WO2019243624A1 - Production d'alcaloïdes de benzylisoquinoline chez des hôtes recombinés - Google Patents

Production d'alcaloïdes de benzylisoquinoline chez des hôtes recombinés Download PDF

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WO2019243624A1
WO2019243624A1 PCT/EP2019/066561 EP2019066561W WO2019243624A1 WO 2019243624 A1 WO2019243624 A1 WO 2019243624A1 EP 2019066561 W EP2019066561 W EP 2019066561W WO 2019243624 A1 WO2019243624 A1 WO 2019243624A1
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seq
set forth
polypeptide
amino acid
acid sequence
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Jens Houghton-Larsen
Esben Halkjaer Hansen
Vincent J.J. Martin
Michael E. Pyne
Lauren NARCROSS
Kaspar KEVVAI
Leanne BOURGEOIS
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Valorbec, Limited Partnership
River Stone Biotech Aps
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • This disclosure relates to recombinant production of benzylisoquinoline alkaloids in recombinant hosts.
  • this disclosure relates to production of (S)-norcoclaurine and derivatives thereof in Saccharomyces cerevisiae.
  • (S)-norcoclaurine synthase is an enzyme involved in committing and rate- limiting steps of benzylisoquinoline (BIA) biosynthesis.
  • BSA benzylisoquinoline
  • 4-hydroxyphenylacetaldehyde (4-HPAA) and dopamine are condensed in a Pictet- Spengler reaction to form (S)-norcoclaurine. See Figure 1.
  • (S)-norcoclaurine synthase is a catalytically inefficient enzyme, having K m values in the double-digit millimolar range.
  • the molar concentration of produced norcoclaurine is about 100 times lower than the concentration of the precursor dopamine. Narcross et al., Trends Biotechnol. 34:228-41 (2016); DeLoache et al., Nat. Chem. Biol. 11 :465-71 (2015).
  • the Ehrlich pathway which involves catabolism of branched-chain amino acids including leucine, valine, and isoleucine; aromatic amino acids including phenylalanine, tyrosine, and tryptophan; and the sulfur-containing amino acid (methionine), leads to the formation of fusel alcohols and fusel acids.
  • S. cerevisiae several dehydrogenase polypeptides and reductase polypeptide are involved.
  • the Ehrlich pathway includes conversion of 4-HPAA to tyrosol.
  • Intracellular availability of 4-HPAA and dopamine in yeast is also highly dependent on the aromatic amino acid biosynthesis pathways, particularly the shikimake pathway, because both precursors are tyrosine derivatives. See Figure 1.
  • benzylisoquinoline alkaloids are of great medical interest because of their pharmacological activity, such as, for example, the antibiotic sanguinarine, the muscle relaxants papaverine and tubocurarine, and the analgesics codeine and morphine. Galanie et al., Science
  • the invention provides a recombinant S. cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine and having reduced expression of:
  • the one or more endogenous transporter genes encode a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72.
  • the one or more endogenous transcription factor genes encode a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 184 or 186.
  • the invention further provides a recombinant host cell comprising reduced expression of the one or more transporter genes encoding the one or more polypeptides having at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:70 or 74, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 76, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:72; and the one or more endogenous transporter genes encoding one or more polypeptides having at least 90% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 86 or 88, or at least 75% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 102, 112, or at least 70% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 78, 80, 98, 114, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 92, 94, 106, or 1
  • the invention further provides a recombinant host cell comprising reduced expression of the transporter gene encoding a polypeptide having at least 75% sequence identity to the amino acid sequence set forth in SEQ ID NO:70 and further comprising:
  • the invention further provides a recombinant host cell comprising reduced expression of the endogenous gene encoding the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
  • the recombinant host cell disclosed herein further comprises reduced expression of:
  • benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in SEQ ID NO:146.
  • the invention further provides a recombinant S. cerevisiae host cell capable of producing one or more benzylisoquinoline alkaloids, comprising a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine, wherein:
  • the gene has a copy number of 2 or more and the host cell further
  • the one or more polypeptides comprises a polypeptide having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO: 140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 154, 152, or 150; or
  • the host cell has reduced expression of one or more endogenous genes encoding one or more NCS-compatible substrate pathway polypeptide or one or more transcription factor genes that regulate expression of the one or more endogenous genes.
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine has a copy number of 2 or more.
  • the polypeptide capable of synthesizing (S)-norcoclaurine comprises a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOs:30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, or 198.
  • the benzylisoquinoline alkaloid precursor is 4-hydroxyphenylacetaldehyde (4-HPAA) or 3,4- dihydroxyphenylacetaldehyde (3,4-DHPAA).
  • the one or more endogenous genes encode the one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor having at least 80% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:124, 160, or 146, or at least 70% sequence identity to the amino acid sequence set forth in SEQ ID NO:140, or at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:154, 152, or 150.
  • the NCS-compatible substrate pathway is a tryptophan biosynthesis pathway or a phenylalanine biosynthesis pathway.
  • the endogenous gene encodes an NCS-compatible pathway polypeptide having at least 65% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs: 166 or 168, or at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO: 164.
  • the recombinant host cell disclosed herein further comprises:
  • the invention further provides a recombinant host cell, comprising:
  • the recombinant host cell disclosed herein further comprises:
  • polypeptide capable of synthesizing phenylpyruvate from prephenate comprising a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in SEQ ID NO:164; and/or at least one endogenous gene encoding a polypeptide capable of synthesizing anthranilate from chorismate comprising a polypeptide having at least 65% sequence identity to the amino acid sequence set forth in SEQ ID NO: 166 or 168.
  • the invention further provides a recombinant host cell, comprising:
  • the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine or (S)-norlaudanosoline.
  • the host cell is further capable of producing a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)- norcoclaurine.
  • the derivative is (S)- reticuline, (R)-reticuline, salutaridinol, thebaine neopinone, and codeinone.
  • the invention further provides a method of producing one or more benzylisoquinoline alkaloids in a cell culture, comprising culturing the host cell disclosed herein in the cell culture, under conditions in which the genes are expressed; wherein the one or more benzylisoquinoline alkaloids is produced by the host cell.
  • the genes are constitutively expressed.
  • the host cell further produces a natural or a non-natural benzylisoquinoline alkaloid derivative of (S)-norcoclaurine,
  • the derivative is (S)-reticuline, (R)- reticuline, berberine, papaverine, morphine, sanguinarine, noscapine, codeine, thebaine, northebaine, oripavine, nororipavine, neopinone, codeinone, oxycodone, or buprenorphine.
  • the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate reduced formation of an acetaldehyde.
  • the cell culture comprises a non- fermentable carbon source, and wherein the cell culture has a total level of acetaldehyde that is lower than the total level of acetaldehyde in a corresponding cell culture including a glucose carbon source and/or a sucrose carbon source.
  • the non-fermentable carbon source is an acetate carbon source or a glycerol carbon source.
  • the method further comprises isolating one or more benzylisoquinoline alkaloids produced by the recombinant host cell.
  • the method further comprises enzymatically or chemically converting one or more benzylisoquinoline alkaloids produced by the recombinant host cell to provide a benzylisoquinoline alkaloid derivative.
  • the benzylisoquinoline alkaloid derivative is berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or buprenorphine.
  • the host cell is cultured in a fermentor with a feed profile and at a temperature for a period of time, wherein the feed profile, the temperature, and the period of time facilitate production of the one or more benzylisoquinoline alkaloids.
  • the one or more benzylisoquinoline alkaloids is (S)-norcoclaurine.
  • the invention further provides a cell culture broth, comprising the host cell disclosed herein and further comprising:
  • supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
  • the one or more benzylisoquinoline alkaloids are present at a concentration of at least 100 mg/liter of the cell culture.
  • the cell culture comprises less than 10 g/L of ethanol.
  • the invention further provides a cell lysate from the host cell disclosed herein grown in the cell culture, comprising:
  • supplemental nutrients comprising trace metals, vitamins, salts, yeast nitrogen base (YNB), nucleobases, and/or amino acids; and optionally,
  • Figure 1 shows a biochemical pathway for producing (S)-norcoclaurine
  • a DAHP synthase polypeptide (Aro4), a pentafunctional AROM polypeptide (Aro1 ), a bifunctional chorismate synthase / flavin reductase polypeptide (Aro2), a chorismate mutase polypeptide (Aro7), a prephenate dehydrogenase polypeptide (Tyr1 ), an aromatic aminotransferase I polypeptide (Aro8), an aromatic aminotransferase II polypeptide (Aro9), a phenylpyruvate decarboxylase polypeptide (Aro10), a tyrosine hydroxylase polypeptide (TyrH), an L-DOPA decarboxylase polypeptide (DODC), an (S)-norcoclaurine synthase polypeptide (NCS), a pre
  • Figure 2 shows representative benzylisoquinoline alkaloids obtainable from (S)- norcoclaurine.
  • Figure 3 shows production of dopamine (left) and (S)-norcoclaurine (right) from a control S. cerevisiae strain (Strain C) and a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 4, below).
  • Figure 4 shows visible- and fluorescence-microscopy images (left) and specific fluorescence measurements of S. cerevisiae strains expressing GFP-tagged polypeptides capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in
  • Figure 5 shows production of dopamine and (S)-norcoclaurine (from left to right for each strain) from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 6 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 7 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 8 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 9 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 10 shows off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 5, below.
  • Figure 1 1 shows off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, as described in Example 5, below.
  • Figure 12 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 13 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 14 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 15 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 16 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 17 shows production of certain off-target condensation products of a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 18 shows production of certain off-target condensation products (products 2, 3, and 4; from bottom to top for each strain) of a polypeptide capable of synthesizing (S)- norcoclaurine from 4-HPAA and dopamine in a series of S. cerevisiae strains as described in Example 5, below.
  • Figure 19 shows accumulation of biomass (OD 6 oo) and ethanol during the fed-batch phases of cultivations of S. cerevisiae strains as described in Example 6, below.
  • Figure 20 shows accumulation of norcoclaurine during the fed-batch phases of cultivations of S. cerevisiae strains as described in Example 6, below.
  • Figure 21 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine and further comprising reduced expression of one or more transporter polypeptides, as described in Example 9, below.
  • Figure 22 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 23 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 24 shows production of (S)-norcoclaurine from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a benzylisoquinoline alkaloid precursor, as described in Example 5, below.
  • Figure 25 shows production of dopamine, tyrosol, 4-HPAC, and (S)-norcoclaurine (from left to right for each strain) from a series of S. cerevisiae strains expressing a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine, further expressing one or more benzylisoquinoline alkaloid biosynthesis pathway genes, and further comprising reduced expression of one or more polypeptides capable of oxidizing or reducing a
  • nucleic acid means one or more nucleic acids.
  • the term“substantially” is utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation.
  • the term“substantially” is also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.
  • Methods well known to those skilled in the art can be used to construct genetic expression constructs and recombinant cells according to this invention. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Green & Sambrook, 2012, MOLECULAR CLONING: A LABORATORY MANUAL, Fourth Edition, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1989, CURRENT
  • nucleic acid can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof, in either single-stranded or double-stranded embodiments depending on context as understood by the skilled worker.
  • the terms“microorganism,”“microorganism host,”“microorganism host cell,”“recombinant host,” and“recombinant host cell” can be used interchangeably.
  • the term“recombinant host” is intended to refer to a Saccharomyces cerevisiae ( S . cerevisiae) host, the genome of which has been augmented by at least one DNA sequence.
  • DNA sequences include but are not limited to genes that are not naturally present, DNA sequences that are not normally transcribed into RNA or translated into a protein (“expressed”), and other genes or DNA sequences which one desires to introduce into a host.
  • introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene.
  • the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis.
  • the term“cell culture” refers to a culture medium comprising one or more recombinant hosts.
  • a cell culture may comprise a single strain of recombinant host, or may comprise two or more distinct host strains.
  • the culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B; etc.
  • recombinant gene refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. “Introduced,” or“augmented” in this context, is known in the art to mean introduced or augmented by the hand of man.
  • a recombinant gene can be a DNA sequence from another species or can be a DNA sequence that originated from or is present in the same species but has been incorporated into a host by recombinant methods to form a recombinant host.
  • a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA.
  • said recombinant genes are encoded by cDNA.
  • recombinant genes are synthetic and/or codon-optimized for expression in S. cerevisiae.
  • the term“engineered biosynthetic pathway” refers to a biosynthetic pathway that occurs in a recombinant host, as described herein. In some aspects, one or more steps of the biosynthetic pathway do not naturally occur in an unmodified host. In some embodiments, a heterologous version of a gene is introduced into a host that comprises an endogenous version of the gene.
  • the term“endogenous” gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell. In some
  • an endogenous S. cerevisiae gene is overexpressed.
  • the term “overexpress” is used to refer to the expression of a gene in an organism at levels higher than the level of gene expression in a wild type organism, i.e., increased expression of a recombinant gene in a host cell relative to a corresponding host cell that does not contain the recombinant gene (see, e.g., Prelich, 2012, Genetics 190:841-54).
  • an endogenous yeast gene is deleted. See, e.g., Giaever & Nislow, 2014, Genetics 197(2):451-65.
  • the terms“deletion,”“deleted,”“knockout,” and“knocked out” can be used
  • heterologous sequence and“heterologous coding sequence” are used to describe a sequence derived from a species other than the recombinant host.
  • a heterologous coding sequence for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant S. cerevisiae host expressing the heterologous sequence.
  • a coding sequence is a sequence that is native to the host.
  • A“selectable marker” can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a detectable change, e.g. a color change.
  • Linearized DNA fragments of the gene replacement vector then are introduced into the cells using methods well known in the art ( see below). Integration of the linear fragments into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, polymerase chain reaction (PCR) or Southern blot analysis. Subsequent to its use in selection, a selectable marker can be removed from the genome of the host cell by, e.g., Cre-LoxP systems (see, e.g., Gossen et al., 2002, Ann. Rev.
  • a gene replacement vector can be constructed in such a way as to include a portion of the gene to be disrupted, where the portion is devoid of any endogenous gene promoter sequence and encodes none, or an inactive fragment of, the coding sequence of the gene.
  • the terms“variant” and“mutant” are used to describe a protein sequence that has been modified, by man or nature, at one or more amino acids, compared to the wild-type sequence of a particular protein.
  • the term“inactive fragment” is a fragment of the gene that encodes a protein having, e.g., less than about 10% ⁇ e.g., less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1 %, or 0%) of the activity of the protein produced from the full-length coding sequence of the gene.
  • Such a portion of a gene is inserted in a vector in such a way that no known promoter sequence is operably linked to the gene sequence, but that a stop codon and a transcription termination sequence are operably linked to the portion of the gene sequence.
  • This vector can be subsequently linearized in the portion of the gene sequence and transformed into a cell. By way of single homologous recombination, this linearized vector is then integrated in the endogenous counterpart of the gene with resulting inactivation thereof.
  • “alkaloid” refers to any of the group of naturally occurring chemical compounds that mostly contain basic nitrogen atoms.
  • “alkaloids” include“true alkaloids” (i.e., amino-acid-derived compounds containing nitrogen in the heterocycle), for example, atropine, nicotine, morphine, ergotamine, coniine, and coniceine; “protoalkaloids” (i.e., amino-acid-derived compounds containing nitrogen), for example, mescaline, adrenaline, and ephedrine;“polyamine alkaloids” (i.e., derivatives of putrescine, spermidine, and spermine);“peptide and cyclopeptide alkaloids”; and“pseudoalkaloids” (i.e., alkaloid-like compounds that do not originate from amino acids), for example, terpene-like alkaloids, steroid-like alkaloids, and purine-like alkaloids (
  • alkaloid also includes non-naturally occurring compounds derived from or otherwise resembling any naturally occurring alkaloid. See M. Hesse,“Alkaloids. Nature’s Curse or Blessing?” (2002); T. Aniszewski,“Alkaloids - Secrets of Life” (2007). Alkaloids can contain asymmetric centers and can thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. Each chiral center can be defined in terms of absolute stereochemistry as (R)- or (S)-. As used herein, the term“alkaloid” includes all such possible isomers, as well as their racemic and optically pure forms.
  • alkaloids can be classified by skeleton (e.g., pyrrolidine, quinoline, benzylisoquinoline, indole, and terpenoid).
  • skeleton e.g., pyrrolidine, quinoline, benzylisoquinoline, indole, and terpenoid.
  • the term“benzylisoquinoline alkaloid” refers to alkaloids having a benzylisoquinoline skeleton, including, for example, (S)-norcoclaurine, coclaurine, berberine, papaverine, morphine, sanguinarine, hydrastine, and noscapine. See Figure 2; see also, Singla et al., “BIAdb: A curated database of benzylisoquinoline alkaloids,” BMC Pharmacol. 10:4 (March 2010)).
  • benzylisoquinoline alkaloid precursor compound are used to refer to intermediate compounds in the benzylisoquinoline alkaloid biosynthetic pathway.
  • Benzylisoquinoline alkaloid precursors include, but are not limited to, phosphoenolpyruvate (PEP), erythrose 4-phosphate (E4P), 3- deoxy-D-arabinoheptulosonate-7-phosphate (DAHP), 5-enolpyruvateshikimate 3-phosphate (EPSP), chorismate, prephenate, L-tyrosine, L-3,4-dihydroxyphenylalaine (L-DOPA), 4-(2- aminoethyl)benzene-1 ,2-diol (dopamine), 4-hydroxyphenylpyruvate (4-HPP), and 4- hydroxyphenylacetaldehyde (4-HPAA). See Figure 1.
  • PEP phosphoenolpyruvate
  • E4P erythrose 4-phosphate
  • DAHP
  • benzylisoquinoline alkaloid precursors are themselves benzylisoquinoline alkaloids.
  • (S)-norcoclaurine is a benzylisoquinoline alkaloid precursor of morphine. See Figure 2.
  • Benzylisoquinoline alkaloids and/or precursors thereof can be produced in vivo, (i.e., in a recombinant host), in vitro (i.e., enzymatically or chemically), or by whole cell bioconversion.
  • the terms“produce” and“accumulate” can be used interchangeably to describe synthesis of benzylisoquinoline alkaloids and precursors thereof in vivo, in vitro, or by whole cell conversion.
  • a culture broth can comprise a carbon source such as dextrose, sucrose, fructose, glycerol, xylose, ethanol, glucose, or acetate; a phosphate source; vitamins; trace elements; salts; yeast nitrogen base (YNB); amino acids; nucleobases; and a nitrogen source.
  • the nitrogen source can include, for example, malt extract, corn steep liquor, casein hydrosylate, yeast extract, urea, amino acids, ammonia and/or ammonium salts, etc.
  • a culture broth can comprise one or more benzylisoquinoline alkaloids added to the medium or produced de novo by a recombinant host, as described herein.
  • a non-fermentable carbon source e.g ., an acetate carbon source or a glycerol carbon source
  • reduced intracellular acetaldehyde limits or eliminates spontaneous condensation of acetaldehyde and dopamine.
  • benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more enzymes involved in the benzylisoquinoline alkaloid biosynthetic pathway in a recombinant S. cerevisiae host.
  • a recombinant S. cerevisiae host For example, a recombinant S.
  • DAHP 3-deoxy- D-arabinoheptulosonate-7-phosphate
  • E4P erythrose 4-phosphate
  • DAHP synthesis polypeptide e.g., SEQ ID NO:2 or SEQ ID NO:4
  • EBP erythrose 4-phosphate
  • a gene encoding a polypeptide capable of synthesizing 5-enolpyruvateshikimate 3-phosphate (EPSP) from DAHP ⁇ e.g., a pentafunctional AROM polypeptide; e.g., SEQ ID NO:6)
  • a gene encoding a polypeptide capable of synthesizing chorismate from EPSP ⁇ e.g., bifunctional chorismate synthase / flavin reductase polypeptide; e.g., SEQ ID NO:8)
  • a gene encoding a polypeptide capable of synthesizing chorismate from EPSP ⁇ e.g., bifunctional chorismate synth
  • production of benzylisoquinoline alkaloids can be accomplished by differential copy numbers of the benzylisoquinoline alkaloid
  • biosynthesis pathway genes in the recombinant cell, differential promoter strengths, and/or by utilizing mutants with increased specificity/activity towards the product of interest.
  • additional copies of a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine in recombinant cells as otherwise described herein can increase the production of (S)-norcoclaurine.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP.
  • the gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing DAHP from E4P and PEP results in a total expression level of genes encoding a polypeptide capable of synthesizing DAHP from E4P and PEP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing DAHP from E4P and PEP, i.e., an overexpression of a polypeptide capable of synthesizing DAHP from E4P and PEP.
  • the polypeptide capable of synthesizing DAHP from E4P and PEP is a feedback-resistant (FBR) polypeptide.
  • a feedback resistant version of and/or overexpression of a benzylisoquinoline alkaloid biosynthesis polypeptide e.g., a polypeptide capable of synthesizing DAHP from E4P and PEP, e.g., a DAHP synthase polypeptide; e.g., a polypeptide capable of synthesizing prephenate from chorismate, e.g., a chorismate mutase polypeptide
  • pathway regulation e.g., by reducing or eliminating endogenous transcriptional regulation of expression.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing prephenate from chorismate.
  • the gene encoding a polypeptide capable of synthesizing prephenate from chorismate is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPH (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPH (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing prephenate from chorismate results in a total expression level of genes encoding a polypeptide capable of synthesizing prephenate from chorismate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing prephenate from chorismate, i.e., an overexpression of a polypeptide capable of synthesizing prephenate from chorismate.
  • the polypeptide capable of synthesizing prephenate from chorismate is a feedback-resistant (FBR) polypeptide.
  • IAA indole-3-acetaldehyde
  • 4-HPAA 4-HPAA for condensation with dopamine
  • dopamine catalyzed, e.g., by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • overexpression of a polypeptide capable of synthesizing prephenate from chorismate can increase the benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from chorismate towards IAA, and accordingly increase the availability of intracellular dopamine for condensation with 4-HPAA.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate.
  • the gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NQ:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate results in a total expression level of genes encoding a polypeptide capable of synthesizing 4-HPP from prephenate that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing 4-HPP from prephenate, i.e., an overexpression of a polypeptide capable of synthesizing 4-HPP from prephenate.
  • PAA phenylacetaldehyde
  • S synthesizing
  • overexpression of a polypeptide capable of synthesizing 4-HPP from prephenate can increase benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from prephenate towards PAA, and accordingly increase the availability of intracellular dopamine for condensation with 4-HPAA.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine.
  • the gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO:177), tADH1 (SEQ ID NO: 178), tTDH 1 (SEQ ID NO:181 ), or tPGK1 (SEQ ID NO: 182).
  • a terminator such as, for example, for example, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO:177), tADH1 (SEQ ID NO: 178), tTDH 1 (SEQ ID NO:181 ), or tPGK1 (SEQ ID NO: 182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine results in a total expression level of genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine, i.e., an overexpression of a polypeptide capable of synthesizing L-DOPA from L-tyrosine.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA.
  • the gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), P ⁇ H 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGM (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), P ⁇ H 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing dopamine from L-DOPA results in a total expression level of genes encoding a polypeptide capable of synthesizing L-DOPA from L-tyrosine that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing dopamine from L- DOPA, i.e., an overexpression of a polypeptide capable of synthesizing dopamine from L-
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP.
  • the gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO: 169), pPDC1 (SEQ ID NO: 169), pTEF2 (SEQ ID NO:170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGH (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • a terminator such as, for example, tPGH (SEQ ID NO:174), tCYC1 (SEQ ID NO:175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 177), tADH1 (SEQ ID NO:178), tTDH 1 (SEQ ID NO: 181 ), or tPGK1 (SEQ ID NO:182).
  • synthesizing 4-HPAA from 4-HPP results in a total expression level of genes encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP, i.e., an overexpression of a polypeptide capable of synthesizing 4-HPAA from 4-HPP.
  • overexpression of a polypeptide capable of synthesizing 4-HPAA from 4-HPP can increase benzylisoquinoline alkaloid biosynthesis flux relative to competing pathways from 4-HPP.
  • a recombinant S. cerevisiae host comprises a gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA.
  • the gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from (S)- norcoclaurine from dopamine and 4-HPAA is a recombinant gene.
  • the recombinant gene comprises a nucleotide sequence native to the host. In other aspects, the recombinant gene comprises a heterologous nucleotide sequence.
  • the recombinant gene is operably linked to a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO: 170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • a promoter such as, for example, pPDC1 (SEQ ID NO:169), pTEF2 (SEQ ID NO: 170), pTDH3 (SEQ ID NO:171 ), pPGK1 (SEQ ID NO:172), pTEF1 (SEQ ID NO:173), pTPM (SEQ ID NO:179), or pCCW12 (SEQ ID NO:180).
  • the recombinant gene is operably linked to a terminator, such as, for example, tPGM (SEQ ID NO: 174), tCYC1 (SEQ ID NO: 175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID NO: 174), tPGM (SEQ ID NO: 174), tCYC1 (SEQ ID NO: 175), tFBA1 (SEQ ID NO:176), tEN02 (SEQ ID
  • expression of a recombinant gene encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA results in a total expression level of genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4- HPAA that is higher than the expression level of endogenous genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA, i.e., an overexpression of a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA.
  • the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA is a truncated polypeptide such as, for example, a truncated NCS polypeptide lacking an N-terminal signal peptide (e.g ., SEQ ID NO:42 or SEQ ID NO:44), a truncated NCS polypeptide lacking a C-terminal transmembrane domain (e.g., SEQ ID NO:52 or SEQ ID NO:54), or a truncated NCS polypeptide lacking an N-terminal signal peptide and a C- terminal transmembrane domain.
  • a truncated NCS polypeptide such as, for example, a truncated NCS polypeptide lacking an N-terminal signal peptide (e.g ., SEQ ID NO:42 or SEQ ID NO:44), a truncated NCS polypeptide lacking a C-terminal transmembr
  • a polypeptide lacking a signal peptide and/or a transmembrane domain, expressed in a recombinant host may have increased solubility in the cytosol relative to a corresponding polypeptide comprising the signal peptide and/or transmembrane domain.
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA has a copy number of two (i.e., is present in the recombinant host in two copies).
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA e.g ., an NCS polypeptide
  • the polypeptide capable of synthesizing DAHP from PEP and E4P comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:2 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 1 ) or SEQ ID NO:4 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:3).
  • the polypeptide capable of synthesizing EPSP from DAHP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:6 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:5).
  • the polypeptide capable of synthesizing chorismate from EPSP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:8 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:7).
  • the polypeptide capable of synthesizing prephenate from chorismate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:10 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:9) or SEQ ID NO:12 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:1 1 ).
  • the polypeptide capable of synthesizing 4-HPP from prephenate comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:14 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 13).
  • the polypeptide capable of synthesizing L-tyrosine from 4-HPP and/or the polypeptide capable of synthesizing 4-HPP from L-tyrosine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:16 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 15) or SEQ ID NO:18 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:17).
  • the polypeptide capable of synthesizing 4-HPAA from 4-HPP comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:20 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO: 19).
  • the polypeptide capable of synthesizing L-DOPA from L-tyrosine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:22 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:21 ) or SEQ ID NO:24 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:23).
  • the polypeptide capable of synthesizing dopamine from L-DOPA comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:26 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:25) or SEQ ID NO:28 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:27).
  • the polypeptide capable of synthesizing (S)-norcoclaurine from 4- HPAA and dopamine comprises a polypeptide having the amino acid sequence set forth in SEQ ID NO:32 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:31 ), or SEQ ID NO:34 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:33), or SEQ ID NO:36 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:35), or SEQ ID NO:38 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:37), or SEQ ID NO:40 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:39), or SEQ ID NO:42 (which can be encoded by the nucleotide sequence set forth in SEQ ID NO:41 ), or SEQ ID NO:44 (which can be encoded by the nucleo
  • benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more enzymes involves in the benzylisoquinoline alkaloid biosynthetic pathway.
  • a recombinant S for example, a recombinant S.
  • cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g ., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having the amino acid sequence set forth in SEQ ID NO: 12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA has a copy number of two, three, four, five, six, seven, eight, or more.
  • (S)-Norcoclaurine can be produced in vivo (i.e., in a recombinant host), in vitro (i.e., enzymatically), or by whole cell bioconversion.
  • the terms“produce” and “accumulate” can be used interchangeably to describe synthesis of (S)-norcoclaurine in vivo, in vitro, or by whole cell bioconversion.
  • a cell is permeabilized to take up a substrate to be modified or to excrete a modified product.
  • a permeabilizing agent can be added to aid the feedstock entering into the host and product getting out.
  • the cells are permeabilized with a solvent such as toluene, or with a detergent such as Triton-X or Tween.
  • the cells are permeabilized with a surfactant, for example a cationic surfactant such as cetyltrimethylammonium bromide (CTAB).
  • CTAB cetyltrimethylammonium bromide
  • the cells are permeabilized with periodic mechanical shock such as electroporation or a slight osmotic shock.
  • a crude lysate of the cultured microorganism can be centrifuged to obtain a supernatant.
  • the resulting supernatant can then be applied to a chromatography column, e.g., a C18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol.
  • the compound(s) can then be further purified by preparative HPLC.
  • (S)-norcoclaurine can be produced by co-culturing of two or more hosts.
  • one or more hosts each expressing one or more enzymes involved in the (S)-norcoclaurine biosynthetic pathway, produce (S)-norcoclaurine.
  • a host expressing a gene encoding a polypeptide capable of polypeptide capable of synthesizing (S)-norcoclaurine; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:2 and 4; a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth in SEQ ID NO:6; a gene encoding a polypeptide having at least 60% sequence identity to the amino acid sequence set forth in SEQ ID NO:8; a gene encoding a polypeptide having at least 50% sequence identity to the amino acid sequence set forth in any of SEQ ID NOs:10 and 12; a gene encoding a polypeptide having at least 55% sequence identity to the amino acid sequence set forth
  • the benzylisoquinoline alkaloid comprises, for example, (S)- norcoclaurine, (S)-reticuline, (R)-reticuline, salutaridinol, thebaine, neopinone, and codeinone.
  • a recombinant S. cerevisiae host as otherwise described herein, further comprising a gene encoding a polypeptide capable of synthesizing (S)-coclaurine from (S)- norcoclaurine (e.g ., a 6-O-methyltransferase (6-OMT) polypeptide; e.g., a polypeptide having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO:201 ), a gene encoding a polypeptide capable of synthesizing 3’-hydroxy-coclaurine from (S)-coclaurine or 3’- hydroxy-A/-methyl-coclaurine from /V-methyl-coclaurine (e.g ., an N-methylcoclaurine 3’- monooxygenase (NMCH) polypeptide; e.g., a polypeptide having at least 70% sequence identity to an amino acid sequence as set forth in SEQ ID NO:202
  • 6-OMT 6-O
  • a recombinant S. cerevisiae host capable of producing (S)-reticuline in vivo (e.g., as described herein), further comprising a gene encoding a polypeptide capable of synthesizing (R)-reticuline from (S)-reticuline (e.g., a 1 ,2- dehydroreticuline synthase-1 , 2-dehydroreticuline reductase (DRS-DRR) polypeptide; e.g., a polypeptide having at least 80% sequence identity to an amino acid sequence as set forth in SEQ ID NO:205), a gene encoding a polypeptide capable of synthesizing salutaridine from (R)- reticuline (e.g., a salutaridine synthase (SAS) polypeptide; e.g., a polypeptide having at least 55% sequence identity to an amino acid sequence set forth in SEQ ID NO:206), a gene encoding a polypeptide capable
  • a recombinant S. cerevisiae host capable of producing thebaine in vivo e.g as described herein), further comprising a gene encoding a polypeptide capable of synthesizing neopinone from thebaine ⁇ e.g., a morphinone reductase polypeptide) and a gene encoding a polypeptide capable of synthesizing hydrocodone from codeinone ⁇ e.g., a thebaine 6-O-demethylase polypeptide), can produce hydrocodone in vivo.
  • benzylisoquinoline alkaloids precursors produced in vivo ⁇ e.g., (S)-norcoclaurine, (S)-reticuline, (R)-reticuline, salutaridinol, thebaine, neopinone, codeinone
  • can be converted in vitro e.g., chemically or enzymatically to derivatives including berberine, papaverine, morphine, sanguinarine, noscapine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, and buprenorphine.
  • codeinone produced by a recombinant host described herein is enzymatically converted to hydrocodone by contacting the codeinone with a polypeptide capable of synthesizing hydrocodone from codeinone ⁇ e.g., a thebaine 6-O-demethylase polypeptide) in vitro.
  • thebaine produced by a recombinant host described herein is chemically converted to buprenorphine in vitro ⁇ see, e.g., WO 2018/21 1331 ; Machara et al., Adv. Synth. Catal. 354(4):613-26 (2012); Werner et al., J. Org.
  • thebaine produced by a recombinant host described herein is chemically or enzymatically converted to morphine, neomorphine, hydrocodone, codeine, oxycodone, oxymorphone, dihydromorphine, or etorphine.
  • the terms“detectable amount,”“detectable concentration,” “measurable amount,” and“measurable concentration” refer to a level of benzylisoquinoline alkaloids or precursors thereof measured in AUC, pM/OD 6 oo, mg/L, mM, or mM.
  • Benzylisoquinoline alkaloid production i.e., total, supernatant, and/or intracellular
  • benzylisoquinoline alkaloid levels can be detected and/or analyzed by techniques generally available to one skilled in the art, for example, but not limited to, liquid chromatography-mass spectrometry (LC-MS), thin layer chromatography (TLC), high-performance liquid
  • HPLC ultraviolet-visible spectroscopy/ spectrophotometry
  • MS mass spectrometry
  • NMR nuclear magnetic resonance spectroscopy
  • the term“undetectable concentration” refers to a level of a compound that is too low to be measured and/or analyzed by techniques such as TLC, HPLC, UV-Vis, MS, or NMR.
  • a compound of an“undetectable concentration” is not present in a composition of benzylisoquinoline alkaloids and/or precursors thereof.
  • one or more benzylisoquinoline alkaloids e.g ., (S)-norcoclaurine
  • precursors thereof are produced in vivo by culturing a recombinant S.
  • cerevisiae host comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:12); a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate (e.g., a
  • the gene encoding the polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA has a copy number of two, three, four, five, six, seven, eight, or more.
  • benzylisoquinoline alkaloids e.g., (S)-norcoclaurine
  • the methods of the present invention can optionally comprise one or more additional steps. These steps can for example be directed toward isolation and/or purification of one or more benzylisoquinoline alkaloids from the fermentation broth or host cells.
  • the isolating steps may comprise: (a) contacting the cell culture comprising the benzylisoquinoline alkaloids with: (i) one or more adsorbent resins in a packed column in order to bind at least a portion of the
  • benzylisoquinoline alkaloids to the resin, thereby isolating the benzylisoquinoline alkaloid compounds; or (ii) one or more ion exchange or reversed-phase chromatography columns in order to bind at least a portion of the benzylisoquinoline alkaloid in the column, thereby isolating the benzylisoquinoline alkaloid; or (b) crystallizing and/or organic solvent extracting the benzylisoquinoline alkaloids from the cell culture, thereby isolating the benzylisoquinoline alkaloids;(i) contacting the cell culture with an organic solvent immiscible with water and separating the organic phase enriched in benzylisoquinoline alkaloids (c) separating the cell culture into a solid phase and a liquid phase, wherein the liquid phase comprises of the benzylisoquinoline alkaloids; and (i) contacting the liquid phase with one or more adsorbent resins in order to
  • the isolating step can comprise separating the solid phase from the liquid phase using a process comprising tangential flow filtration with diafiltration membranes to generate a permeate stream comprising the benzylisoquinoline alkaloids, wherein the membranes used in the tangential flow filtration are ultrafiltration or nanofiltration membranes.
  • the permeate stream is extracted by an organic solvent which phase-separates from the aqueous phase to generate an extracted benzylisoquinoline alkaloids in the organic solvent.
  • the permeate stream containing the benzylisoquinoline alkaloids product could be concentrated by some evaporation to produce a crystallized benzylisoquinoline alkaloid.
  • the aqueous permeate or the concentrate can be extracted by an organic solvent which phase-separates from the aqueous phase.
  • the solvent extraction could be performed in a counter-current extraction centrifuge such as a Podbelniak extractor, or in a counter-current extraction column such as a Karr or Scheibel column. This yields the benzylisoquinoline alkaloids products in an organic solvent suitable for subsequent purification processing.
  • the isolating step can also comprise separating a liquid phase of the cell culture from a solid phase of the cell culture to obtain a supernatant comprising the produced one or more benzylisoquinoline alkaloids, and (a) contacting the supernatant with one or more adsorbent resins in order to obtain at least a portion of the produced one or more
  • organic solvent extraction can be replaced with a series of process operations which yield a similar organic solution of benzylisoquinoline alkaloids.
  • the series of process operations would include (a) precipitation of benzylisoquinoline alkaloids from the aqueous concentrate produced by addition of acid until acidic pH; (b) filtration and optionally water-washing of the resulting solids; and (c) dissolution of the filtered benzylisoquinoline alkaloids containing solids into an organic solvent suitable for further purification.
  • the organic extract can be contacted with carbon to adsorb impurities and color bodies.
  • the carbon contacting can be done by mixing carbon in the organic extract and filtering the carbon out of the resulting suspension, or by feeding the organic extract to a column or filter containing a fixed bed of carbon and collecting a purified effluent stream.
  • the organic extract can be crystallized by concentrating the solution evaporatively.
  • the resulting benzylisoquinoline alkaloids products crystals can be filtered, washed, and dried to yield a high-purity
  • the reaction mixture can be filtered in order to remove the solid in the media (cell debris etc.).
  • the resulting aqueous solution can be extracted repeatedly with an organic solvent not miscible with water (this can be chloroform, toluene, dichloromethane, ethyl acetate, or the like).
  • the resulting organic phase can be concentrated into small quantity (resulting into a syrup).
  • the aqueous phase can be discarded.
  • the resulting residue benzylisoquinoline alkaloid crude material can be then crystallized from any short chain alcohol, such as methanol or it can be purified with other suitable purification technique such as chromatography or other standard techniques.
  • Another possible procedure to extract the alkaloids from the fermentation broth or cells can be a caustic wash of the broth/cells followed by a filtration in order to remove the biological material and other solids.
  • the alkaloids can then be precipitated from the basic solution as salt after adjusting the pH to acidic with addition of acid (for example, sulphuric acid or hydrochloric acid, etc.).
  • acid for example, sulphuric acid or hydrochloric acid, etc.
  • the benzylisoquinoline alkaloids can be extracted from the cells/broth trough percolation via an organic solvent.
  • the resulting organics can be concentrated into small quantities.
  • the resulting residue can be purified with other suitable purification technique such as crystallization and/or chromatography or other standard techniques.
  • the terms“or” and“and/or” are used to describe multiple components in combination or exclusive of one another.
  • “x, y, and/or z” can refer to“x” alone,“y” alone,“z” alone,“x, y, and z,”“(x and y) or z,”“x or (y and z),” or“x or y or z.”
  • “and/or” is used to refer to the exogenous nucleic acids that a recombinant cell comprises, wherein a recombinant cell comprises one or more exogenous nucleic acids selected from a group.
  • “and/or” is used to refer to production of benzylisoquinoline alkaloids and/or benzylisoquinoline alkaloid precursors.
  • “and/or” is used to refer to production of benzylisoquinoline alkaloids, wherein one or more benzylisoquinoline alkaloids are produced.
  • “and/or” is used to refer to production of benzylisoquinoline alkaloids, wherein one or more benzylisoquinoline alkaloids are produced through the following steps: culturing a recombinant S. cerevisiae host, synthesizing one or more benzylisoquinoline alkaloids in a recombinant S. cerevisiae host, and/or isolating one or more benzylisoquinoline alkaloids.
  • Functional homologs of the polypeptide described above are also suitable for use in producing benzylisoquinoline alkaloids and/or precursors thereof in a recombinant S. cerevisiae host.
  • a functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide.
  • a functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs.
  • Variants of a naturally occurring functional homolog can themselves be functional homologs.
  • Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally occurring polypeptides (“domain swapping”).
  • Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs.
  • the term“functional homolog” is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.
  • Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of benzylisoquinoline alkaloid biosynthesis polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using, for example, the amino acid sequence of an NCS polypeptide as the reference sequence. An amino acid sequence is, in some instances, deduced from a corresponding nucleotide sequence.
  • nucleic acids and polypeptides are identified from transcriptome data based on expression levels rather than by using BLAST analysis.
  • conserveed regions can be identified by locating a region within the primary amino acid sequence of a benzylisoquinoline alkaloid biosynthesis polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer et al., Nucl.
  • conserveed regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.
  • polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions.
  • conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g ., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity).
  • a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.
  • polypeptides suitable for producing norcoclaurine in a recombinant S. cerevisiae host include NCS polypeptides and functional homologs thereof.
  • a candidate sequence typically has a length that is from 80% to 250% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250% of the length of the reference sequence.
  • a functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 1 10, 1 15, or 120% of the length of the reference sequence, or any range between.
  • a % sequence identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows.
  • a reference sequence e.g., a nucleic acid sequence or an amino acid sequence described herein
  • Clustal Omega version 1.2.1 , default parameters
  • Clustal Omega calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments.
  • word size 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5.
  • gap opening penalty 10.0; gap extension penalty: 5.0; and weight transitions: yes.
  • word size 1 ; window size: 5; scoring
  • the Clustal Omega output is a sequence alignment that reflects the relationship between sequences.
  • Clustal Omega can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European
  • % sequence identity of a candidate nucleic acid or amino acid sequence to a reference sequence
  • the sequences are aligned using Clustal Omega, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100.
  • the% sequence identity value can be rounded to the nearest tenth. For example, 78.1 1 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.
  • a nucleic acid sequence encoding a benzylisoquinoline alkaloid biosynthesis polypeptide can include a tag sequence that encodes a“tag” designed to facilitate subsequent manipulation (e.g ., to facilitate purification or detection), solubility, secretion, or localization of the encoded polypeptide.
  • Tag sequences can be inserted in the nucleic acid sequence encoding the polypeptide such that the encoded tag is located at either the carboxyl or amino terminus of the polypeptide.
  • Non-limiting examples of encoded tags include green fluorescent protein (GFP), human influenza hemagglutinin (HA), glutathione S transferase (GST), polyhistidine-tag (HIS tag), disulfide oxidoreductase (DsbA), maltose binding protein (MBP), N-utilization substance (NusA), small ubiquitin-like modifier (SUMO), and FlagTM tag (Kodak, New Haven, CT).
  • Other examples of tags include a chloroplast signal peptide, a mitochondrial signal peptide, an amyloplast peptide, signal peptide, or a secretion tag.
  • a benzylisoquinoline alkaloid biosynthesis polypeptide is a protein altered by domain swapping.
  • domain swapping is used to describe the process of replacing a domain of a first protein with a domain of a second protein.
  • the domain of the first protein and the domain of the second protein are functionally identical or functionally similar.
  • the structure and/or sequence of the domain of the second protein differs from the structure and/or sequence of the domain of the first protein.
  • an NCS polypeptide e.g., a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA is altered by domain swapping.
  • benzylisoquinoline alkaloid biosynthesis polypeptides can include additional amino acids that are not involved in the enzymatic activities carried out by the polypeptides.
  • benzylisoquinoline alkaloid biosynthesis polypeptides are fusion proteins.
  • the terms“chimera,”“fusion polypeptide,” “fusion protein,”“fusion enzyme,”“fusion construct,”“chimeric protein,” “chimeric polypeptide,” “chimeric construct,” and“chimeric enzyme” can be used interchangeably herein to refer to proteins engineered through the joining of two or more genes that code for different proteins.
  • a protein is altered by circular permutation, which involves covalent attachment of the ends of a protein that would be opened elsewhere afterwards.
  • circular permutation involves covalent attachment of the ends of a protein that would be opened elsewhere afterwards.
  • the order of the sequence is altered without causing changes in the amino acids of the protein.
  • a targeted circular permutation can be produced, for example but not limited to, by designing a spacer to join the ends of the original protein. Once the spacer has been defined, there are several possibilities to generate permutations through generally accepted molecular biology techniques, for example but not limited to, by producing
  • concatemers by means of PCR and subsequent amplification of specific permutations inside the concatemer or by amplifying discrete fragments of the protein to exchange to join them in a different order.
  • the step of generating permutations can be followed by creating a circular gene by binding the fragment ends and cutting back at random, thus forming collections of permutations from a unique construct.
  • Modification of transport systems in a recombinant S. cerevisiae host that are involved in transport of benzylisoquinoline alkaloids and/or precursors thereof can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts.
  • the terms“transport of a benzylisoquinoline alkaloid (precursor),” “benzylisoquinoline alkaloid (precursor) transport,”“excretion of a benzylisoquinoline alkaloid (precursor),” and“benzylisoquinoline alkaloid (precursor) excretion” can be used
  • transporter also referred to as a membrane transport protein refers to a membrane protein involved in the movement of small molecules, macromolecules (such as carbohydrates), and ions across a biological membrane.
  • Transporters span the membrane in which they are localized and across which they transport substances. Transporter proteins can assist in the movement (i.e., transport or excretion) of a substance from the intracellular space to the culture medium, or from a vacuolar space to the intracellular space. Transporters are known to function as passive transport systems, carrying molecules down their concentration gradient, or as active transport systems, using energy to carry molecules uphill against their concentration gradient. Active transport is mediated by carriers which couple transport directly to the use of energy derived from hydrolysis of an ATP molecule or by carriers which make use of a pre-established electrochemical ion gradient to drive co-transport of the nutrient molecule and a co-transported ion. The latter category comprises symporters and antiporters, which carry the ion in the same or opposite direction, respectively, as the transported substrate.
  • Transport proteins have been classified according to various criteria at the
  • Transporter Classification Database (on the world-wide web at tcdb.org). See Saier Jr. et al., Nucl. Acids Res., 42(1 ):D251-258 (2014).
  • Non-limiting examples thereof include, among others, the family of Multiple Drug Resistance (MDR) plasma membrane transporters that is thought to be ubiquitous among living organisms.
  • MDR transporter superfamily can be further subdivided according to the mode of operation by which the substrate is transported from one side of the membrane to the other. Transporters can operate to move substances across membranes in response to chemiosmotic ion gradients or by active transport.
  • ABC transporters are transmembrane proteins that utilize the energy of adenosine triphosphate (ATP) hydrolysis to carry out translocation of various substrates across membranes. They can transport a wide variety of substrates across the plasma membrane and intracellular membranes, including metabolic products, lipids and sterols, and drugs.
  • endogenous ABC transporter genes include PDR5, PDR10, PDR12, PDR15, PDR18, SNQ2, YDR061W, YOR1 , YOL075C, MDL2,
  • ABC transporters transport benzylisoquinoline alkaloid precursors and/or benzylisoquinoline alkaloids.
  • MFS transporters are monomeric polypeptides that can transport small solutes in response to proton gradients.
  • the MFS transporter family is sometimes referred to as the uniporter-symporter-antiporter family.
  • MFS transporters function in, inter alia, in sugar uptake and drug efflux systems.
  • MFS transporters typically comprise conserved MFS-specific motifs.
  • Non-limiting examples of endogenous MFS transporter genes include TP01 , TP02, TP03, TP04, QDR1 , QDR2, QDR3, FLR1 , DTR1 , YHK8, SE01 , YBR241 C, VBA3, FEN2, SNF3, STL1 , HXT10, AZR1 , MPH3, VBA5, GEX2, SNQ1 , AQR1 , MCH1 , MCH5, ATG22, HXT15, MPH2, ITR1 , SIT 1 , VPS73, HXT5, SOA1 , HXT9, YMR279C, YIL166C, HOL1 , and ENB1 (or a functional homolog thereof).
  • MFS transporters transport benzylisoquinoline alkaloid precursors and/or benzylisoquinoline alkaloids.
  • cerevisiae host that involve one or more benzylisoquinoline alkaloids precursors can allow for more effective production of benzylisoquinoline alkaloids in recombinant hosts.
  • oxidoreductases in yeast can reduce the level of reduction of 4-HPAA to tyrosol and thereby improve norcoclaurine or norlaudanosoline yields in recombinant strains expressing a norcoclaurine synthase (NCS) enzyme, with ARM (YGL157W) being the most prominent, with clear indication in the presented data from that ADH6 disruption also has an effect.
  • NCS norcoclaurine synthase
  • ARM YGL157W
  • WO 2018/029282 Kristy Hawkins,“Metabolic engineering of Saccharomyces cerevisiae for the production of benzylisoquinoline alkaloids” 1 January 2009. Caltech Thesis, XP55361294, pp. 1-154.
  • the fold-increase yield demonstrated by these approaches are still not high enough to make the process commercially viable since much 4-HPAA are still lost to Tyrosol and yields of norcoclaurine still are in pg/L or low mg/L scale
  • S. cerevisiae comprises a number of endogenous genes encoding dehydrogenases and reductases that act on substrates similar to one or more benzylisoquinoline alkaloid precursors such as, for example, 4-HPAA.
  • S. cerevisiae comprises five aldehyde dehydrogenase genes (ALD2, ALD3, ALD4, ALD5, and ALD6) and seven primary alcohol dehydrogenase genes (ADH1 , ADH2, ADH3, ADH4, ADH5, ADH6, and ADH7).
  • S. cerevisiae further includes a formaldehyde dehydrogenase gene (SFA1 ), and an aldehyde reductase gene (ARM ).
  • Other endogenous genes similar to ARI1 include YGL039W, YDR541C, YPR1 , GCY1 , GRE2, AAD3, AAD4, and AAD14.
  • tyrosine e.g ., by expressing one or more pathway polypeptides such as Aro10, Aro7, and/or Tyr1 from a constitutive, non-native promoter, and/or by expression one or more feedback-resistant pathway polypeptides such as Aro7-FBR (SEQ ID NO: 12) or Aro4-FBR (SEQ ID NO:4) to remove feedback inhibition from tyrosine results in a downregulated, reinforced tyrosine pathway.
  • pathway polypeptides such as Aro10, Aro7, and/or Tyr1
  • a feedback-resistant pathway polypeptides such as Aro7-FBR (SEQ ID NO: 12) or Aro4-FBR (SEQ ID NO:4)
  • S. cerevisiae comprises an endogenous biosynthetic pathway towards
  • phenylalanine of which the intermediate PAA can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • the phenylalanine pathway involves endogenous gene PHA2.
  • S. cerevisiae also comprises an endogenous biosynthetic pathway towards tryptophan, on which the intermediate IAA can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • the tryptophan pathway involves endogenous genes TRP2 and TRP3.
  • acetaldehyde (AA), 2-methylbutanal (2-MB) 3-methylbutanal (3-MB) (collectively, methylbutanal (MB)), acetoin, and pyruvate can, in certain embodiments, be condensed with dopamine by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • AA, IAA, PAA, 2-MB, 3-MB, acetoin, and pyruvate are intermediates in various biosynthetic pathways (e.g., an endogenous biosynthetic pathway towards tyrosine, leucine, isoleucine, valine, or methionine).
  • NCS-compatible substrate includes any aldehyde other than 4-HPAA that can be condensed with dopamine by a polypeptide also capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine (e.g., an (S)-norcoclaurine synthase (NCS) polypeptide; e.g., SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:52, SEQ ID NO:54) such as, for example, AA, IAA, PAA, 2-MB, 3-MB, acetoin, and pyruvate.
  • S S-norcoclaurine synthase
  • NCS-compatible substrate pathway refers to any biosynthetic pathway that includes an NCS- compatible substrate as an intermediate or final product, such as, for example, the aromatic acid biosynthesis pathways endogenous to S. cerevisiae.
  • recombinant S. cerevisiae host cells capable of producing one or more benzylisoquinoline alkaloids and/or precursors thereof comprise one or more inactivated endogenous genes.
  • An endogenous gene is typically inactivated by disrupting expression of the gene or introducing a mutation to reduce or even completely eliminate endogenous gene activity in a host comprising the mutation.
  • a disruption in one or more endogenous transporter genes reduces or deletes transport expression or activity for the transporter encoded by the disrupted gene(s).
  • reduced expression refers to any level of expression that is less than that of a corresponding gene not having reduced expression, including“repressed expression,”“lowered expression,”“no expression,” and“deletion.”
  • reduced expression can be produced in a host cell by disrupting or deleting the gene locus of the one or more endogenous genes.
  • recombinant S. cerevisiae host cells comprising reduced expression of at least one endogenous transporter gene or a transcription factor that regulates expression of at least one endogenous transporter gene are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine.
  • reducing endogenous transporter activity of a recombinant S. cerevisiae host cell increases the intracellular accumulation of one or more benzylisoquinoline alkaloid precursors (e.g ., dopamine) by the recombinant host, which can subsequently increase production of one or more benzylisoquinoline alkaloids derived therefrom.
  • benzylisoquinoline alkaloid precursors e.g ., dopamine
  • Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene, as disclosed herein, can include one or more
  • benzylisoquinoline alkaloid biosynthesis genes as disclosed herein, such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from 4- HPP; a gene encoding a polypeptide capable of synthesizing 4-HPP from L-tyrosine; a gene encoding a polypeptide capable of synthesizing 4-HPAA from 4-HPP; a gene encoding a polypeptide capable of synthesizing L-DOPA from L-
  • endogenous genes encoding one or more polypeptides capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor e.g., 4-HPAA
  • a disruption in one or more endogenous genes encoding one or more polypeptides capable of synthesizing 4-hydroxyphenylethanol (tyrosol) or 4- hydroxyphenylacetate (4-HPAC) from 4-HPAA reduces expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • recombinant S As set forth herein, recombinant S.
  • cerevisiae host cells comprising reduced expression (i.e., repressed expression or deletion) of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing 4-HPAA, as disclosed herein, are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine.
  • cerevisiae host cell increases the intracellular accumulation of one or more benzylisoquinoline alkaloid precursors (e.g., 4-HPAA) by the recombinant host, which can subsequently increase production of one or more benzylisoquinoline alkaloids derived therefrom.
  • benzylisoquinoline alkaloid precursors e.g., 4-HPAA
  • Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., 4-HPAA), as disclosed herein, can include one or more benzylisoquinoline alkaloid biosynthesis genes as disclosed herein, such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from
  • endogenous genes encoding one or more NCS-compatible substrate pathway polypeptides reduces or deletes expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • a disruption in one or more endogenous genes encoding one or more polypeptides involved in tyrosine reduces or deletes expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • phenylalanine, tryptophan, leucine, isoleucine, valine, or methionine biosynthetic reduces expression or activity for the one or more polypeptides encoded by the disrupted gene(s).
  • recombinant S. cerevisiae host cells comprising reduced expression (i.e., repressed expression or deletion) of at least one NCS-compatible substrate pathway
  • polypeptides disclosed herein are capable of producing at least one benzylisoquinoline alkaloid such as, for example, (S)-norcoclaurine.
  • reducing activity of one or more NCS-compatible substrate pathway polypeptides of a recombinant S. cerevisiae host cell decreases the intracellular accumulation of NCS-compatible substrates (e.g., AA, PAA, IA, 2- MB, 3-MP, acetoin, pyruvate) by the recombinant host, decreasing competition with 4-HPAA, and increasing the relative amount of intracellular dopamine available for condensation with 4- HPAA by a polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine.
  • NCS-compatible substrates e.g., AA, PAA, IA, 2- MB, 3-MP, acetoin, pyruvate
  • Recombinant S. cerevisiae hosts comprising reduced expression of at least one endogenous gene encoding one or more NCS-compatible substrate pathway polypeptides or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, as disclosed herein, can include one or more benzylisoquinoline alkaloid biosynthesis genes such as a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P; a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP; a gene encoding a polypeptide capable of synthesizing prephenate from chorismate; a gene encoding a polypeptide capable of synthesizing 4-HPP from prephenate; a gene encoding a polypeptide capable of synthesizing L-tyrosine from
  • Endogenous genes can be reduced, or endogenous genes can be deleted by mutations that disrupt the gene.
  • a gene replacement vector can be constructed in such a way to include a selectable marker gene flanked at both the 5' and 3' ends by portions of the gene of sufficient length to mediate homologous recombination.
  • the selectable marker can be one of any number of genes that complement host cell auxotrophy, provide antibiotic resistance, or result in a color change.
  • Linearized DNA fragments of the gene replacement vector, containing no plasmid DNA or an element, are then introduced into cells using known methods. Integration of the linear fragment into the genome and the disruption of the gene can be determined based on the selection marker and can be verified by, for example, Southern blot analysis.
  • the resulting cells contain an inactivated mutant gene, due to insertion of the selectable marker at the locus for the polypeptide.
  • a deletion-disruption gene replacement vector can be constructed in a similar way using known techniques and, by way of homologous recombinant, integrated in the endogenous gene, thereby inactivating it.
  • the selectable marker can be removed from the genome of the host cell after determining that the desired disruption mutation has been introduced. See, e.g., Gossen et al. (2002) Ann. Rev. Genetics 36:153-173.
  • endogenous genes can also be reduced, or endogenous genes can be deleted by utilizing CRISPR systems (see, e.g., Adli,“The CRISPR tool kit for genome editing and beyond,” Nature Communications 9:1911 (2016)), transcription activator-like effector nucleases (TALENs) (see, e.g., Joung & Sander,“TALENs: a widely applicable technology for targeted genome editing,” Nat. Rev. Mol. Cell. Biol. 14(1 ):49-55 (2013)), or modified zinc finger nucleases (see, e.g., Carroll,“Genome Engineering With Zinc-Finger Nucleases,” Genetics 188(4):773-82 (2011 ) to introduce desired insertion or deletion mutations.
  • CRISPR systems see, e.g., Adli,“The CRISPR tool kit for genome editing and beyond,” Nature Communications 9:1911 (2016)
  • TALENs transcription activator-like effector nucleases
  • Joung & Sander “TALENs:
  • an endogenous gene is inactivated by introducing a mutation that results in insertions of nucleotides, deletions of nucleotides, or transition or transversion point mutations in the wild-type gene sequence.
  • Other types of mutations that may be introduced in a gene include duplications and inversions in the wild-type sequence. Mutations can be made in the coding sequence at a locus for the polypeptide, as well as in noncoding sequences such as regulatory regions, introns, and other untranslated sequences. Mutations in the coding sequence can result in insertions of one or more amino acids, deletions of one or more amino acids, and/or non-conservative amino acid substitutions in the corresponding gene product. In some cases, the sequence of a gene comprises more than one mutation or more than one type of mutation. Insertion or deletion of amino acids in a coding sequence can, for example, disrupt the conformation of a substrate-binding pocket of the resulting gene product.
  • Amino acid insertions or deletions can also disrupt catalytic sites important for gene product activity. It is known in the art that the insertion or deletion of a larger number of contiguous amino acids is more likely to render the gene product non-functional, compared to a smaller number of inserted or deleted amino acids.
  • Non-conservative substitutions can make a substantial change in the charge or hydrophobicity of the gene product.
  • Non-conservative amino acid substitutions can also make a substantial change in the bulk of the residue side chain, e.g., substituting an alanine residue for an isoleucine residue. Examples of non conservative substitutions include a basic amino acid for a non-polar amino acid, or a polar amino acid for an acidic amino acid.
  • a mutation in a gene may result in no amino acid changes but, although not affecting the amino acid sequence of the encoded polypeptide, may alter transcriptional levels (e.g., increasing or decreasing transcription), decrease translational levels, alter secondary structure of DNA or mRNA, alter binding sites for transcriptional or translational machinery, or decrease tRNA binding efficiency.
  • Mutations in loci for polypeptides can be generated by site-directed mutagenesis of the transporter gene sequence in vitro, followed by homologous recombination to introduce the mutation into the host genome as described above.
  • mutations can also be generated by inducing mutagenesis in cells of the host, using a mutagenic agent to induce genetic mutations within a population of cells. Mutagenesis is particularly useful for those species or strains for which in vitro mutagenesis and homologous recombination is less well established or is inconvenient.
  • the dosage of the mutagenic chemical or radiation for a particular species or strain is determined experimentally such that a mutation frequency is obtained that is below a threshold level characterized by lethality or reproductive sterility.
  • Modification of transcription factor expression can also be used to reduce or eliminate gene expression.
  • the yeast transcriptions factors PDR1 and/or PDR3 regulate expression of the genes encoding ABC transporters PDR5, SNQ2 and YOR1.
  • a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous transporter gene or a transcription factor gene that regulates expression of at least one endogenous transporter gene.
  • the endogenous transporter gene is PDR5, PDR12, PDR15, and/or SNQ2.
  • the endogenous transporter genes are PDR5 and one or more of PDR12, PDR15 and SNQ2.
  • the endogenous transporter genes are one or more of PDR5, PDR12, PDR15, and/or SNQ2 (e.g., PDR5 and PDR12), and one or more of AUS1 , PDR10, YOR1 , TP01 , TP02, TP03, TP04, QDR1 , QDR2, QDR3, FLR1 , YOL075C, PDR18, DTR1 , YHK8, NFT1 , STE6, YCF1 , YBT1 , BPT1 , and VMR1.
  • PDR5 and PDR12 e.g., PDR5 and PDR12
  • a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor, or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor.
  • the at least one endogenous gene encodes a polypeptide capable of synthesizing tyrosol or 4-HPAC from 4-HPAA, such as, for example, ARM , ALD4, ADH6, YPR1 , or YDR541 C.
  • the endogenous gene is ARM , ALD4, ADH6, YPR1 , YDR541 C, YGL039W,
  • a benzylisoquinoline alkaloid-producing host comprises reduced expression of at least one endogenous gene encoding one or more NCS-compatible substrate pathway polypeptides.
  • the NCS-compatible substrate pathway is an aromatic amino acid biosynthesis pathway, such as, for example, tryptophan (e.g., involving TRP2 and TRP3) or phenylalanine (e.g., involving PHA2) biosynthesis.
  • the NCS-compatible substrate pathway polypeptide is capable of synthesizing phenylpyruvate from prephenate (e.g., PHA2), capable of synthesizing anthranilate from chorismate (e.g., TRP2, and/or TRP3).
  • prephenate e.g., PHA2
  • chorismate e.g., TRP2, and/or TRP3
  • the PDR5 gene which can have the nucleotide sequence set forth in SEQ ID NO:69, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:70.
  • the PDR12 gene which can have the nucleotide sequence set forth in SEQ ID NO:71 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:72.
  • the PDR15 gene which can have the nucleotide sequence set forth in SEQ ID NO:73) encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:74.
  • the SNQ2 gene which can have the nucleotide sequence set forth in SEQ ID NO:75 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:76.
  • the PDR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:183, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 184.
  • the PDR3 gene which can have the nucleotide sequence set forth in SEQ ID NO: 185, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:186.
  • the AUS1 gene which can have the nucleotide sequence set forth in SEQ ID NO:77 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:78.
  • the PDR10 gene which can have the nucleotide sequence set forth in SEQ ID NO:79, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:80.
  • the YOR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:81 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:82.
  • the TP01 gene which can have the nucleotide sequence set forth in SEQ ID NO:83 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:84.
  • the TP02 gene which can have the nucleotide sequence set forth in SEQ ID NO:85 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:86.
  • the TP03 gene which can have the nucleotide sequence set forth in SEQ ID NO:87, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:88.
  • the TP04 gene which can have the nucleotide sequence set forth in SEQ ID NO:89, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:90.
  • the QDR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:91 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:92.
  • the QDR2 gene which can have the nucleotide sequence set forth in SEQ ID NO:93, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:94.
  • the QDR3 gene which can have the nucleotide sequence set forth in SEQ ID NO:95, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:96.
  • the FLR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:97, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:98.
  • the YOL075C gene which can have the nucleotide sequence set forth in SEQ ID NO:99, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:100.
  • the PDR18 gene which can have the nucleotide sequence set forth in SEQ ID NO:101 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:102.
  • the DTR1 which can have the nucleotide sequence set forth in SEQ ID NO: 103, gene encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:104.
  • the YHK8 gene which can have the nucleotide sequence set forth in SEQ ID NO:105, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:106.
  • the NFT 1 gene which can have the nucleotide sequence set forth in SEQ ID NO:107, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 108.
  • the STE6 gene which can have the nucleotide sequence set forth in SEQ ID NO:109, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 10.
  • the YCF1 gene which can have the nucleotide sequence set forth in SEQ ID NO:1 1 1 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 12.
  • the YBT 1 gene which can have the nucleotide sequence set forth in SEQ ID NO: 113 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 1 14.
  • the BPT2 gene which can have the nucleotide sequence set forth in SEQ ID NO:1 15 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:1 16.
  • the VMR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:1 17 encodes a polypeptide having an amino acid sequence set as forth in SEQ ID NO:1 18.
  • the ARI1 gene which can have the nucleotide sequence set forth in SEQ ID NO:145, encodes a polypeptide having an amino acid sequence set forth in SEQ ID NO: 146.
  • the ALD4 gene which can have the nucleotide sequence set forth in SEQ ID NO:123, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:124.
  • the ADH6 gene which can have the nucleotide sequence set forth in SEQ ID NO: 139, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 140.
  • the YPR1 gene which can have the nucleotide sequence set forth in SEQ ID NO:153, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 154.
  • the YDR541 C gene which can have the nucleotide sequence set forth in SEQ ID NO:149 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 150.
  • the PHA2 gene which can have the nucleotide sequence set forth in SEQ ID NO: 163 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 164.
  • the TRP2 gene which can have the nucleotide sequence set forth in SEQ ID NO:165 encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 166.
  • the TRP3 gene which can have the nucleotide sequence set forth in SEQ ID NO:167, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:168.
  • the AAD3 gene which can have the nucleotide sequence set forth in SEQ ID NO:159, encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO:160.
  • the GRE2 gene which can have the nucleotide sequence set forth in SEQ ID NO: 151 , encodes a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 152.
  • benzylisoquinoline alkaloids and/or precursors thereof are produced in vivo through expression of one or more recombinant genes encoding one or more benzylisoquinoline biosynthesis polypeptides in a recombinant host comprising reduced expression of one or more endogenous genes.
  • a recombinant S for example, a recombinant S.
  • cerevisiae host comprising reduced expression of at least one endogenous transporter gene (e.g ., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene (e.g., PDR1 and/or PDR3), and further comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephen
  • the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or each of PDR5 (SEQ ID NO:69, SEQ ID NO:70), PDR12 (SEQ ID NO:71 , SEQ ID NO:72), PDR15 (SEQ ID NO:73, SEQ ID NO:74), and SNQ2 (SEQ ID NO:75, SEQ ID NO:76).
  • the polypeptide capable of synthesizing (S)- norcoclaurine from 4-HPAA and dopamine has the amino acid sequence set forth in any of SEQ ID NO:42 or 30.
  • a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3,
  • GRE2 GRE2, and/or YDR541 C
  • a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor, and further comprising a gene encoding a polypeptide capable of synthesizing DAHP from PEP and E4P (e.g., a recombinant gene encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO:4); a gene encoding a polypeptide capable of synthesizing EPSP from DAHP; a gene encoding a polypeptide capable of synthesizing chorismate from EPSP (e.g., a recombinant gene having an amino acid sequence as set forth in SEQ ID NO:8); a gene encoding a polypeptide capable of synthesizing prephenate from chorismate (e.g., a recombinant gene encoding
  • the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or four, or five, or six, or each of ARM (SEQ ID NO:145, SEQ ID NO:146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO:140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), AAD3 (SEQ ID NO:159, SEQ ID NO: 160), GRE2 (SEQ ID NO: 151 , SEQ ID NO:152), and YDR541 C (SEQ ID NO:149, SEQ ID NO:150).
  • ARM SEQ ID NO:145, SEQ ID NO:146
  • ALD4 SEQ ID NO:123, SEQ ID NO:124
  • ADH6 SEQ ID NO:139, SEQ ID NO:140
  • YPR1 SEQ ID NO:153, SEQ ID NO:154
  • AAD3 SEQ ID NO:159,
  • the recombinant S. cerevisiae host comprises reduced expression of one, or two, or three, or four, or five, or six, or each of ARM (SEQ ID NO:145, SEQ ID NO: 146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO:140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), AAD3 (SEQ ID NO: 159, SEQ ID NO:160), GRE2 (SEQ ID NO: 151 , SEQ ID NO:152), and YDR541C (SEQ ID NO: 149, SEQ ID NO:150), and further comprises reduced expression of one, or two, or each of TRP2 (SEQ ID NO:165, SEQ ID NO: 166), TRP3 (SEQ ID NO: 167, SEQ ID NO: 168), and PHA2 (SEQ ID NO: 163, SEQ ID NO:164).
  • ARM SEQ ID NO:
  • the polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine is a truncated polypeptide (e.g., lacking an N-terminal signal peptide; e.g. having an amino acid sequence as set forth in SEQ ID NO:42) .
  • the polypeptide capable of synthesizing (S)-norcoclaurine from 4-HPAA and dopamine is a polypeptide having an amino acid sequence as set forth in SEQ ID NO:30.
  • a recombinant S. cerevisiae host comprising reduced expression of at least one endogenous transporter gene (e.g ., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene, further comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3, GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor and/or reduced expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide (e.g., PHA2, TRP2, and/or T
  • expression of one or more recombinant genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA e.g., a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198
  • S synthesizing
  • cerevisiae host comprising reduced expression of one or more endogenous transporter genes (e.g., PDR5, PDR12, PDR15, and/or SNQ2) or a transcription factor that regulates expression of at least one endogenous transporter gene (e.g., PDR1 and/or PDR3) increases the amount of (S)- norcoclaurine produced by the host cell by at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%.
  • endogenous transporter genes e.g., PDR5, PDR12, PDR15, and/or SNQ2
  • a transcription factor that regulates expression of at least one endogenous transporter gene e.g., PDR1 and/or
  • expression of one or more recombinant genes encoding a polypeptide capable of synthesizing (S)-norcoclaurine from dopamine and 4-HPAA e.g ., a polypeptide having an amino acid sequence set forth in any of SEQ ID NOs: 30, 32, 34, 36, 38, 40, 42, 44, 52, 54, 188, 190, 192, 194, 196, and 198
  • S synthesizing
  • cerevisiae host comprising reduced expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor (e.g., ARM , ALD4, ADH6, YPR1 , AAD3, GRE2, and/or YDR541 C) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a polypeptide capable of reducing or oxidizing a benzylisoquinoline alkaloid precursor and/or reduced expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide (e.g., PHA2, TRP2, and/or TRP3) or a transcription factor gene that regulates expression of at least one endogenous gene encoding a NCS-compatible substrate pathway polypeptide, increases the amount of (S)-norcoclaurine produced by the host cell by at least about 50%, or at least about 100%, or at least about 200%, or at least about
  • a recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired.
  • a coding sequence and a regulatory region are considered to be operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.
  • the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid.
  • the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism.
  • a native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct.
  • stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found.
  • Regulatory region refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product.
  • Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 ' and 3 ' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof.
  • a regulatory region typically comprises at least a core (basal) promoter.
  • a regulatory region also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR).
  • a regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence.
  • the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter.
  • a regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.
  • regulatory regions The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region may be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.
  • One or more genes can be combined in a recombinant nucleic acid construct in “modules” useful for a discrete aspect of production of benzylisoquinoline alkaloids and/or precursors thereof. Combining a plurality of genes in a module, particularly a polycistronic module, facilitates the use of the module in a variety of species.
  • a recombinant construct typically also contains an origin of replication, and one or more selectable markers for maintenance of the construct in appropriate species.
  • nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid.
  • codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular host is obtained, using appropriate codon bias tables for that host ⁇ e.g.,
  • these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs.
  • Recombinant S. cerevisiae hosts can be used to express polypeptides as otherwise described herein for producing benzylisoquinoline alkaloids.
  • the recombinant host is grown in a fermenter at a temperature for a period of time, wherein the temperature and period of time facilitate production of one or more benzylisoquinoline alkaloids.
  • the constructed and genetically engineered S. cerevisiae hosts provided by the invention can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, semi-continuous fermentations such as draw and fill, continuous perfusion fermentation, and continuous perfusion cell culture.
  • Levels of substrates and intermediates e.g., E4P, DAHP, EPSP, chorismate, prephenate, 4-HPP, 4-HPAA, L-tyrosine, L-DOPA, and dopamine, can be determined by extracting samples from culture media for analysis according to published methods.
  • Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host cell to facilitate growth and/or production of the benzylisoquinoline alkaloids.
  • suitable carbon sources include sucrose, fructose, xylose, ethanol, glycerol, and glucose.
  • the carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during a fed-batch phase.
  • the present invention further relates to a fermentation process, including but not limited to small-scale or batch and/or fed-batch fermentation process and a large-scale fermentation process, for the production of (S)-norcoclaurine, as described herein, whereby the cultivation regime of the process comprises at least one production stage fermentation phase.
  • the fermentation process is a batch cultivation.
  • the fermentation process is a fed-batch process.
  • a base medium supports initial growth and production, and a continuously or periodically supplied feeding medium prevents depletion of nutrients and sustains the production stage.
  • the media can be selected to accommodate the distinct metabolic requirements during different cultivation phases.
  • Process parameters including feeding strategy and control parameters define the chemical and physical environments suitable for cell growth and/or (S)-norcoclaurine production.
  • Benzylisoquinoline alkaloids and precursors thereof can be isolated from a cell culture using a method described herein. For example, following fermentation, cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose. After 72-96 h growth, metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice- cold 100% acetonitrile (ACN; 80% final concentration). Cells were incubated for 5 minutes and subsequently diluted with 417 mI of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract was utilized for LC-MS analysis. Cell broth was diluted 13.34-fold using this method.
  • Fluorescence levels were measured after cultivating strains overnight in SC with 2% glucose. Overnight cultures were diluted 10* into fresh media and incubated for an additional 4 h to obtain log phase cells. Fluorescence was measured from cell suspension using a microplate reader and normalized against OD 6 oo for three biological replicates. Fluorescence was detected by the TECAN M200 plate reader using an excitation wavelength of 485 nm and an emission wavelength of 525 nm. Gain was adjusted for each sample. A background strain lacking GFP was used to correct for autofluorescence generated by cells and media.
  • Dopamine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a feedback-resistant variant of DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:21 , SEQ ID NO:22), and a recombinant gene encoding an L-DOPA decarboxylase polypeptide
  • a dopamine-producing S. cerevisiae strain as described in Example 3 was transformed with a vector comprising a codon-optimized nucleotide sequence encoding an NCS polypeptide, optionally N-terminal truncated to remove a signal peptide, C-terminal truncated to remove a transmembrane domain, or C-terminal domain-swapped, and/or optionally green fluorescent protein (GFP)-tagged, operably linked to a pTEF1 promoter (SEQ ID NO:173 and a tPGM terminator (SEQ ID NO:174), as summarized in Table 1 , below.
  • GFP green fluorescent protein
  • Dopamine-producing S. cerevisiae cells were cultivated in YPD medium (10 g/L yeast extract, 20 g/L tryptone, 20 g/L dextrose; Thermo Fisher Scientific) for routine strain growth and maintenance.
  • YPD was supplemented, if necessary, with 200 pg/ml G418 or 200 pg/nnl hygromycin B. G418 concentration was increased to 400 pg/ml for selection on agar- solidified YPD.
  • E. coli DH5a was utilized for routine plasmid maintenance and propagation and was cultivated in Lysogeny Broth (LB) (Sigma Aldrich). Ampicillin or kanamycin at
  • Colonies were picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates.
  • the present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%.
  • cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose.
  • metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice-cold 100% acetonitrile (ACN; 80% final concentration).
  • a dopamine-producing S. cerevisiae strain as described in Example 3, further engineered to express at least one (S)-norcoclaurine synthase as described in Example 4 (SEQ ID NO:31 , SEQ ID NO:32; SEQ ID NO:41 , SEQ ID NO:42) was engineered to downregulate expression of one or more native genes encoding dehydrogenase and reductase polypeptides having potential activity on one or more benzylisoquinoline alkaloid precursors, and optionally to overexpress one or more benzylisoquinoline alkaloid biosynthesis polypeptides, as summarized in Table 4, below.
  • Colonies were picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates.
  • the present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%.
  • cultures were diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose.
  • metabolites were extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice-cold 100% acetonitrile (ACN; 80% final concentration).
  • S. cerevisiae genes with potential activity for 4-HPAA were targeted for deletion (strains 20-40) including all 5 aldehyde dehydrogenase genes (ALD2, ALD3, ALD4, ALD5, and ALD6), all 7 primary alcohol dehydrogenase genes (ADH1-ADH7), the gene encoding formaldehyde dehydrogenase (SFA1 ), and 7 genes similar to ARI1 (YGL039W, YDR541C, YPR1 , GCY1 , GRE2, AAD3, and AAD14).
  • S formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenase
  • SFA1 formaldehyde dehydrogenas
  • Strain 32 showed a 3.5-fold increase in (S)-norcoclaurine production.
  • Strains 21 and 23 demonstrated increased dopamine formation; strain 21 (lacking ALD6) showed a 2.8-fold increase.
  • Strain 40 produced no dopamine or (S)-norcoclaurine.
  • strains further overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, YPR1 showed no additional improvement over strain 66, similarly overexpressing N-terminal truncated NdNCS and lacking the combination of ARM , ALD4, ADH6, YPR1 , and YDR541 C.
  • strain 66 demonstrated a 121-fold increase of (S)-norcoclaurine production. See Figure 9.
  • LC-MS spectra were examined for peaks indicating off-target NCS condensation products by first searching for 35 unique m/z values derived from condensation of dopamine with 63 possible endogenous aldehydes and ketones, including aliphatic, aromatic, and cyclic species. From this analysis, six putative Pictet-Spengler-competent carbonyl species were identified. See Figure 10. A major peak consistent with salsolinol (1 ), derived from condensation of dopamine with acetaldehyde, was observed and was larger than the (S)-norcoclaurine peak in all LC-MS spectra.
  • PAA and IAA aromatic Ehrlich pathway aldehydes
  • deletion of ALD4 enhanced formation of all condensation products by a factor of 2.2-10, likely due to increased levels of dopamine and presumably higher flux through the aromatic amino acid (AAA) pathway.
  • strain 87 a strain having inactivated phenylalanine and tryptophan biosynthesis polypeptides was further engineered to express eight copies of the recombinant gene encoding N-terminal truncated NdNCS of Example 4 (strain 87). Further overexpression of a chorismate synthase polypeptide in strain 88 led to an increase in production of (S)-norcoclaurine relative to strain 87. Of strains 89-93, further engineered to delete, respectively, ADH5, AAD14, ADH3, YGL029W, or AAD3, strain 93 showed the greatest increase in production of (S)-norcoclaurine relative to strain 88. See Figures 22-23.
  • Strains overexpressing N-terminal truncated NdNCS, Tyr1 , Aro7, Aro10, and Aro2, and lacking ARM , ALD4, ADH6, YPR1 , YDR541C, PHA2, TRP3, and AAD3, were further engineered to delete AAD4, ADH7, SFA1 , YGL039, AAD14, GRE2, GCY1 , or ALD2 and ALD3 to provide, respectively, strains 94-101.
  • strain 99 demonstrated an 82% decrease in tyrosol production and a 97% increase in (S)-norcoclaurine production relative to strain 93. See Figures 24-25.
  • aldo-keto reductases e.g., ARM , ADH6, YPR1 , YDR541C, AAD3, GRE2
  • tyrosol formation i.e., from 4-HPAA
  • deletion of genes not directly related tyrosol formation ⁇ e.g., ALD4, PHA2, TRP3
  • ALD4, PHA2, TRP3 can further increase carbon flux to L-tyrosine.
  • Strains 67 and 68 of Example 5 were each cultivated in a fed-batch bioreactor. Controlled fed-batch cultivations were carried out in a 3 L BioBundle fermenter (Applikon) equipped with d0 2 , pH, and temperature sensors. Cultivation was controlled using an EZ- Control bio-controller and BioXpert cultivation control software (Applikon). A variable speed pump (Watson-Marlow) was used to supply the feeding medium. Culture pH was maintained at 4.5 by titration with 3 M NaOH and 3 N H 2 S0 4 . Dissolved oxygen was maintained at 10 % of air saturation by automatically adjusting the stirring rate (aeration rate 0.5 L/min). Cultivation temperature was maintained at 30 °C.
  • the batch medium contained per L: 2.5 g KH 2 P0 4 , 5 g NH CI, 1 g MgS0 4 -7H 2 0, 5 ml vitamin stock, 5 ml microelement stock, 1.6 g yeast synthetic drop-out medium without leucine, 380 mg L-leucine, and (i) 40 g sucrose or (ii) 30 g glycerol.
  • Vitamin stock contained per L: 5 mg biotin, 100 mg calcium pantothenate, 100 mg nicotinic acid, 2,500 mg myo-inositol, 100 mg thiamine hydrochloride, 100 mg pyridoxine, and 20 mg p-aminobenzoic acid.
  • Microelement stock contained per L: 15 g disodium EDTA, 2.9 g CaCI 2 , 9.2 g ZnS0 4 -7H 2 0, 5.1 g FeS0 4 -7H 2 0, 0.5 g CuS0 4 , 0.43 g MnS0 4 H 2 0, 0.47 g CoCI 2 , and 0.48 g Na 2 Mo0 4 .
  • the culture was grown in batch mode for 16 to 96 hours.
  • the fed-batch phase was carried out with a constant feeding rate of 3.5 mL/h.
  • the feeding medium contained per L: 10 g KH 2 P0 4 , 40 g NH 4 CI, 4 g
  • MgS0 -7H 2 0, 10 ml vitamin stock, 10 mL microelement stock, 400 mg yeast synthetic drop-out medium without leucine, 2.5 g L-leucine, 810 mg L-histidine, 570 mg L-methionine, 800 mg uracil, and 240 g sucrose.
  • the total cultivation time was seven to ten days and samples (5 mL) were collected daily.
  • Cell densities were determined by dilution of fermentation broth with water followed by measurement of absorption at 600 nm (OD 6 oo). Dry cell weight (g/L) was calculated using a conversion factor of 0.59 g/L per OD 6 oo (determined gravimetrically). Culture broths were analyzed for metabolite production according to Example 1.
  • Results are shown in Figures 19-20.
  • the batch medium for strain 67 contained sucrose as a carbon source.
  • the cells were unable to metabolize the ethanol produced during the batch phase of the cultivation, ultimately leading to growth arrest. See Figure 19. Under these conditions, this strain produced 215 mg/L (S)-norcoclaurine. See Figure 20.
  • glycerol was used as a carbon source in the batch medium of strain 68. Ethanol levels remained low during the fed-batch phase, and the strain produced 383 mg/L (S)- norcoclaurine. See Figure 20.
  • An (S)-norcoclaurine stock solution was prepared in DMSO at a concentration of 10 mM. Standard solutions were prepared at concentrations of 4 mM, 2 mM, 1 mM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, and 10 nM from the stock solution. Samples were injected into an Agilent 1290 UPLC coupled to an Ultivo Triple Quadrupole. The LC-MS method was as follows: Mobile Phase A. H 2 0 + 0.1 % Formic acid; Mobile Phase B: Acetonitrile + 0.1 % Formic acid; Column: Phenomenex Kinetex 1.7 pm XB-C18 100A, 2.1x100mm.
  • Table 5 shows the elution gradient and the LC-MS conditions are given in Table 6.
  • Table 7 shows the mass spectrometer source and detector parameters and Table 8 shows the target compound, retention time, parent ion, transition ions (MRM) as well as dwell time, fragmentor voltage and collision energy used.
  • MRM transition ions
  • (S)-norcoclaurine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), operably linked to a pPDC1 promoter (SEQ ID NO:169) and a tCYC1 terminator (SEQ ID NO:175); a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:SEQ ID NO:23, SEQ ID NO:24), operably linked to a pTEF2 promoter (SEQ ID NO:170) and a tFBA1 terminator (SEQ ID NO: 176); a recombinant gene encoding an L-DOPA decarboxylase polypeptide (SEQ ID NO:SEQ ID NO:27, SEQ ID NO:28), operably linked to a pTDH3 promoter (SEQ ID NO
  • Yeast transformants were grown in 96-deep well plates in 500 pL liquid synthetic complete media for 3 days at 30 °C with shaking at 230 rpm in a Kuhner Climo-Shaker ISF1-X.
  • Culture samples for LC-MS were prepared by extraction as follows: 96% ethanol and culture sample were mixed 1 :1 and incubated on a heating block at 80 °C for 10 minutes. After heating, cells were pelleted in an Eppendorff tabletop centrifuge and the supernatant was then transferred to a new tube and diluted 1 :10 in water.
  • (S)-norcoclaurine-producing S. cerevisiae strains comprising and expressing a recombinant gene encoding a DAHP synthase polypeptide (SEQ ID NO:3, SEQ ID NO:4), a recombinant gene encoding a tyrosine hydroxylase polypeptide (SEQ ID NO:21 , SEQ ID NO:22; SEQ ID NO:23, SEQ ID NO:24), a recombinant gene encoding an L-DOPA decarboxylase polypeptide (SEQ ID NO:25, SEQ ID NO:26; SEQ ID NO:27, SEQ ID NO:28), a recombinant gene encoding a prephenate dehydrogenase (SEQ ID NO:13, SEQ ID NO:14), a recombinant gene encoding a phenylpyruvate decarboxylase (SEQ ID NO:19, SEQ ID NO:20), and/or
  • strains are further engineered to comprise reduced expression of one or more endogenous transporter genes PDR5 (SEQ ID NO:69, SEQ ID NO:70), PDR12 (SEQ ID NO:71 , SEQ ID NO:72), PDR15 (SEQ ID NO:73, SEQ ID NO:74), SNQ2 (SEQ ID NO:75, SEQ ID NO:76), and/or one or more endogenous transporter genes AUS1 (SEQ ID NO:77, SEQ ID NO:78), PDR10 (SEQ ID NO:79, SEQ ID NO:80), YOR1 (SEQ ID NO:81 , SEQ ID NO:82),
  • TP01 (SEQ ID NO:83, SEQ ID NO:84), TP02 (SEQ ID NO:85, SEQ ID NO:86), TP03 (SEQ ID NO:87, SEQ ID NO:88), TP04 (SEQ ID NO:89, SEQ ID NO:90), QDR1 (SEQ ID NO:91 , SEQ ID NO:92), QDR2 (SEQ ID NO:93, SEQ ID NO:94), QDR3 (SEQ ID NO:95, SEQ ID NO:96), FLR1 (SEQ ID NO:97, SEQ ID NO:98), YOL075C (SEQ ID NO:99, SEQ ID NO:100), PDR18 (SEQ ID NO: 101 , SEQ NO:102), DTR1 (SEQ ID NO:103, SEQ ID NO:104), YHK8 (SEQ ID NO:105, SEQ ID NQ:106), NFT1 (SEQ ID NQ:107, SEQ ID NQ: 108), STE6 (SEQ ID NQ:109
  • BPT1 SEQ ID NO:1 15, SEQ ID NO:1 16
  • VMR1 SEQ ID NO:1 17, SEQ ID NO: 1 18
  • the strains are further engineered to comprise reduced expression of one or more endogenous genes ARI1 (SEQ ID NO:145, SEQ ID NO:146), ALD4 (SEQ ID NO:123, SEQ ID NO:124), ADH6 (SEQ ID NO:139, SEQ ID NO: 140), YPR1 (SEQ ID NO:153, SEQ ID NO:154), YDR541C (SEQ ID NO: 149, SEQ ID NO:150), PHA2 (SEQ ID NO:163, SEQ ID NO: 164), TRP2 (SEQ ID NO: 165, SEQ ID NO:166), and TRP3 (SEQ ID NO: 167, SEQ ID NO:168).
  • ARI1 SEQ ID NO:145, SEQ ID NO:146
  • ALD4 SEQ ID NO:123, SEQ ID NO:124
  • ADH6 SEQ ID NO:139, SEQ ID NO: 140
  • YPR1 SEQ ID NO:153, SEQ ID NO:154
  • YDR541C S
  • Colonies of the (S)-norcoclaurine-producing strains as described in Example 10 are picked in triplicate and inoculated into 500 pi of 2* synthetic complete (SC) medium containing 4% glucose or sucrose in 96-well two ml deep well plates.
  • SC synthetic complete
  • the present inventors have determined that sucrose, in place of glucose, increases production of metabolites derived from the shikimate pathway by 50%.
  • cultures are diluted 50* into 500 pi of fresh 2*SC+4% glucose or sucrose.
  • metabolites are extracted from cells by combining 50 pi of culture broth (cells+medium) with 200 pi of ice- cold 100% acetonitrile (ACN; 80% final concentration). Cells are incubated for 5 minutes and subsequently diluted with 417 pi of 0.16% formic acid (FA) to give a final concentration of 30% ACN and 0.1 % FA. The resulting extract is utilized for LC-MS analysis according to Example 1. Cell broth is diluted 13.34-fold using this method.
  • Strains as described in Example 10 are each cultivated in a fed-batch bioreactor. Controlled fed-batch cultivations are carried out in a 3 L BioBundle fermenter (Applikon) equipped with d0 2 , pH, and temperature sensors. Cultivation is controlled using an EZ-Control bio-controller and BioXpert cultivation control software (Applikon). A variable speed pump (Watson-Marlow) s used to supply the feeding medium. Culture pH is maintained at 4.5 by titration with 3 M NaOH and 3 N H 2 SO 4 . Dissolved oxygen is maintained at 10 % of air saturation by automatically adjusting the stirring rate (aeration rate 0.5 L/min). Cultivation temperature is maintained at 30 °C.
  • the batch medium contains per L: 2.5 g KH 2 PO 4 , 5 g NH 4 CI, 1 g MgS0 4 -7H 2 0, 5 ml vitamin stock, 5 ml microelement stock, 1.6 g yeast synthetic drop-out medium without leucine, 380 mg L-leucine, and (i) 40 g sucrose or (ii) 30 g glycerol.
  • Vitamin stock contains per L: 5 mg biotin, 100 mg calcium pantothenate,
  • Microelement stock contains per L: 15 g disodium EDTA, 2.9 g CaCh, 9.2 g ZnS0 4 -7H 2 0, 5.1 g FeS0 4 -7H 2 0, 0.5 g CuS0 4 , 0.43 g MnS0 4 H 2 0, 0.47 g C0CI 2 , and 0.48 g Na 2 Mo0 4 .
  • the culture is grown in batch mode for 16 to 96 hours.
  • the fed-batch phase is carried out with a constant feeding rate of 3.5 mL/h.
  • the feeding medium contains per L: 10 g KH 2 PO 4 , 40 g NH 4 CI, 4 g MgS0 4 -7H 2 0, 10 ml vitamin stock, 10 mL microelement stock, 400 mg yeast synthetic drop-out medium without leucine, 2.5 g L-leucine, 810 mg L-histidine, 570 mg L-methionine, 800 mg uracil, and 240 g sucrose.
  • the total cultivation time is seven to ten days and samples (5 mL) are collected daily. Cell densities are determined by dilution of fermentation broth with water followed by measurement of absorption at 600 nm (OD 6 oo). Dry cell weight (g/L) is calculated using a conversion factor of 0.59 g/L per OD 6 OO (determined gravimetrically). Culture broths are analyzed for metabolite production according to Example 1.
  • NCS enzymes inherently are promiscuous and can perform the Pictet-Spengler reaction with aldehydes other than 4-HPAA, resulting in unwanted side products and carbon loss.
  • GLINDPDVNN TFNINKGLQS ARQLFVNLTN IGLP IGSEML DTISPQYLAD LVSFGAIGAR 180
  • MAPVTIEKFV NQEERHLVSN RSATIPFGEY IFKRLLSIDT KSVFGVPGDF NLSLLEYLYS 60 PSVESAGLRW VGTCNELNAA YAADGYSRYS NKIGCLITTY GVGELSALNG IAGSFAENVK 120 VLHIVGVAKS IDSRSSNFSD RNLHHLVPQL HDSNFKGPNH KVYHDMVKDR VACSVAYLED 180 IETACDQVDN VIRDIYKYSK PGYIFVPADF ADMSVTCDNL VNVPRISQQD CIVYPSENQL 240 SDI INKITSW IYSSKTPAIL GDVLTDRYGV SNFLNKLICK TGIWNFSTVM GKSVIDESNP 300 TYMGQYNGKE GLKQVYEHFE LCDLVLHFGV DINEINNGHY TFTYKPNAKI IQFHPNYIRL 360 VDTRQGNEQM FKGINFAP IL KELYKRIDV
  • MDHATLAMIL AILFISFHFI KLLFSQQTTK LLPPGPKPLP I IGNILEVGK KPHRSFANLA 60 KIHGPLISLR LGSVTTIVVS SADVAKEMFL KKDHPLSNRT IPNSVTAGDH HKLTMSWLPV 120 SPKWRNFRKI TAVHLLSPQR LDACQTFRHA KVQQLYEYVQ ECAQKGQAVD IGKAAFTTSL 180 NLLSKLFFSV ELAHHKSHTS QEFKELIWNI MEDIGKPNYA DYFP ILGCVD PSGIRRRLAC 240 SFDKLIAVFQ GIICERLAPD SSTTTTTTTD DVLDVLLQLF KQNELTMGEI NHLLVDIFDA 300 GTDTTSSTLE WVMTELIRNP EMMEKAQEEI KQVLGKDKQI QESDIINLPY LQAI IKETLR 360 LHPPTVFLLP RKADTDVELY GYIVPKDAQI LVNLWAIG
  • MDHATLAMIL AILFISFHFI KLLFSQQTTK LLPPGPKPLP I IGNILEVGK KPHRSFANLA 60 KIHGPLISLR LGSVTTIVVS SADVAKEMFL KKDHPLSNRT IPNSVTAGDH HKLTMSWLPV 120 SPKWRNFRKI TAVHLLSPQR LDACQTFRHA KVQQLYEYVQ ECAQKGQAVD IGKAAFTTSL 180 NLLSKLFFSV ELAHHKSHTS QEFKELIWNI MEDIGKPNYA DYFP ILGCVD PSGIRRRLAC 240 SFDKLIAVFQ GIICERLAPD SSTTTTTTTD DVLDVLLQLF KQNELTMGEI NHLLVDIFDA 300 GTDTTSSTLE WVMTELIRNP EMMEKAQEEI KQVLGKDKQI QESDIINLPY LQAI IKETLR 360 LHPPTVFLLP RKADTDVELY GYIVPKDAQI LVNLWAIG
  • KIVLENKAKH KGFIEIGMSK GEELFTGWP ILVELDGDVN GHKFSVSGEG EGDATYGKLT 240
  • Atgtcatcag atatcagaga cgtagaggaa cgaaattcgc ggagctcgag ctcaagctcg 60 agctcgaact ctgccgccca atccattgga cagcatccat accgcggttt cgacagcgaa 120 gccgcggaaa gggtgcatga gttggctaga acactcacat cgcagagttt actatacact 180 gctaactcaa acaatagctc ttccagcaac cataatgcgc acaatgcgga ctcgagatcc 240 gtattttcta cggacatgga aggtgtgaac ccggtgttca ctaacccgga caccccggga 300 tacaatccca a
  • MSSDIRDVEE RNSRSSSSSS SSNSAAQSIG QHPYRGFDSE AAERVHELAR TLTSQSLLYT 60 ANSNNSSSSN HNAHNADSRS VFSTDMEGVN PVFTNPDTPG YNPKLDPNSD QFSSTAWVQN 120 MANICTSDPD FYKPYSLGCV WKNLSASGDS ADVSYQSTFA NIVPKLLTKG LRLLKPSKEE 180 DTFQILKPMD GCLNPGELLV VLGRPGSGCT TLLKSISSNS HGFKIAKDSI VSYNGLSSSD 240 IRKHYRGEW YNAESDIHLP HLTVYQTLFT VARMKTPQNR IKGVDREAYA NHVTEVAMAT 300 YGLSHTRDTK VGNDLVRGVS GGERKRVSIA EVAICGARFQ CWDNATRGLD SATALEFIRA 360 LKTQAD IGKT AATVAIYQCS QDAYDLFDKV CVLDDGYQLY F
  • MSQQENGDVA TELIENRLSF SRIPRISLHV RDLSIVASKT NTTLVNTFSM DLPSGSVMAV 60 MGGSGSGKTT LLNVLASKIS GGLTHNGSIR YVLEDTGSEP NETEPKRAHL DGQDHPIQKH 120 VIMAYLPQQD VLSPRLTCRE TLKFAADLKL NSSERTKKLM VEQLIEELGL KDCADTLVGD 180 NSHRGLSGGE KRRLSIGTQM ISNPSIMFLD EPTTGLDAYS AFLVIKTLKK LAKEDGRTFI 240 MSIHQPRSDI LFLLDQVCIL SKGNWYCDK MDNTIPYFES IGYHVPQLVN PADYFIDLSS 300 VDSRSDKEEA ATQSRLNSLI DHWHDYERTH LQLQAESYIS NATEIQIQNM TTRLPFWKQV 360 TVLTRRNFKL NFSDYVTLIS TFAEPLI IGT VCGWIYYKPD KSS
  • GVEMANSSEF GLGSGIETES LSTGLKVAKM LKAGTVWINT YNDFDSRVPF
  • HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQWKSIS IVGSYVGNRA 300
  • HGI INVSVSE AAIEASTRYC RANGTWLVG LPAGAKCSSD VFNHWKSIS IVGSYVGNRA 300
  • PAWWSAANY YATSYGKTPF SIYQGKWNVL NRDFERDIIP MARHFGMALA PWDVMGGGRF 240
  • HGVINVSVSE AAIEASTRYV RANGTTVLVG MPAGAKCCSD VFNQWKSIS IVGSYVGNRA 300

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Abstract

L'invention concerne des micro-organismes recombinés, ainsi que des procédés de production d'alcaloïdes de benzylisoquinoline et de leurs précurseurs.
PCT/EP2019/066561 2018-06-22 2019-06-21 Production d'alcaloïdes de benzylisoquinoline chez des hôtes recombinés WO2019243624A1 (fr)

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WO2022234005A1 (fr) 2021-05-07 2022-11-10 River Stone Biotech Aps Opioïdes glycosylés
WO2024100063A1 (fr) 2022-11-08 2024-05-16 River Stone Biotech Aps Cellules hôtes produisant des alcaloïdes de benzylisoquinoline génétiquement modifiées avec expression génique de transporteur d'efflux modifiée

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