE or BBE-like polypeptides of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in various forms including but not limited to crude extracts (e.g., cell-free lysates), powders (e.g., shake-flask powders), lyophilizates, frozen stocks made with glycerol or another

cryoprotectant, and substantially pure preparations (e.g., DSP powders).

[00504] In some embodiments, the BBE or BBE-like polypeptides of the disclosure expressed in the modified host cells of the disclosure can be prepared and used in purified form. Generally, conditions for purifying a particular BBE or BBE-like polypeptide will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art.

EXAMPLES OF NON-LIMITING EMBODIMENTS OF THE DISCLOSURE

[00505] Embodiments of the present subject matter disclosed herein may be beneficial alone or in combination with one or more other embodiments. Without limiting the foregoing description, certain non-limiting embodiments of the disclosure, numbered 1-1 to 1-96 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered embodiments may be used or combined with any of the preceding or following individually numbered embodiments. This is intended to provide support for all such combinations of embodiments and is not limited to combinations of embodiments explicitly provided below.

[00506] Some embodiments of the disclosure are of Embodiment I:

[00507] Embodiment 1-1. A modified host cell for expressing a Berberine bridge enzyme (BBE) or BBE-like polypeptide, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides or a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, or a combination of any of the foregoing, and wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE- like polypeptide.

[00508] Embodiment 1-2. The modified host cell of Embodiment 1-1, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

[00509] Embodiment 1-3. The modified host cell of Embodiment 1-2, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), or one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing.

[00510] Embodiment 1-4. The modified host cell of Embodiment 1-3, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides.

[00511] Embodiment 1-5. The modified host cell of Embodiment 1-4, wherein the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide, a JEM1 polypeptide, a LHSl polypeptide, a SIS1 polypeptide, a SSB1 polypeptide, a CNE1 polypeptide, a CNS1 polypeptide, a PFD2s polypeptide, a PFDl polypeptide, a SSA1 polypeptide, a YDJ1 polypeptide, a SIL1 polypeptide, a SCJ1 polypeptide, a ROTl polypeptide, a CPR5 polypeptide, or a FPR1 polypeptide, or a combination of any of the foregoing.

[00512] Embodiment 1-6. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a JEM1 polypeptide and a KAR2 polypeptide.

[00513] Embodiment 1-7. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a LHS1 polypeptide and a KAR2 polypeptide.

[00514] Embodiment 1-8. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a SIL1 polypeptide and a KAR2 polypeptide.

[00515] Embodiment 1-9. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a CNE1 polypeptide and a KAR2 polypeptide. [00516] Embodiment I- 10. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a SIS1 polypeptide and a KAR2 polypeptide.

[00517] Embodiment 1-11. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a SSB1 polypeptide and a KAR2 polypeptide.

[00518] Embodiment 1-12. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a ROT1 polypeptide and a KAR2 polypeptide.

[00519] Embodiment 1-13. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide.

[00520] Embodiment 1-14. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a PFD1 polypeptide, a PFD2s polypeptide, and a KAR2 polypeptide.

[00521] Embodiment 1-15. The modified host cell of Embodiments 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide.

[00522] Embodiment 1-16. The modified host cell of any one of Embodiment 1-5 to 1-15, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[00523] Embodiment 1-17. The modified host cell of any one of Embodiments 1-5 to 1-15, wherein the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

[00524] Embodiment 1-18. The modified host cell of Embodiment 1-5, wherein the one or more chaperone or co-chaperone polypeptides comprise a SSB1 polypeptide.

[00525] Embodiment 1-19. The modified host cell of any one of Embodiments 1-3 to 1-18, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response.

[00526] Embodiment 1-20. The modified host cell of Embodiment 1-19, wherein the one or more polypeptides involved in unfolded protein response comprise an IRE1 polypeptide or a HACls polypeptide, or a combination of any of the foregoing. [00527] Embodiment 1-21. The modified host cell of Embodiment 1-20, wherein the one or more polypeptides involved in unfolded protein response comprise an IRE1 polypeptide.

[00528] Embodiment 1-22. The modified host cell of Embodiment 1-20 or

Embodiment 1-21, wherein the one or more polypeptides involved in unfolded protein response comprise a HACls polypeptide.

[00529] Embodiment 1-23. The modified host cell of Embodiment 1-19, wherein the one or more polypeptides involved in unfolded protein response comprise a transcription factor polypeptide or a lumenal sensor polypeptide that binds to an unfolded protein response element.

[00530] Embodiment 1-24. The modified host cell of any one of Embodiments 1-3 to 1-23, wherein the modified host cell comprises a synthetic polypeptide that binds to an unfolded protein response element.

[00531] Embodiment 1-25. The modified host cell of any one of Embodiments 1-3 to 1-24, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides.

[00532] Embodiment 1-26. The modified host cell of Embodiment 1-25, wherein the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

[00533] Embodiment 1-27. The modified host cell of any one of Embodiments 1-3 to 1-26, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides.

[00534] Embodiment 1-28. The modified host cell of Embodiment 1-27, wherein the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide.

[00535] Embodiment 1-29. The modified host cell of any one of Embodiments 1-3 to 1-28, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides. [00536] Embodiment 1-30. The modified host cell of Embodiment 1-29, wherein the one or more thiol oxidase polypeptides comprise an EROl polypeptide or an ERV2 polypeptide, or a combination of any of the foregoing.

[00537] Embodiment 1-31. The modified host cell of Embodiment 1-30, wherein the one or more thiol oxidase polypeptides comprise an EROl polypeptide.

[00538] Embodiment 1-32. The modified host cell of Embodiment 1-30 or

Embodiment 1-31, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide.

[00539] Embodiment 1-33. The modified host cell of any one of Embodiments 1-30 to 1-32, wherein the one or more thiol oxidase polypeptides comprise an ERV2 polypeptide.

[00540] Embodiment 1-34. The modified host cell of any one of Embodiments 1-1 to 1-33, wherein the modified host cell comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

[00541] Embodiment 1-35. The modified host cell of Embodiment 1-34, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, or one or more genes encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing.

[00542] Embodiment 1-36. The modified host cell of Embodiment 1-35, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides.

[00543] Embodiment 1-37. The modified host cell of Embodiment 1-36, wherein the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene or a MNS1 gene, or a combination of any of the foregoing.

[00544] Embodiment 1-38. The modified host cell of Embodiment 1-37, wherein the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

[00545] Embodiment 1-39. The modified host cell of any one of Embodiments 1-35 to 1-38, wherein the one or more genes encoding one or more secretory pathway

polypeptides deleted or downregulated comprise one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism. [00546] Embodiment 1-40. The modified host cell of Embodiment 1-39, wherein the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene.

[00547] Embodiment 1-41. The modified host cell of any one of Embodiments 1-35 to 1-40, wherein the one or more genes encoding one or more secretory pathway

polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides.

[00548] Embodiment 1-42. The modified host cell of Embodiment 1-41, wherein the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, a PRB1 gene, or a PRC1 gene, or a combination of any of the foregoing.

[00549] Embodiment 1-43. The modified host cell of Embodiment 1-42, wherein the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

[00550] Embodiment 1-44. The modified host cell of Embodiment 1-2, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in EIPR, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, wherein the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and a SSB1 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in EIPR comprise a HACls polypeptide, and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FADl polypeptide.

[00551] Embodiment 1-45. The modified host cell of Embodiment 1-1, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co- chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, and one or more genes encoding one or more glycosidase polypeptides, wherein the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

[00552] Embodiment 1-46. The modified host cell of any one of Embodiments 1-1 to 1-45, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide.

[00553] Embodiment 1-47. The modified host cell of any one of Embodiments 1-1 to 1-46, wherein the nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide is codon-optimized.

[00554] Embodiment 1-48. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue. [00555] Embodiment 1-49. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a fungal cell.

[00556] Embodiment 1-50. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a bacterial cell.

[00557] Embodiment 1-51. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a tetrahydrocannabinolic acid synthase polypeptide.

[00558] Embodiment 1-52. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a cannabichromenic acid synthase polypeptide.

[00559] Embodiment 1-53. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a cannabidiolic acid synthase polypeptide.

[00560] Embodiment 1-54. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE polypeptide from Eschscholzia californica.

[00561] Embodiment 1-55. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum.

[00562] Embodiment 1-56. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans.

[00563] Embodiment 1-57. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a daurichromenic acid synthase polypeptide from Rhododendron dauricum.

[00564] Embodiment 1-58. The modified host cell of any one of Embodiments 1-1 to 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

[00565] Embodiment 1-59. The modified host cell of any one of Embodiments 1-1 to 1-58, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprises a signal sequence polypeptide. [00566] Embodiment 1-60. The modified host cell of Embodiment 1-59, wherein the signal sequence polypeptide is a secretory signal sequence polypeptide.

[00567] Embodiment 1-61. The modified host cell of Embodiment 1-60, wherein the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide.

[00568] Embodiment 1-62. The modified host cell of Embodiment 1-60, wherein the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

[00569] Embodiment 1-63. The modified host cell of Embodiment 1-60, wherein the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide.

[00570] Embodiment 1-64. The modified host cell of Embodiment 1-60, wherein the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide.

[00571] Embodiment 1-65. The modified host cell of Embodiment 1-60, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide.

[00572] Embodiment 1-66. The modified host cell of Embodiment 1-65, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

[00573] Embodiment 1-67. The modified host cell of Embodiment 1-65, wherein the vacuolar localization signal sequence polypeptide is a PRClt polypeptide.

[00574] Embodiment 1-68. The modified host cell of Embodiment 1-60, wherein the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide.

[00575] Embodiment 1-69. The modified host cell of Embodiment 1-60, wherein the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide.

[00576] Embodiment 1-70. The modified host cell of Embodiment 1-51, wherein the tetrahydrocannabinolic acid synthase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:92.

[00577] Embodiment 1-71. The modified host cell of Embodiment 1-52, wherein the cannabichromenic acid synthase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:94. [00578] Embodiment 1-72. The modified host cell of Embodiment 1-53, wherein the cannabidiolic acid synthase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 108 or SEQ ID NO: 132.

[00579] Embodiment 1-73. The modified host cell of Embodiment 1-54, wherein the BBE polypeptide from Eschscholzia californica comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 116.

[00580] Embodiment 1-74. The modified host cell of Embodiment 1-55, wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 120.

[00581] Embodiment 1-75. The modified host cell of Embodiment 1-56, wherein the BBE-like 6-hydroxy -D-nicotine oxidase polypeptide from Paenarthrobacter

nicotinovorans comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 122.

[00582] Embodiment 1-76. The modified host cell of Embodiment 1-57, wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 118.

[00583] Embodiment 1-77. The modified host cell of Embodiment 1-58, wherein the xylooligosaccharide oxidase polypeptide from Myceliophthor a thermophila comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 124.

[00584] Embodiment 1-78. The modified host cell of any one of Embodiments 1-1 to 1-77, wherein the modified host cell is a eukaryotic cell.

[00585] Embodiment 1-79. The modified host cell of Embodiment 1-78, wherein the eukaryotic cell is a yeast cell.

[00586] Embodiment 1-80. The modified host cell of Embodiment 1-79, wherein the yeast cell is Saccharomyces cerevisiae.

[00587] Embodiment 1-81. The modified host cell of Embodiment 1-80, wherein the Saccharomyces cerevisiae is a protease-deficient strain of Saccharomyces cerevisiae.

[00588] Embodiment 1-82. The modified host cell of any one of Embodiments 1-1 to 1-81, wherein at least one of the one or more heterologous nucleic acids are integrated into the chromosome of the modified host cell.

[00589] Embodiment 1-83. The modified host cell of any one of Embodiments 1-1 to 1-81, wherein at least one of the one or more heterologous nucleic acids are maintained extrachromosomally. [00590] Embodiment 1-84. The modified host cell of any one of Embodiments 1-1 to 1-83, wherein at least one of the one or more heterologous nucleic acids are operably- linked to an inducible promoter.

[00591] Embodiment 1-85. The modified host cell of any one of Embodiments 1-1 to 1-83, wherein at least one of the one or more heterologous nucleic acids are operably- linked to a constitutive promoter.

[00592] Embodiment 1-86. The modified host cell of any one of Embodiments 1-1 to 1-85, wherein the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding an Internal Ribosome Entry Site (IRES) element.

[00593] Embodiment 1-87. The modified host cell of any one of Embodiments 1-1 to 1-85, wherein the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding a T2A element.

[00594] Embodiment 1-88. The modified host cell of any one of Embodiments 1-1 to 1-87, wherein the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding a green fluorescent protein (GFP) polypeptide.

[00595] Embodiment 1-89. A method of making a modified host cell of any one of Embodiments 1-1 to 1-88, the method comprising introducing into a host cell: a) one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

[00596] Embodiment 1-90. A method of making a modified host cell of any one of Embodiments 1-1 to 1-88, the method comprising introducing into a host cell: a) one or more vectors comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more vectors comprising one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

[00597] Embodiment 1-91. The method of Embodiment 1-89 or Embodiment 1-90, the method comprising introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides. [00598] Embodiment 1-92. A method for expressing a Berberine bridge enzyme (BBE) or BBE-like polypeptide, the method comprising culturing a modified host cell of any one of Embodiments 1-1 to 1-88 in a culture medium.

[00599] Embodiment 1-93. The method of Embodiment 1-92, wherein the culture medium comprises a fermentable sugar.

[00600] Embodiment 1-94. The method of Embodiment 1-92, wherein the culture medium comprises a pretreated cellulosic feedstock.

[00601] Embodiment 1-95. The method of Embodiment 1-92, wherein the culture medium comprises a non-fermentable carbon source.

[00602] Embodiment 1-96. The method of Embodiment 1-95, wherein the non- fermentable carbon source comprises ethanol.

[00603] Provided in Table 1 are amino acid and nucleotide sequences disclosed herein. Where a genus and/or species is noted, the sequence should not be construed to be limited only to the specified genus and/or species, but also includes other genera and/or species expressing said sequence. Orthologs of the sequences disclosed in Table 1 may also be encompassed by this disclosure. Nucleotide sequences indicated as codon optimized in Table 1 are codon optimized for expression in S. cerevisiae. In Table 1,“*” used as the end of a sequence denotes a stop codon.

Table 1: Amino acid and nucleotide sequences of the disclosure

EXAMPLES

[00604] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); bp, base pair(s); nt, nucleotide(s); and the like.

General Methods of the Examples

Yeast transformation methods

[00605] Each DNA construct comprising one or more heterologous nucleic acids disclosed herein (e.g., constructs detailed in Table 3) was integrated into Saccharomyces cerevisiae (CEN.PK2, Strain S4) with standard molecular biology techniques in an optimized lithium acetate (LiAc) transformation. Briefly, cells were grown overnight in yeast extract peptone dextrose (YPD) media at 30 °C with shaking (200 rpm), diluted to an OD600 of 0.1 in 100 mL YPD, and grown to an OD600 of 0.6 - 0.8. For each

transformation, 5 mL of culture was harvested by centrifugation, washed in 5 mL of sterile water, spun down again, resuspended in 1 mL of 100 mM Li Ac, and transferred to a microcentrifuge tube. Cells were spun down (13,000 x g) for 30 seconds, the supernatant was removed, and the cells were resuspended in a transformation mix consisting of 240 pL 50% PEG, 36 pL 1M LiAc, 10 pL boiled salmon sperm DNA, and 74 pL of donor DNA. Following a heat shock at 42 °C for 40 minutes, cells were recovered overnight in YPD media before plating on selective media. DNA integration was confirmed by colony PCR with primers specific to the integrations.

Yeast culturing conditions

[00606] Yeast colonies verified to contain the expected DNA assembly comprising one or more heterologous nucleic acids disclosed herein, modified host cells, were picked into 96-well microtiter plates containing 360 pL of YPD (10 g/L yeast extract, 20 g/L Bacto peptone, 20 g/L dextrose (glucose)) and sealed with a breathable film seal. Cells were cultured at 30 °C in a high capacity microtiter plate incubator shaking at 1000 rpm and 80% humidity for 2 days until the cultures reached carbon exhaustion. The growth-saturated cultures were subcultured into fresh plates containing YPGAL and optionally olivetolic acid if the modified host cells express a cannabinoid synthase polypeptide (10 g/L yeast extract, 20 g/L Bacto peptone, 20 or 40 g/L galactose, 1 g/L glucose and optionally 1 mM olivetolic acid), by taking 14.4 pL from the saturated cultures and diluting into 360 pL of fresh media and sealed with a breathable film seal. Providing modified host cells expressing cannabinoid synthase polypeptides olivetolic acid will afford cannabinoids such as CBDA and THCA. Modified host cells in the production media were cultured at 30 °C in a high capacity microtiter plate shaker at 1000 rpm and 80% humidity for an additional 3, 5, or 7 days prior to extraction and analysis. Upon completion, 100 pL of whole cell broth from the cultures provided olivetolic acid was diluted into 900 pL of methanol, sealed with a foil seal, and shaken at 1500 rpm for 60 seconds to extract the cannabinoids. After shaking, the plate was centrifuged at 1000 x g for 60 seconds to remove any solids. After centrifugation, 12 pL of supernatant was transferred to a fresh assay plate containing 228 pL of methanol (or more to maintain samples in the assay linear range), sealed with a foil seal, shaken for 60 seconds at 900 rpm, and analyzed by LC-MS.

Analytical methods

[00607] Samples were analyzed by LC-MS mass spectrometer (Agilent 6470) using a Phenomenex Kinetex Phenyl-Hexyl 2.1 x 30 mm, 2.6 pm analytical column with the following gradient (Mobile Phase A: LC-MS grade water with 0.1% formic acid; Mobile Phase B: LC-MS grade acetonitrile with 0.1% formic acid):

[00608] The mass spectrometer was operated in negative ion multiple reaction monitoring mode. Each cannabinoid or cannabinoid derivative was identified by retention time, determined from an authentic standard, and multiple reaction monitoring (MRM) transition:

Yeast cultivation in a bioreactor

[00609] Single yeast colonies comprising modified host cells disclosed herein are grown in 15 mL of Verduyn medium (originally described by Verduyn et al, Yeast 8(7): 501-17) with 50 mM succinate (pH 5.0) and 2% glucose in a 125 mL flask at 30 °C, with shaking at 200 rpm to an OD600 between 4 to 9. Glycerol is then added to the culture to a concentration of 20% and 1 mL vials of the modified host cell suspension are stored at -80 °C. One to two vials of modified host cells are thawed and grown in Verduyn medium with 50 mM succinate (pH 5.0) and 4% sucrose for 24 hours, then sub-cultured to an OD600 reading of 0.1 in the same media. After 24 hours of growth at 30 °C with shaking, 65 mL of culture is used to inoculate a 1.3-liter fermenter (Eppendorf DASGIP Bioreactor) with 585 mL of Verduyn fermentation media containing 20 g/L galactose optionally supplemented with olivetolic acid (1 mM, provided if the modified host cells express a cannabinoid synthase polypeptide). A poly-alpha-olefm may be added to the fermenter as an extractive agent. The fermenter is maintained at 30 °C and pH 5.0 with addition of NH₄OH. In an initial batch phase, the fermenter is aerated at 0.5 volume per volume per minute air (VVM) and agitation ramped to maintain 30% dissolved oxygen. After the initial sugar is consumed, the rise in dissolved oxygen triggers feeding of galactose + optionally olivetolic acid (800 g galactose per liter + 9.28 g olivetolic acid per liter) at 10 g galactose per liter per hour in pulses of 10 g galactose per liter doses.

[00610] Between pulses, the feed rate is lowered to 5 g galactose per liter per hour. Upon a 10% rise in dissolved oxygen, the feed rate is resumed at 10 g L^-1 hour^-1. As modified host cell density increases, dissolved oxygen is allowed to reach 0%, and the pulse dose is increased to 50 g galacose per liter. Oxygen transfer rate is maintained at rates representative of full-scale conditions of 100 mM per liter per hour by adjusting agitation as volume increased. Feed rate is adjusted dynamically to meet demand using an algorithm that alternates between a high feed rate and low feed rate. During the low feed rate, modified host cells should consume galactose and optionally olivetolic acid (if the modified host cells express a cannabinoid synthase polypeptide), and any overflow metabolites accumulated during the high feed rate. A rise in dissolved oxygen triggers the high feed rate to resume. The length of time spent in the low feed rate reflects the extent to which modified host cells are over- or under-fed in the prior high feed rate pulse; this information is then monitored and used to tune the high feed rate up or down, keeping the low feed rate within a defined range.

[00611] Over time, the feed rate matches sugar and optionally olivetolic acid (if the modified host cells express a cannabinoid synthase polypeptide) demand from modified host cells. This algorithm ensures minimal net accumulation of fermentation products other than cannabinoids in cultures of modified host cells expressing a cannabinoid synthase polypeptide with olivetolic acid present; biomass; and CO2. In some embodiments, the process continues for 5 to 14 days. In certain such embodiments, accumulated broth is removed daily and assayed for biomass and, in cultures of modified host cells expressing a cannabinoid synthase polypeptide with olivetolic acid, cannabinoid concentration. A concentrated solution of NH4H2PO4, trace metals and vitamins are added periodically to maintain steady state concentrations.

Example 1 : Assay of Expression of Cannabinoid Synthase Polypeptides

[00612] To examine the relationship between synthase codon-optimization and functional expression in naive or secretory pathway modified host strains, strains were constructed containing a CBDAS polypeptide fused to a GFP polypeptide via a cleavable peptide linker, T2A. The CBDAScoX-T2A-GFP cassettes (where X indicates the codon- optimization) express from a single promoter and terminator (pGALl and tTDHl, respectively) but T2A-mediated cleavage results in two separate proteins. In this manner the GFP polypeptide level can be used as a proxy for CBDAS polypeptide level, and the relationships among expression and activity could be investigated. The various CBDAS codon-optimized sequences fused to GFP via T2A were introduced into either a secretory engineering-naive parent (S29) or a secretory engineered parent (S228) and assayed both for GFP polypeptide expression and CBDA titer (Table 2 and FIGS. 29 and 30).

[00613] Strains containing CBDAScoX-T2A-GFP (in locus i7, from promoter pGALl, and terminator tTDHl; where X refers to codon-optimized sequences 1 (SEQ ID NO: 107), 2 (SEQ ID NO: 109), 3 (SEQ ID NO: 110), 4 (SEQ ID NO: 111), 5 (SEQ ID

NO: l 12), 7 (SEQ ID NO: l 13), or 8 (SEQ ID NO:l 14) were grown in 96-well plates in non inducing media (360 pL YPD) for two days. Then, these cultures were back-diluted into two plates, one for measuring GFP polypeptide level (360 pL YPG, assayed after 24 hrs of growth) and one for measuring titer (360 pL YPG+olivetolic acid, assayed after 72 hrs of growth). The GFP plate was assayed with a flow analyzer (BD Accuri C6plus) configured for detecting GFP polypeptide. The production plate was assayed with an LC-MS as described under the General Methods of the Examples. Representative strains comprising codon-optimized nucleotide sequences encoding CBDAS polypeptides are shown in Table 2. These codon-optimized nucleotide sequences lack the stop codon, enabling read-through and expression of the GFP polypeptide.

Table 2: Expression level and titer from CBDAS codon-optimizations (codon-opts) fused to GFP via cleavable T2A linker

Table 2 Legend: CBDAS codon-optimized sequences fused to GFP via cleavable T2A linker allow for measurement of CBDAS polypeptide expression level via GFP polypeptide level as a proxy. These cassettes were tested in a non-engineered CBGA producer (S29) or in a secretory-engineered CBGA producer (S228). The data are calculated from four to eight replicate cultures. Data shown graphically in FIG. 29.

[00614] To explore the solubility and subcellular expression of the different synthase codon-optimization variants in naive or secretory-engineered host strains, strains were constructed comprising a CBDAS polypeptide fused to GFP polypeptide via a non-cleavable flexible linker, GS12. The resulting fusion polypeptide can be used to examine how engineering the secretory pathway affects synthase functional expression.

[00615] Strains containing CBDAScoX-GSl2-GFP (in locus i7, from promoter pGALl, and terminator tTDHl) were grown in 96-well plates in non-inducing media (360 pL YPD) for two days. Then, these cultures were back-diluted (360 pL YPG) and assayed after 24 hrs of growth. Cultures were washed and resuspended in lx PBS and imaged on a Zeiss LSM 710 confocal system mounted on a Zeiss inverted microscope with a 63 ^c objective and processed using Zeiss Zen software (FIG. 28).

[00616] Strains expressing other BBE or BBE-like polypeptides could be tested in the same manner as the strains detailed above. Table 3: Constructs and strains used in the Examples

* If a strain has a parent strain, it is a child strain. All of the constructs present in the parent strain are also all present in the child strain.

** S4 is CEN.PK113-1 A with genotype MATalpha; UR A3; TRP1; LEU2; HIS3; MAL2-8C; SUC2

*** S228 has no BBE or BBE-like polypeptide but is a parent strain for several strains.

**** S280 is a“base strain” for integrating the different BBE or BBE-like polypeptide codon optimizations into a strain with secretory pathway engineering. In S280, the nucleotide sequence encoding a GFP polypeptide is added to locus i7, creating a strain without synthase. Subsequently, the BBE or BBE-like polypeptide codon optimization variant is then added on top of the nucleotide sequence encoding the GFP polypeptide, replacing it.

***** S369 is a“base strain” with extensive secretory pathway engineering used to test different cannabinoid synthases and codon optimizations. In S369, the nucleotide sequence encoding a GFP polypeptide is added and the synthase polypeptide in parent S270 is deleted, reverting the locus to its wild type state and creating a strain without synthase. Subsequently, the THCAS or other cannabinoid synthase codon optimization variant replaces the nucleotide sequence encoding the GFP polypeptide.

Table 4: List of Regulatory and Other Elements

are listed in Table 4. Flanking Homology regions direct recombination at specific genomic loci. Flanking homology upstream sequences are denoted with a“u”, and downstream with a "d"'.“I” indicates an intergenic integration site, e.g., ui7, di7 are the regions flanking intergenic region 7. Integrations that delete an open reading frame have flanking homology with the deleted gene indicated, e.g., uPEP4, dPEP4 are the regions flanking the PEP4 gene. Synthetic Recombination Sequences (SRS) direct internal recombination of two DNA constructs targeted for integration at the same locus. Linkers are short sequences used in assembly the DNA constructs, they are intervening between the indicated parts. Linkers Gl, G7, G10, RG1, and LTTDH1 contain the last 36 bp of the upstream DNA part; in cases where these linkers are used assume that the linker reconstitutes sequence omitted from the upstream part to create a seamless junction with the downstream part. Linkers DO and D9 are terminal linkers that direct entry of the DNA constructs into cloning vectors, and are not integrated into the genome. Where no linker is shown between parts, the junction is also seamless.

[00617] Although the present disclosure has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the disclosure. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present disclosure. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMS What is claimed is:

1. A modified host cell for expressing a Berberine bridge enzyme (BBE) or BBE-like polypeptide, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides or a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, or a combination of any of the foregoing, and wherein the modified host cell comprises one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide.

2. The modified host cell of claim 1, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

3. The modified host cell of claim 2, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more glycosidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), or one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing.

4. The modified host cell of claim 3, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway

polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides.

5. The modified host cell of claim 4, wherein the one or more chaperone or co- chaperone polypeptides comprise a KAR2 polypeptide, a JEM1 polypeptide, a LHS1 polypeptide, a SIS1 polypeptide, a SSB1 polypeptide, a CNE1 polypeptide, a CNS1 polypeptide, a PFD2s polypeptide, a PFD1 polypeptide, a SSA1 polypeptide, a YDJ1 polypeptide, a SIL1 polypeptide, a SCJ1 polypeptide, a ROT1 polypeptide, a CPR5 polypeptide, or a FPR1 polypeptide, or a combination of any of the foregoing.

6. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a JEM1 polypeptide and a KAR2 polypeptide.

7. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a LHS1 polypeptide and a KAR2 polypeptide.

8. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a SIL1 polypeptide and a KAR2 polypeptide.

9. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a CNE1 polypeptide and a KAR2 polypeptide.

10. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a SIS1 polypeptide and a KAR2 polypeptide.

11. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a SSB1 polypeptide and a KAR2 polypeptide.

12. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a ROT1 polypeptide and a KAR2 polypeptide.

13. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide.

14. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a PFD1 polypeptide, a PFD2 polypeptide, and a KAR2 polypeptide.

15. The modified host cell of claim 5, wherein the one or more chaperone or co- chaperone polypeptides comprise a KAR2 polypeptide.

16. The modified host cell of any one of claims 5-15, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

17. The modified host cell of any one of claims 5-15, wherein the modified host cell comprises three or more heterologous nucleic acids comprising a nucleotide sequence encoding a KAR2 polypeptide.

18. The modified host cell of claim 5, wherein the one or more chaperone or co chaperone polypeptides comprise a SSB1 polypeptide.

19. The modified host cell of any one of claims 3-18, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response.

20. The modified host cell of claim 19, wherein the one or more polypeptides involved in unfolded protein response comprise an IRE1 polypeptide or a HACls polypeptide, or a combination of any of the foregoing.

21. The modified host cell of claim 20, wherein the one or more polypeptides involved in unfolded protein response comprise an IRE1 polypeptide.

22. The modified host cell of claim 20 or claim 21, wherein the one or more polypeptides involved in unfolded protein response comprise a HACls polypeptide.

23. The modified host cell of claim 19, wherein the one or more polypeptides involved in unfolded protein response comprise a transcription factor polypeptide or a lumenal sensor polypeptide that binds to an unfolded protein response element.

24. The modified host cell of any one of claims 3-23, wherein the modified host cell comprises a synthetic polypeptide that binds to an unfolded protein response element.

25. The modified host cell of any one of claims 3-24, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides.

26. The modified host cell of claim 25, wherein the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

27. The modified host cell of any one of claims 3-26, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides.

28. The modified host cell of claim 27, wherein the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide.

29. The modified host cell of any one of claims 3-28, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides.

30. The modified host cell of claim 29, wherein the one or more thiol oxidase polypeptides comprise an EROl polypeptide or an ERV2 polypeptide, or a combination of any of the foregoing.

31. The modified host cell of claim 30, wherein the one or more thiol oxidase polypeptides comprise an EROl polypeptide.

32. The modified host cell of claim 30 or claim 31, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding an EROl polypeptide.

33. The modified host cell of any one of claims 30-32, wherein the one or more thiol oxidase polypeptides comprise an ERV2 polypeptide.

34. The modified host cell of any one of claims 1-33, wherein the modified host cell comprises a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

35. The modified host cell of claim 34, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, or one or more genes encoding one or more vacuolar proteinase polypeptides, or a combination of any of the foregoing.

36. The modified host cell of claim 35, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more glycosidase polypeptides.

37. The modified host cell of claim 36, wherein the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene or a MNS1 gene, or a combination of any of the foregoing.

38. The modified host cell of claim 37, wherein the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

39. The modified host cell of any one of claims 35-38, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism.

40. The modified host cell of claim 39, wherein the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene.

41. The modified host cell of any one of claims 35-40, wherein the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides.

42. The modified host cell of claim 41, wherein the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, a PRB1 gene, or a PRC1 gene, or a combination of any of the foregoing.

43. The modified host cell of claim 42, wherein the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene.

44. The modified host cell of claim 2, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides, wherein the one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more protein disulfide isomerase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in EIPR, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, wherein the one or more chaperone or co-chaperone polypeptides comprise a KAR2 polypeptide and a SSB1 polypeptide, the one or more protein disulfide isomerase polypeptides comprise a PDI1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in EIPR comprise a HACls polypeptide, and the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide.

45. The modified host cell of claim 1, wherein the modified host cell comprises one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides and a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides, wherein the one or more

heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides comprise one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more chaperone or co-chaperone polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more flavin adenine dinucleotide (FAD) synthetase polypeptides, one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more thiol oxidase polypeptides, and one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more polypeptides involved in unfolded protein response (UPR), and the one or more genes encoding one or more secretory pathway polypeptides deleted or downregulated comprise one or more genes encoding one or more vacuolar proteinase polypeptides, one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism, and one or more genes encoding one or more glycosidase polypeptides, wherein the one or more chaperone or co-chaperone polypeptides comprise a CPR5 polypeptide and a KAR2 polypeptide, the one or more flavin adenine dinucleotide (FAD) synthetase polypeptides comprise a FAD1 polypeptide, the one or more thiol oxidase polypeptides comprise an EROl polypeptide, the one or more polypeptides involved in unfolded protein response (UPR) comprise an IRE1 polypeptide, the one or more genes encoding one or more vacuolar proteinase polypeptides comprise a PEP4 gene, the one or more genes encoding one or more polypeptides involved in regulation of lipid metabolism comprise an OPI1 gene, and the one or more genes encoding one or more glycosidase polypeptides comprise a ROT2 gene.

46. The modified host cell of any one of claims 1-45, wherein the modified host cell comprises two or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide.

47. The modified host cell of any one of claims 1-46, wherein the nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide is codon-optimized.

48. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a plant cell within a plant secretory tissue.

49. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a fungal cell.

50. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a Berberine bridge enzyme (BBE) or BBE-like polypeptide produced in a bacterial cell.

51. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a tetrahydrocannabinolic acid synthase polypeptide.

52. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a cannabichromenic acid synthase polypeptide.

53. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a cannabidiolic acid synthase polypeptide.

54. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE polypeptide from Eschscholzia californica.

55. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum.

56. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans .

57. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a daurichromenic acid synthase polypeptide from Rhododendron dauricum.

58. The modified host cell of any one of claims 1-47, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide is a xylooligosaccharide oxidase polypeptide from Myceliophthora thermophila.

59. The modified host cell of any one of claims 1-58, wherein the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprises a signal sequence polypeptide.

60. The modified host cell of claim 59, wherein the signal sequence polypeptide is a secretory signal sequence polypeptide.

61. The modified host cell of claim 60, wherein the secretory signal sequence polypeptide is a native secretory signal sequence polypeptide.

62. The modified host cell of claim 60, wherein the secretory signal sequence polypeptide is a synthetic secretory signal sequence polypeptide.

63. The modified host cell of claim 60, wherein the secretory signal sequence polypeptide is an endoplasmic reticulum retention signal sequence polypeptide.

64. The modified host cell of claim 60, wherein the secretory signal sequence polypeptide is a vacuolar localization signal sequence polypeptide.

65. The modified host cell of claim 60, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide or a PRClt polypeptide.

66. The modified host cell of claim 65, wherein the vacuolar localization signal sequence polypeptide is a PEP4t polypeptide.

67. The modified host cell of claim 65, wherein the vacuolar localization signal sequence polypeptide is a PRClt polypeptide.

68. The modified host cell of claim 60, wherein the secretory signal sequence polypeptide is a plasma membrane localization signal sequence polypeptide.

69. The modified host cell of claim 60, wherein the secretory signal sequence polypeptide is a peroxisome targeting signal sequence polypeptide.

70. The modified host cell of claim 51, wherein the tetrahydrocannabinolic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:92.

71. The modified host cell of claim 52, wherein the cannabichromenic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:94.

72. The modified host cell of claim 53, wherein the cannabidiolic acid synthase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 108 or SEQ ID NO: 132.

73. The modified host cell of claim 54, wherein the BBE polypeptide from Eschscholzia californica comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 116.

74. The modified host cell of claim 55, wherein the BBE-like nicotine bridge enzyme polypeptide from Nicotiana tabacum comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 120.

75. The modified host cell of claim 56, wherein the BBE-like 6-hydroxy-D-nicotine oxidase polypeptide from Paenarthrobacter nicotinovorans comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 122.

76. The modified host cell of claim 57, wherein the daurichromenic acid synthase polypeptide from Rhododendron dauricum comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 118.

77. The modified host cell of claim 58, wherein the xylooligosaccharide oxidase polypeptide from Myceliophthor a thermophila comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 124.

78. The modified host cell of any one of claims 1-77, wherein the modified host cell is a eukaryotic cell.

79. The modified host cell of claim 78, wherein the eukaryotic cell is a yeast cell.

80. The modified host cell of claim 79, wherein the yeast cell is Saccharomyces cerevisiae.

81. The modified host cell of claim 80, wherein the Saccharomyces cerevisiae is a protease-deficient strain of Saccharomyces cerevisiae.

82. The modified host cell of any one of claims 1-81, wherein at least one of the one or more heterologous nucleic acids are integrated into the chromosome of the modified host cell.

83. The modified host cell of any one of claims 1-81, wherein at least one of the one or more heterologous nucleic acids are maintained extrachromosomally.

84. The modified host cell of any one of claims 1-83, wherein at least one of the one or more heterologous nucleic acids are operably-linked to an inducible promoter.

85. The modified host cell of any one of claims 1-83, wherein at least one of the one or more heterologous nucleic acids are operably-linked to a constitutive promoter.

86. The modified host cell of any one of claims 1-85, wherein the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding an Internal Ribosome Entry Site (IRES) element.

87. The modified host cell of any one of claims 1-85, wherein the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding a T2A element.

88. The modified host cell of any one of claims 1-87, wherein the one or more heterologous nucleic acids comprising a nucleotide sequence encoding the Berberine bridge enzyme (BBE) or BBE-like polypeptide comprise a nucleotide sequence encoding a green fluorescent protein (GFP) polypeptide.

89. A method of making a modified host cell of any one of claims 1-88, the method comprising introducing into a host cell: a) one or more heterologous nucleic acids comprising nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

90. A method of making a modified host cell of any one of claims 1-88, the method comprising introducing into a host cell: a) one or more vectors comprising one or more heterologous nucleic acids comprising a nucleotide sequence encoding a Berberine bridge enzyme (BBE) or BBE-like polypeptide and b) one or more vectors comprising one or more heterologous nucleic acids comprising nucleotide sequences encoding one or more secretory pathway polypeptides.

91. The method of claim 89 or claim 90, the method comprising introducing into the host cell a deletion or downregulation of one or more genes encoding one or more secretory pathway polypeptides.

92. A method for expressing a Berberine bridge enzyme (BBE) or BBE-like polypeptide, the method comprising culturing a modified host cell of any one of claims 1-88 in a culture medium.

93. The method of claim 92, wherein the culture medium comprises a fermentable sugar.

94. The method of claim 92, wherein the culture medium comprises a pretreated cellulosic feedstock.

95. The method of claim 92, wherein the culture medium comprises a non-fermentable carbon source.

96. The method of claim 95, wherein the non-fermentable carbon source comprises ethanol.