WO2024015152A1 - Tetrahydropapaverine-producing microbes and methods of making and using the same - Google Patents

Tetrahydropapaverine-producing microbes and methods of making and using the same Download PDF

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WO2024015152A1
WO2024015152A1 PCT/US2023/023869 US2023023869W WO2024015152A1 WO 2024015152 A1 WO2024015152 A1 WO 2024015152A1 US 2023023869 W US2023023869 W US 2023023869W WO 2024015152 A1 WO2024015152 A1 WO 2024015152A1
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thp
plant cell
amino acid
cell
enzyme
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PCT/US2023/023869
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French (fr)
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Christina D. Smolke
Osman K. JAMIL
James T. PAYNE
Aaron CRAVENS
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The Board Of Trustees Of The Leland Stanford Junior University
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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • 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.)
    • C12N9/1003—Transferases (2.) transferring one-carbon groups (2.1)
    • C12N9/1007—Methyltransferases (general) (2.1.1.)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y201/00—Transferases transferring one-carbon groups (2.1)
    • C12Y201/01—Methyltransferases (2.1.1)
    • C12Y201/01117—(S)-Scoulerine 9-O-methyltransferase (2.1.1.117)

Definitions

  • Plant secondary metabolism is a rich source of structurally unique and functionally diverse bioactive compounds. These secondary metabolites, called plant natural products (PNPs), serve crucial functions in plant development, communication, and defense(5).
  • PNPs plant natural products
  • Many PNPs exhibit therapeutic activities and have been used as antibiotics, analgesics, antivirals, neurocognitive therapies, and chemotherapeutics(6).
  • Isolation of PNPs from plant biomass requires laborious extraction and purification procedures and the yields are subject to both seasonal and long-term instability due to weather and climate change.
  • the supply of plant-derived PNPs is less able to respond to sudden shocks in demand.
  • 1 -Benzylisoquinoline alkaloids (1 -Bl As) are a family of PNPs with diverse structures and significant medicinal value, including the analgesics morphine and codeine, the antimicrobials sanguinarine and berberine, and the cough suppressant noscapine(7). Many widely used medicinal compounds are still produced through extraction of the active ingredient, or its precursors, from commercially grown plant material(8).
  • (S)- Tetrahydropapaverine (THP) and papaverine are 1-BIAs with established clinical significance that are derived from the opium poppy (Papaver somniferum).
  • THP is a direct precursor to papaverine and a precursor in the production of the neuromuscular blocking agents atracurium and cisatracurium(l ).
  • Atracurium and cisatracurium are often administered during anesthesia to facilitate intubation(9). Due to increased incidence of patient intubation during the COVID-19 pandemic, atracurium and cisatracurium have experienced recent global supply shortages(I O). Papaverine is used directly in the clinic as a vasodilator and antispasmodic(11-13). In addition, recent studies have demonstrated potential clinical applications of papaverine due to its anti-cancer(14) and antiviral 5 activities. Despite the availability of several chemical syntheses of papaverine dating back to the early 1900s(16-18), shortages of papaverine in the 2010s forced many vascular surgeons to seek replacements for the drug due to the disruption in supply(2).
  • Saccharomyces cerevisiae (baker’s yeast) has served as a platform for enzyme characterization, pathway elucidation(23), and de novo metabolite production for various plant natural products(24).
  • S. cerevisiae can be more readily engineered and provides a cleaner secondary metabolite background compared to plants(25) and is able to functionally express eukaryotic cytochromes P450 with higher efficacy than prokaryotic microbes(3, 26).
  • S. cerevisiae has been used as a platform for the construction of several complex plant natural product pathways, including opioids(27), noscapinoids(23, 28), artemisinin(29), strictosidine(30), and tropane alkaloids(31 ).
  • Significant work on benzylisoquinoline alkaloids has already been accomplished in yeast, including the de novo production of key intermediates of THP and papaverine production(32).
  • Non-plant cells that produce tetrahydropapaverine (THP) via an engineered THP- biosynthetic pathway are provided.
  • the engineered THP- biosynthetic pathway is a norreticuline mediated pathway.
  • methods of producing THP using the cells as well as methods of producing papaverine, e.g., via oxidation of THP, as well as other produces from THP, e.g., atracurium and cisatracurium.
  • FIG. 1 De novo production of THP is achieved following the N- desmethylated route with enzymes from four kingdoms, overexpressed native yeast genes, and multiple engineered variants of plant enzymes.
  • Light gray arrows native yeast enzymes
  • dark gray arrows native yeast enzymes that have been modified to improve activity in the context of this pathway or that are overexpressed with the addition of multiple copies to the strain
  • purple arrows mammalian enzymes
  • light green arrows wildtype plant enzymes
  • dark green arrows plant enzymes that have been modified to improve their activity on a non-native substrate
  • orange enzymes bacterial enzymes.
  • Engineered enzymes modified from their wild-type sequence are noted with a superscript identifying the mutation if single mutation or with an abbreviation if multiple mutations. Moieties added by a single reaction in the pathway are highlighted in blue.
  • FIG. 2 Structure-guided semi-targeted mutagenesis helps identify NMCH variant with improved activity on the N-desmethylated substrate coclaurine.
  • FIG. 3 Multiple protein engineering strategies were applied to generate variants of TfS9OMT with an improved capacity to produce de novo THP.
  • THP THP.
  • CSY1174 was grown with a high-copy plasmid encoding expression of either the DS variant of T/S9OMT (blue) or with GFP as a negative control (gray), and the media was analyzed for the presence of THP. Traces shown show a single sample which was representative of tests in triplicate.
  • TfS9OMT DS Relative THP concentration produced by CSY1354 when expressing different TfS9OMT variants: TfS9OMT DS ; the best single mutant identified, TfS9OMT F296L ; the DNA shuffled variant, TfS9OMT SI "*; the final optimized mutant incorporating additional NNK library selections, TfS9OMT OPT .
  • Error bars represent the standard deviation of triplicate samples.
  • FIG. 4 Multi-drug resistance transporter knockouts affect the transport of pathway intermediates into the media and contribute to higher THP producing strains.
  • Both strains contain a low-copy plasmid expressing TfS9OMT OPT . Intracellular concentration is reported as relative to the extracellular concentration of that metabolite in the same sample.
  • FIG. 5 Chemical oxidation of biosynthesized THP enables semi-synthetic papaverine production.
  • Biosynthesized THP reaction used spent media from CSY1354 grown with TfS9OMT OPT expressed from a high-copy plasmid.
  • the reactions had a media-to-buffer ratio of 40:150 and had a final Tris-HCI concentration of 75 mM. Error bars represent the standard deviation of triplicate samples in all panels.
  • N-methylated and N-desmethylated routes of papaverine production Structures and names of each molecule in the proposed N- methylated (blue background) and N-desmethylated (green background) routes to papaverine production.
  • the nitrogen molecule, whose methylation or lack thereof distinguishes the two paths from one another is highlighted in blue.
  • Truncated enzyme names accompany the arrows between molecules and presently uncharacterized enzymes are highlighted in red.
  • Enzyme names coclaurine N-methyltransferase (CNMT), Coclaurine hydroxylase (CocH), N-methylcoclaurine hydroxylase (NCMH), 4’-O-methyltransferase (4’OMT), 7-O-methyltransferase (7OMT), 3'-O-methyltransferase (3’OMT), laudanosine demethylase (LdM), and dihydrobenzophenanthridine oxidase (DBOX).
  • CNMT coclaurine N-methyltransferase
  • CocH Coclaurine hydroxylase
  • NCMH N-methylcoclaurine hydroxylase
  • 4’OMT 4’-O-methyltransferase
  • 7OMT 7-O-methyltransferase
  • 3’OMT 3'-O-methyltransferase
  • LdM laudanosine demethylase
  • DBOX dihydrobenzophenanthridine oxidas
  • Supplementary Figure 5 Growth curves for CSY1174 and CSY1354 cultured in SD media. Both strains harbor a low-copy plasmid expressing TfS9OMT OPT . Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples. Supplementary Figure 6: Integration area of norcoclaurine measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a high-copy plasmid expressing T/S9OMT OPT . Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 7 Integration area of coclaurine measured in the spent media. Integration area is used as a proxy for concentration.
  • CSY1174 black diamonds
  • CSY1354 white squares harbor a low-copy plasmid expressing TfS9OMT OPT . Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 8 Integration area of 3’-hydroxycoclaurine measured in the spent media. Integration area is used as a proxy for concentration.
  • CSY1174 black diamonds
  • CSY1354 white squares
  • Measurements were taken at approximately six-hour intervals.
  • Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 9 Integration area of norreticuline measured in the spent media. Integration area is used as a proxy for concentration.
  • CSY1174 black diamonds
  • CSY1354 white squares harbor a low-copy plasmid expressing TfS9OMT OPT . Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 10 Integration area of norcodamine measured in the spent media. Integration area is used as a proxy for concentration.
  • CSY1174 black diamonds
  • CSY1354 white squares harbor a low-copy plasmid expressing TfS9OMT OPT . Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 11 Integration area of norlaudanine measured in the spent media. Integration area is used as a proxy for concentration.
  • CSY1174 black diamonds
  • CSY1354 white squares
  • Measurements were taken at approximately six-hour intervals.
  • Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 12 Integration area of THP measured in the spent media. Integration area is used as a proxy for concentration.
  • CSY1174 black diamonds
  • CSY1354 white squares harbor a low-copy plasmid expressing TfS9OMT OPT . Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 ⁇ L were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 14 THP titer produced by CSY1361 with different media bases and carbon sources. All carbon sources in the media were present at 2% except when indicated otherwise. Dex/Raf indicates 2% dextrose and 2% raffinose. 2x synthetic complete (2x SC) and 4x synthetic complete (4x SC) indicate twice and four times the standard concentration of amino acid supplements when preparing synthetic complete media. Error bars represent the standard deviation of triplicate samples.
  • THP titer produced by CSY1361 with different media bases and carbon sources All carbon sources were present at 2% unless otherwise indicated (including samples with multiple carbon sources.
  • Dex indicates dextrose
  • Tre indicates trehalose
  • Gly indicates glycerol
  • Sue indicates sucrose
  • Gal indicates galactose.
  • 2x synthetic complete (2x SC) and 4x synthetic complete (4x SC) indicate twice and four times the standard concentration of amino acid supplements when preparing synthetic complete media. Error bars represent the standard deviation of triplicate samples.
  • Supplementary Figure 17 THP titer produced by CSY1354 harboring a high- copy plasmid encoding an expression cassette for TfS9OMT OPT grown in different media bases and carbon sources.
  • 2x synthetic complete (2x SC) indicates twice the standard concentration of amino acid supplements were used when preparing synthetic complete media.
  • SC 2x carbon indicates synthetic complete prepared with 4% (w/v) of the indicated carbon source. 2% (w/v) of the carbon source was used in the media for all other samples. Error bars represent the standard deviation of triplicate samples.
  • THP normalized molar concentration of THP and papaverine in chemical oxidation reactions with 0.5% mM hydrogen peroxide at 85°C using biosynthesized THP.
  • the percent yield of papaverine is indicated above the marker corresponding with the highest production of papaverine.
  • the concentration of THP and papaverine are normalized with 100 being equal to the molar concentration of THP at the start of the reaction. These conditions resulted in the highest yield of papaverine using biosynthesized THP. Error bars represent the standard deviation of triplicate samples.
  • determining As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
  • polypeptide refers to a polymeric form of amino acids of any length, including peptides that range from 2-50 amino acids in length and polypeptides that are greater than 50 amino acids in length.
  • polypeptide and protein are used interchangeably herein.
  • polypeptide includes polymers of coded and noncoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones.
  • a polypeptide may be of any convenient length, e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids.
  • “Peptides” may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, peptides are between 5 and 30 amino acids in length.
  • isolated refers to an moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the moiety is associated with prior to purification.
  • the term “encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of 3 or more amino acids, such as 5 or more, 8 or more, 10 or more, 15 or more, or 20 or more amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed by the term are polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence.
  • a “vector” is capable of transferring gene sequences to target cells.
  • vector construct As used herein, the terms, “vector construct,” “expression vector,” and “gene transfer vector,” are used interchangeably to mean any nucleic acid construct capable of directing the expression of a gene of interest and which may transfer gene sequences to target cells, which is accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element.
  • the term includes cloning, and expression vehicles, as well as integrating vectors.
  • An “expression cassette” includes any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassette is constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into target ceils.
  • vector vector construct
  • expression vector vector
  • gene transfer vector gene transfer vector
  • a “plurality” contains at least 2 members.
  • a plurality may have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 106 or more, 107 or more, 108 or more, or 109 or more members.
  • a plurality can have 2-20 members.
  • each step may be performed after a predetermined amount of time has elapsed between steps, as desired.
  • the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and including 5 hours or more.
  • each subsequent step is performed immediately after completion of the previous step.
  • a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.
  • Non-plant cells that produce tetrahydropapaverine (THP) via an engineered THP- biosynthetic pathway are provided.
  • the engineered THP- biosynthetic pathway is a norreticuline mediated pathway.
  • methods of producing THP using the cells as well as methods of producing papaverine, e.g., via oxidation of THP, as well as other produces from THP, e.g., atracurium and cisatracurium.
  • Tetrahydropapaverine (THP), along with and papaverine, are plant natural products with clinically significant roles.
  • THP is a precursor in the production of the drugs atracurium and cisatracurium.
  • Papaverine is used as an anti-spasmodic during vascular surgery.
  • metabolic engineering advances have enabled the production of other natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products.
  • THP and papaverine have not been achieved to date, in part, because multiple pathway enzymes have not been elucidated.
  • the production of THP is achieved through the heterologous expression of two novel enzyme variants with activity on non-native substrates.
  • the first of the two novel enzyme variants is a protein engineered variant of M-methylcoclaurine hydroxylase with activity on coclaurine enabling de novo norreticuline biosynthesis.
  • the second of the two novel enzyme variants is a protein engineered variant of scoulerine 9-O-methyltransferase capable of O-methylating 1 - benzylisoquinoline alkaloids at the 3’ position enabling de novo THP biosynthesis.
  • strain engineering aspects were also developed so as to increase flux through the heterologous pathway for the production of THP.
  • flux through the heterologous pathway was improved by knocking out yeast multi-drug resistance transporters and optimization of media conditions.
  • strain engineering increased the concentration of biosynthesized THP 600-fold to 121 pg/L.
  • embodiments provide for production of papaverine.
  • some embodiments of provide for papaverine semi-synthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce de novo semi-synthesized papaverine through the oxidation of biosynthesized THP.
  • THP tetrahydropapaverine
  • methods for semi-synthesis of papaverine using microbially biosynthesized THP demonstrates the ability to use protein homologs and protein engineering to replace the activity of unknown enzymes in heterologous biosynthetic pathways.
  • methods are provided herein for improving THP pathway flux by knocking out two yeast multi-drug resistance (MDR) transporters, which reduces the export of pathway intermediates. MDR knockouts may be applied to increase flux through other heterologous pathways.
  • MDR yeast multi-drug resistance
  • the strain engineering as disclosed herein provides methods for fermentation-based production of clinically significant molecules THP and papaverine, which have experienced recent supply chain shortages.
  • THP tetrahydropapaverine
  • THP-producing cell is meant to include cells that are engineered to produce THP from a starting compound via an engineered synthetic pathway.
  • the host cells are non-plant cells. In some instances, the host cells may be characterized as microbial cells. In certain cases, the host cells are insect cells, mammalian cells, bacterial cells, fungal cells or yeast cells. Any convenient type of host cell may be utilized in producing the subject cells producing the subject THP-producing cells, see, e.g., US2008/0176754; WO/2012/039438; WO2013136057; US2017/0253898;
  • Host cells of interest include, but are not limited to, bacterial cells, such as Bacillus subtilis, Escherichia coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium
  • the host cells are yeast cells or E. coli cells. In some cases, the host cell is a yeast cell. Any of the host cells described in e.g., US2008/0176754; WO/2012/039438; WO2013136057; US2017/0253898; US2018/0163241 and WO 2020/185626, as well as U.S. Patent Nos. 9,534,241 ; 11 ,124,814 and 10,752,903; may be adapted for use in the subject cells and methods. In certain embodiments, the yeast cells may be of the species Saccharomyces cerevisiae (S. cerevisiae).
  • the yeast cells may be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cells may be of the species Pichia pastoris. Yeast is of interest as a host cell because cytochrome P450 proteins are able to fold properly into the endoplasmic reticulum membrane so that their activity is maintained. In examples, cytochrome P450 proteins are involved in some biosynthetic pathways of interest.
  • Yeast strains of interest that find use in the invention include, but are not limited to, CEN.PK (Genotype: MATa/a ura3-52/ura3-52 trp 1-289/trp 1-289 leu2-3_ 112/leu2-3_ 112 his3 A 1/his3 A 1 MAL2-8C/MAL2- 8C SUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, £1278B, AB972, SK1 , and FL100.
  • the yeast strain is any of S288C (MATa; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1 ), BY4741 (MATa; his3A1 ; leu2A0; met15A0; ura3A0), BY4742 (MATa; his3A1 ; leu2A0; lys2A0; ura3A0), BY4743 (MATa/MATa; his3A1/his3A1 ; leu2A0/leu2A0; met15A0/MET15; LYS2/lys2A0; ura3A0/ura3A0), and WAT11 or ⁇ N(R), derivatives of the W303-B strain (MATa; ade2-1 ; his3-11 , -15; leu2-3,-112; ura3-1 ; canR; cyr+) which express the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeast NAD
  • the yeast cell is W303alpha (MATa; his3-11 ,15 trp1-1 leu2-3 ura3-1 ade2-1 ).
  • the identity and genotype of additional yeast strains of interest may be found at EUROSCARF (web.uni- frankfurt.de/fb15/mikro/euroscarf/col_index.html).
  • the host cell is a fungal cell.
  • the fungal cells may be of the Aspergillus species and strains include Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624, ATCC 20542) and Aspergillus nidulans (FGSC A4).
  • the host cells may be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of THP.
  • a modification is a genetic modification, such as a mutation, addition, or deletion of a gene or fragment thereof, or transcription regulation of a gene or fragment thereof.
  • the term "mutation" refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence or motif. The mutation may be incorporated as a directed mutation to the native gene at the original locus.
  • the mutation may be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy on an episomal vector such as a 2p or centromeric plasmid.
  • the substrate inhibited copy of the enzyme is under the native cell transcriptional regulation.
  • the substrate inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.
  • the object of one or more modifications may be a native gene.
  • the object of one or more modifications may be a non-native gene.
  • a non-native gene may be inserted into a host cell.
  • a non-native gene may be altered by one or more modifications prior to being inserted into a host cell.
  • An engineered host cell may overproduce one or more products.
  • overproduce is meant that the cell has an improved or increased production of a product relative to a control cell (e.g., an unmodified cell).
  • improved or increased production is meant both the production of some amount of the THP product where the control has no THP product production, as well as an increase of about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, such as 2-fold or more, such as 5-fold or more, including 10-fold or more in situations where the control has some THP product production.
  • THP-producing cells of embodiments of the invention include an engineered THP-biosynthetic pathway.
  • engineered THP-biosynthetic pathway is meant a metabolic pathway that produces THP as a product and includes one or more enzymatic activities exogenous to the host cell, i.e., one or more heterologous coding sequences.
  • the one or more exogeneous enzymatic activities may be naturally occurring, e.g., wild type, or synthetic, e.g., mutant.
  • the THP-biosynthetic pathway may vary, where examples of THP-biosynthetic pathways that may be present cells of the invention include those that synthesize THP via the NH route involving norreticuline and the NCH 3 route involving reticuline.
  • the engineered THP-biosynthetic pathway is a norreticuline mediated pathway (i.e., a pathway that synthesizes THP via the NH route).
  • the host cell includes one or more heterologous coding sequences encoding at least one enzyme involved in the norreticuline mediated pathway, such as two or more heterologous coding sequences encoding two or more enzymes involved in the norreticuline mediated pathway.
  • the non-plant cell includes one or more heterologous coding sequences encoding at least one enzyme involved in conversion of coclaurine to THP in the norreticuline mediated pathway, such as the conversion of coclaurine to 3'-OH Coclaurine.
  • the pathway includes a heterologous coding sequence for a N-methylcoclaurine hydroxylase (NMCH).
  • NMCH N-methylcoclaurine hydroxylase
  • the heterologous coding sequence for NMCH may be a wild-type or mutant NMCH coding sequence from any convenient source, e.g., Eschscholzia californica, Papaver somniferum, Papaver bracteatum, Coptis japonica, and the like.
  • the NMCH coding sequence encodes a mutant NMCH that is capable of accepting the non-native substrate (S)-coclaurine to produce (S)-3’-hydroxy-coclaurine at a higher concentration as compared to the wild-type enzyme, such as 5-fold or higher concentration, 10-fold or higher concentration, 25-fold or higher concentration and in some instances 40-fold or higher concentration.
  • Specific mutant or variant NMCH enzymes that may be employed in embodiments of the invention include, but are not limited to: EcNMCH L203S , PsNMCH L203S , PbNMCH L203S , C/NMCH 12035 , and the like.
  • the THP-biosynthetic pathway includes at least one enzyme having an activity that is capable of O-methylating norreticuline at the 7' and 3' positions to produce THP, such as first and second O-methylating enzymes capable of O-methylating norreticuline at the 7' and 3' positions, respectively, to produce THP.
  • the first O-methylating enzyme is capable of O-methylating norreticuline at the 3' position, such as an O-methyl transferase (OMT).
  • OMT O-methyl transferase
  • Any convenient OMT may be employed, where examples of suitable OMTs include, but are not limited to scoulerine 9-0- methyltransferases and the like.
  • the pathway includes a heterologous coding sequence for a scoulerine 9-0-methyltransferases (S90MT).
  • S90MT scoulerine 9-0-methyltransferases
  • the heterologous coding sequence for S90MT may be a wild-type or mutant S90MT coding sequence from any convenient source, e.g., Thalictrum flavum, Papaver somniferum, Papaver bracteatum, Coptis japonica, Coptis chinensis, and the like.
  • the S90MT coding sequence encodes a mutant S90MT that is capable of accepting the non-native substrates, e.g., norreticuline to produce norcodamine and/or norlaudanine to produce THP, to ultimately produce THP at a higher concentration as compared to a suitable control, such as 2-fold or higher concentration, e.g., 3-fold or higher concentration.
  • the non-native substrates e.g., norreticuline to produce norcodamine and/or norlaudanine to produce THP
  • Specific mutant or variant S90MT enzymes that may be employed in embodiments of the invention include, but are not limited to: TfS9OMT OPT , TfS9OMT F296L , T/S9OMT T83A C98P A " 9L 77ggQMT T8 3A C98P.A I I9LV28 I y- ⁇ ggQ
  • the THP biosynthetic pathway may include a second O- methylating enzyme, where the second O-methylating enzyme is capable of O-methylating norreticuline at the 7' position.
  • the second O-methylating enzyme is a norreticuline 7-O-methyltransferase (N7OMT).
  • the pathway includes a heterologous coding sequence for a N7OMT).
  • the heterologous coding sequence for N70MT may be a wild-type or mutant N7OMT coding sequence from any convenient source, e.g., Glaucium flavum, Papaver somniferum, Papaver armeniacum, Eschscholzia californica, and the like.
  • one or more (such as two or more, three or more, or four or more) additional modifications may be present, where such modifications may be selected from: a substrate inhibition alleviating mutation in a biosynthetic enzyme gene; a product inhibition alleviating mutation in a biosynthetic enzyme gene; a cofactor recovery promoting mechanism; a feedback inhibition alleviating mutation in a biosynthetic enzyme gene; and a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme gene. Further details regarding such modifications may be found in Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
  • the engineered host cells are cells that include one or more substrate inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell.
  • the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell).
  • the one or more biosynthetic enzyme genes are non-native to the cell.
  • substrate inhibition alleviating mutation refers to a mutation that alleviates a substrate inhibition control mechanism of the cell.
  • a mutation that alleviates substrate inhibition reduces the inhibition of a regulated enzyme in the cell of interest relative to a control cell and provides for an increased level of the regulated compound or a downstream biosynthetic product thereof.
  • alleviating inhibition of the regulated enzyme is meant that the IC 5 o of inhibition is increased by 2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or even more.
  • increased level is meant a level that is 110% or more of that of the regulated compound in a control cell or a downstream product thereof, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more or even more of the regulated compound in the engineered host cell or a downstream product thereof.
  • the engineered host cell may include one or more substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes.
  • the one or more mutations may be located in any convenient biosynthetic enzyme genes where the biosynthetic enzyme is subject to regulatory control.
  • the engineered host cell may include one or more substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
  • the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more substrate inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell.
  • the engineered host cells are cells that include one or more cofactor recovery promoting mechanisms (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell.
  • the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell).
  • the one or more biosynthetic enzyme genes are non-native to the cell.
  • cofactor recovery promoting mechanism refers to a mechanism that promotes a cofactor recovery control mechanism of the cell.
  • the one or more cofactors of interest for recovery include but are not limited to S-adenosyl methionine, nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide, tetrahydrobiopterin, and flavin adenine dinucleotide.
  • the engineered host cell may include one or more cofactor recovery promoting mechanism in one or more biosynthetic enzyme genes.
  • the engineered host cell may include one or more cofactor recovery promoting mechanisms in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
  • the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more cofactor recovery promoting mechanisms such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cofactor recovery promoting mechanisms in one or more biosynthetic enzyme genes within the engineered host cell.
  • the engineered host cells are cells that include one or more product inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell.
  • the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell).
  • the one or more biosynthetic enzyme genes are non-native to the cell.
  • product inhibition alleviating mutation refers to a mutation that alleviates a short term and/or long term product inhibition control mechanism of an engineered host cell. Short term product inhibition is a control mechanism of the cell in which there is competitive binding at a cosubstrate binding site. Long term product inhibition is a control mechanism of the cell in which there is irreversible binding of a compound away from a desired pathway.
  • a mutation that alleviates product inhibition reduces the inhibition of a regulated enzyme in the cell of interest relative to a control cell and provides for an increased level of the regulated compound or a downstream biosynthetic product thereof.
  • alleviating inhibition of the regulated enzyme is meant that the IC 5 o of inhibition is increased by 2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or even more.
  • increased level is meant a level that is 110% or more of that of the regulated compound in a control cell or a downstream product thereof, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more or even more of the regulated compound in the engineered host cell or a downstream product thereof.
  • the engineered host cell may include one or more product inhibition alleviating mutations in one or more biosynthetic enzyme genes.
  • the mutation may be located in any convenient biosynthetic enzyme genes where the biosynthetic enzyme is subject to regulatory control.
  • the engineered host cell includes one or more product inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
  • the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more product inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 product inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell.
  • the engineered host cells are cells that include one or more feedback inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell.
  • the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell). Additionally or alternatively, in some examples the one or more biosynthetic enzyme genes are non-native to the cell.
  • the term “feedback inhibition alleviating mutation” refers to a mutation that alleviates a feedback inhibition control mechanism of an engineered host cell.
  • Feedback inhibition is a control mechanism of the cell in which an enzyme in the synthetic pathway of a regulated compound is inhibited when that compound has accumulated to a certain level, thereby balancing the amount of the compound in the cell.
  • a mutation that alleviates feedback inhibition reduces the inhibition of a regulated enzyme in the engineered host cell relative to a control cell. In this way, the engineered host cell provides for an increased level of the regulated compound or a downstream biosynthetic product thereof.
  • the IC 5 o of inhibition is increased by 2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300- fold or more, 1000-fold or more, or even more.
  • increased level is meant a level that is 110% or more of that of the regulated compound in a control cell or a downstream product thereof, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, such as at least 3- fold or more, at least 5-fold or more, at least 10-fold or more or even more of the regulated compound in the host cell or a downstream product thereof.
  • the host cell may include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell.
  • the one or more mutations may be located in any convenient biosynthetic enzyme genes where the biosynthetic enzyme is subject to regulatory control.
  • the engineered host cell may include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
  • any convenient numbers and types of mutations may be utilized to alleviate a feedback inhibition control mechanism.
  • the term "mutation" refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence or motif.
  • the mutation may be incorporated as a directed mutation to the native gene at the original locus.
  • the mutation may be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy on an episomal vector such as a 2g or centromeric plasmid.
  • the feedback inhibited copy of the enzyme is under the native cell transcriptional regulation.
  • the feedback inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.
  • the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell.
  • the host cells may include one or more transcriptional modulation modifications (such as two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell.
  • the one or more biosynthetic enzyme genes are native to the cell.
  • the one or more biosynthetic enzyme genes are non-native to the cell. Any convenient biosynthetic enzyme genes of the cell may be targeted for transcription modulation.
  • transcription modulation is meant that the expression of a gene of interest in a modified cell is modulated, e.g., increased or decreased, enhanced or repressed, relative to a control cell (e.g., an unmodified cell).
  • transcriptional modulation of the gene of interest includes increasing or enhancing expression.
  • increasing or enhancing expression is meant that the expression level of the gene of interest is increased by 2-fold or more, such as by 5-fold or more and sometimes by 25-, 50-, or 100-fold or more and in certain embodiments 300- fold or more or higher, as compared to a control, i.e., expression in the same cell not modified (e.g., by using any convenient gene expression assay).
  • the expression level of the gene of interest is considered to be increased if expression is increased to a level that is easily detectable.
  • transcriptional modulation of the gene of interest includes decreasing or repressing expression.
  • decreasing or repressing expression is meant that the expression level of the gene of interest is decreased by 2-fold or more, such as by 5-fold or more and sometimes by 25-, 50-, or 100- fold or more and in certain embodiments 300-fold or more or higher, as compared to a control. In some cases, expression is decreased to a level that is undetectable.
  • the transcriptional modulation modification may include a substitution of a strong promoter for a native promoter of the one or more biosynthetic enzyme genes or the expression of an additional copy(ies) of the gene or genes under the control of a strong promoter.
  • the promoters driving expression of the genes of interest may be constitutive promoters or inducible promoters, provided that the promoters may be active in the host cells.
  • the genes of interest may be expressed from their native promoters. Additionally or alternatively, the genes of interest may be expressed from non-native promoters. Although not a requirement, such promoters may be medium to high strength in the host in which they are used. Promoters may be regulated or constitutive.
  • promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, may be used.
  • suitable promoters examples of which include promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding fructose biphosphate aldolase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase) (Bitter G. A., Meth. Enzymol. 152:673 684 (1987)).
  • promoters of interest include, but are not limited to, the ADHI promoter of baker's yeast (Ruohonen L., et al, J. Biotechnol. 39:193 203 (1995)), the phosphate-starvation induced promoters such as the PHO5 promoter of yeast (Hinnen, A., et al, in Yeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989), the alkaline phosphatase promoter from B. licheniformis (Lee. J. W. K., et al., J. Gen. Microbiol. 137:1127 1133 (1991 )), GPD1 , and TEF1 .
  • ADHI promoter of baker's yeast Ruohonen L., et al, J. Biotechnol. 39:193 203 (1995)
  • the phosphate-starvation induced promoters such as the PHO5 promoter of
  • Yeast promoters of interest include, but are not limited to, inducible promoters such as Gall -10, Gall , GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3- phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation f actor- 1 -alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1 ), MRP7 promoter, etc.
  • GPD1 glyceraldehyde 3- phosphate dehydrogenase promoter
  • ADH alcohol dehydrogenase promoter
  • TEF cytochrome c-oxidase promoter
  • MRP7 promoter MRP7 promoter
  • Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE), see e.g., those promoters described in U.S. Pat. No. 7,045,290.
  • Vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • any promoter/enhancer combination as per the Eukaryotic Promoter Data Base EPDB
  • any convenient promoters specific to the host cell may be selected, e.g., E. coli. In some cases, promoter selection may be used to optimize transcription, and hence, enzyme levels to maximize production while minimizing energy resources.
  • the engineered host cells may include one or more inactivating mutations to an enzyme of the cell (such as two or more, three or more, four or more, five or more, or even more).
  • the inclusion of one or more inactivating mutations may modify the flux of a synthetic pathway of an engineered host cell to increase the levels of a BIA of interest or a desirable enzyme or precursor leading to the same.
  • the one or more inactivating mutations are to an enzyme native to the cell. Additionally or alternatively, the one or more inactivating mutations are to an enzyme non-native to the cell.
  • inactivating mutation is meant one or more mutations to a gene or regulatory DNA sequence of the cell, where the mutation(s) inactivates a biological activity of the protein expressed by that gene of interest.
  • the gene is native to the cell.
  • the gene encodes an enzyme that is inactivated and is part of or connected to the synthetic pathway of a BIA of interest produced by the host cell.
  • an inactivating mutation is located in a regulatory DNA sequence that controls a gene of interest.
  • the inactivating mutation is to a promoter of a gene. Any convenient mutations (e.g., as described herein) may be utilized to inactivate a gene or regulatory DNA sequence of interest.
  • inactivated or “inactivates” is meant that a biological activity of the protein expressed by the mutated gene is reduced by 10% or more, such as by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, relative to a control protein expressed by a non-mutated control gene.
  • the protein is an enzyme and the inactivating mutation reduces the activity of the enzyme.
  • the engineered host cell includes an inactivating mutation in an enzyme native to the cell. Any convenient enzymes may be targeted for inactivation. Enzymes of interest may include, but are not limited to those enzymes, described in Table 1 of Published United States Patent Application Publication No. 2017/0253898 (the disclosure of which is herein incorporated by reference), whose action in the synthetic pathway of the engineered host cell tends to reduce the levels of THP products.
  • the engineered host cells harbor one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more) which encode activity(ies) that enable the engineered host cells to produce THP, e.g., as described herein.
  • heterologous coding sequence is used to indicate any polynucleotide that codes for, or ultimately codes for, a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism and may be expressed in the host cell under proper conditions.
  • heterologous coding sequences includes multiple copies of coding sequences that are normally present in the host cell, such that the cell is expressing additional copies of a coding sequence that are not normally present in the cells.
  • the heterologous coding sequences may be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or a hybrid of RNA/DNA. Coding sequences of interest include, but are not limited to, full-length transcription units that include such features as the coding sequence, introns, promoter regions, 3'-UTRs, and enhancer regions.
  • the engineered host cells may also be modified to possess one or more genetic alterations to accommodate the heterologous coding sequences.
  • Alterations of the native host genome include, but are not limited to, modifying the genome to reduce or ablate expression of a specific protein that may interfere with the desired pathway. The presence of such native proteins may rapidly convert one of the intermediates or final products of the pathway into a metabolite or other compound that is not usable in the desired pathway. Thus, if the activity of the native enzyme were reduced or altogether absent, the produced intermediates would be more readily available for incorporation into the desired product.
  • ablation of expression of a protein may be of interest, as in proteins involved in the pleiotropic drug response, including, but not limited to, ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, e.g., PDR5, SNG2, and associated transcription factors.
  • ABSC ATP-binding cassette
  • MDR multidrug resistance
  • the host cells may be modified to include a variety of plant proteins that provide for a desirable activity or property. Any convenient plant proteins related to the synthesis of THP or precursor thereof may be utilized in the engineered host cells, such as enzymes, chaperones, co-factors, and the like.
  • the host cell includes a plant chaperone protein.
  • the plant chaperone may facilitate the action of an enzyme of interest in the host cell, thereby providing for an improved production of THP or precursor thereof.
  • Plant chaperones of interest include, but are not limited to, binding immunoglobulin protein (BiP), DnaJ protein, glucose regulated protein (GRP) 94, binding protein (BiP), protein disulphide isomerase (PDI), cyclophilin, and calnexin.
  • Heterologous coding sequences include but are not limited to sequences that encode enzymes, either wild-type or equivalent sequences, that are normally responsible for the production of noscapinoid products in plants.
  • the enzymes for which the heterologous sequences code may be any of the enzymes in the 1 -BIA pathway, and may be from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequences for the particular synthetic pathway may be selected based upon the desired product.
  • the host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 heterologous coding sequences.
  • heterologous coding sequences also includes the coding portion of the peptide or enzyme, i.e., the cDNA or mRNA sequence, of the peptide or enzyme, as well as the coding portion of the full-length transcriptional unit, i.e., the gene including introns and exons, as well as "codon optimized” sequences, truncated sequences or other forms of altered sequences that code for the enzyme or code for its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein.
  • Such equivalent amino acid sequences may have a deletion of one or more amino acids, with the deletion being N-terminal, C-terminal, or internal. Truncated forms are envisioned as long as they have the catalytic capability indicated herein. Fusions of two or more enzymes are also envisioned to facilitate the transfer of metabolites in the pathway, provided that catalytic activities are maintained.
  • Operable fragments, mutants, or truncated forms may be identified by modeling and/or screening. In some cases, this is achieved by deletion of, for example, N-terminal, C- terminal, or internal regions of the protein in a step-wise fashion, followed by analysis of the resulting derivative with regard to its activity for the desired reaction compared to the original sequence. If the derivative in question operates in this capacity, it is considered to constitute an equivalent derivative of the enzyme proper.
  • Any convenient enzyme of interest may be mutated or engineered to provide for a desirable biological activity in the engineered host cell. In some cases, the mutant enzyme is engineered to facilitate the correct folding of the enzyme. In certain instances, the mutant enzyme is engineered to increase a desirable activity or property of the enzyme relative to a non-mutated enzyme.
  • the mutant enzyme is engineered to decrease an undesirable activity or property of the enzyme relative to a non-mutated enzyme.
  • the cell includes one or more heterologous coding sequences that encode one or more mutant enzymes. Aspects of the invention also relate to heterologous coding sequences that code for amino acid sequences that are equivalent to the native amino acid sequences for the various enzymes.
  • An amino acid sequence that is "equivalent" is defined as an amino acid sequence that is not identical to the specific amino acid sequence, but rather contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein as compared to a similar activity of the specific amino acid sequence, when used for a desired purpose.
  • Equivalent sequences are also meant to include those which have been engineered and/or evolved to have properties different from the original amino acid sequence. Mutable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc.
  • an "equivalent" amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98%, and 99% identity, at the amino acid level.
  • the amino acid sequence may be identical but the DNA sequence is altered such as to optimize codon usage for the host organism, for example.
  • each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and/or production of THP.
  • additional gene copies i.e., multiple copies
  • Embodiments of the invention include increased production of THP in a host cell through simultaneous expression of multiple species variants of a single or multiple enzymes.
  • additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient methods may be utilized including multiple copies of a heterologous coding sequence for an enzyme in the host cell.
  • the engineered host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies.
  • the engineered host cell includes multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc.
  • the multiple copies of the heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell.
  • the engineered host cell may include multiple copies of one heterologous coding sequence, where each of the copies is derived from a different source organism. As such, each copy may include some variations in explicit sequences based on inter-species differences of the enzyme of interest that is encoded by the heterologous coding sequence.
  • heterologous coding sequences are as reported in GENBANK.
  • a list of enzymes of interest is disclosed herein as well as in Table 1 of Published United States Patent Application Publication No. US20170253898; the disclosure of which is herein incorporate reference.
  • the host cells of the invention may include any combination of the listed enzymes, from any source.
  • Accession numbers disclosed herein refer to GenBank. Some accession numbers refer to the Saccharomyces genome database (SGD), which is available on the world-wide web at www.yeastgenome.org.
  • the cell is a norreticuline producing cell.
  • the cell is a modified version of cells described in Published United States Patent Application Publication No. US20170253898; the disclosure of which is herein incorporate reference.
  • the cells may be modified to produce norreticuline instead of reticuline, e.g., by removing the PsCNMT activity.
  • the cell includes a coding sequence for producing a 6-pyruvoyl tetrahydrobiopterin synthase (PTPS), such as Rattus norvegicus PTPS (RnPTPS).
  • PTPS 6-pyruvoyl tetrahydrobiopterin synthase
  • RnPTPS Rattus norvegicus PTPS
  • the cell includes a coding sequence for producing a sepiapterin reductase (SepR), such as RnSepR.
  • the cell includes a coding sequence for producing a pterin carbinolamine dehydratase (PCD), such as F?nPCD.
  • the cell includes a coding sequence for producing a quinonoid dihydropteridine reductase (QDHPR), such as RnQDHPR.
  • the cell includes a coding sequence for producing a dihydrofolate reductase (DHFR), such as RnDHFR.
  • DHFR dihydrofolate reductase
  • the cell includes a coding sequence for producing a dihydrofolate reductase a tyrsosine hydroxylase (TyrH), such as RnTyrH, including a mutant thereof, e.g., F?nTyrH WR .
  • the cell includes a coding sequence for producing a norcoclaurine synthase (NOS), such as Coptis japonica (CyNCS).
  • the cell includes a coding sequence for producing a DOPA decarboxylase (DODC), such as form Pseudomonas putida (PpDODC).
  • the host cell includes a heterologous coding sequence for a CPR enzyme.
  • the CPR enzyme is an Arabidopsis thaliana P450 Reductase (ATR), e.g., ATR1 , or CPR enzyme from Papaver somniferum (PsCPR).
  • ATR Arabidopsis thaliana P450 Reductase
  • the cell includes a coding sequence for producing a norcoclaurine 6-0-methyltransferase (6OMT), such as ps6OMT.
  • the cell includes a coding sequence for producing a 4'-0-methyltransferase (4'OMT), such as ps4'OMT.
  • the cell includes a coding sequence for producing a coclaurine-N-methyltransferase (CNMT), such as PsCNMT.
  • the cell includes a coding sequence for producing a 3-deoxy-D-arabino-2-heptulosonic acid 7- phosphate synthase (ARO4), such as Aro4p Q166K .
  • the cell includes a coding sequence for producing a chorismate mutase (ARO7), such as Aro7p T2261 .
  • the cell includes a coding sequence for producing a phenylpyruvate decarboxylase (ARO10).
  • the cell includes a coding sequence for producing a transketolase (TKL1 ).
  • the cell may produce THP using one or more enzymes that provide for derivatization in the cell.
  • the cell includes one or more heterologous coding sequences for one or more enzymes selected from a P450, a halogenase, a glycosylase, a methyltransferase, an acetyltransferase, a short-chain dehydrogenase, a carboxylesterase, and a prenyltransferase.
  • the host cell e.g., a yeast strain
  • the host cell is engineered for selective production of THP, or a precursor thereof, by localizing one or more enzymes to a compartment in the cell. Any convenient compartments or structures of a cell may be targeted for localization of an enzyme of interest.
  • the cell includes an enzyme that is spatially localized to a compartment in the yeast cell, wherein the compartment is selected from mitochondrion, endoplasmic reticulum (ER), golgi, vacuole, nucleus, plasma membrane, peroxisome, and periplasm.
  • an enzyme is localized to the yeast endoplasmic reticulum by fusing an ER targeting sequence to the N- terminus of the protein.
  • an enzyme of interest is spatially localized to the outside of the compartment in the yeast cell.
  • an enzyme of interest is spatially localized to the inside of the compartment in the yeast cell.
  • an enzyme may be located in the host cell such that the compound produced by this enzyme spontaneously rearranges, or is converted by another enzyme to a desirable metabolite before reaching a localized enzyme that may convert the compound into an undesirable metabolite.
  • the spatial distance between two enzymes may be selected to prevent one of the enzymes from acting directly on a compound to make an undesirable metabolite, and restrict production of undesirable end products (e.g., an undesirable opioid by-product).
  • any of the enzymes described herein, either singularly or together with a second enzyme may be localized to any convenient compartment in the host cell, including but not limited to, an organelle, endoplasmic reticulum, golgi, vacuole, nucleus, plasma membrane, or the periplasm.
  • the host cell includes one or more of the enzymes that include a localization tag.
  • Any convenient localization tags may be utilized.
  • the localization tag is a peptidic sequence that is attached at the N-terminal and or C- terminal of the enzyme. Any convenient methods may be utilized for attaching a tag to the enzyme.
  • the localization tag is derived from an endogenous yeast protein. Such tags may provide routing to a variety of yeast organelles, including but not limited to, the endoplasmic reticulum (ER), mitochondria (MT), plasma membrane (PM), and vacuole (V).
  • the tag includes or is derived from, a transmembrane domain from within the tail-anchored class of proteins.
  • the localization tag locates the enzyme on the outside of an organelle. In certain embodiments, the localization tag locates the enzyme on the inside of an organelle. In some instances, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and ultimately THP production. Embodiments of the invention include increased THP production in a host cell through simultaneous expression of multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient methods may be utilized including multiple copies of a heterologous coding sequence for an enzyme in the host cell.
  • the host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies. In certain embodiments, the host cell include multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc.
  • the multiple copies of the heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell.
  • the host cell may include multiple copies of one heterologous coding sequence, where each of the copies is derived from a different source organism.
  • the copies are derived from P. somniferum and E. californica source organisms.
  • each copy may include some variations in explicit sequences based on inter-species differences of the enzyme of interest that is encoded by the heterologous coding sequence.
  • the host cell includes multiple heterologous coding sequences that each encode an enzyme and are each derived from a different source organisms as compared to the host cell.
  • the host cell includes copies of an enzyme derived from two or more different source organisms as compared to the host cell.
  • the engineered host cell medium may be sampled and monitored for the production of THP.
  • the THP may be observed and measured using any convenient methods.
  • Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein) where a sample of interest is analyzed by comparison with a known amount of a standard compound. Additionally, there are other ways that THP may be observed and/or measured. Examples of alternative ways of observing and/or measuring BIAs include GC- MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, capillary electrophoresis, among others.
  • Identity may be confirmed, e.g., by m/z and MS/MS fragmentation patterns, and quantitation or measurement of the compound may be achieved via LG trace peaks of know retention time and/or EIC MS peak analysis by reference to corresponding LC-MS analysis of a known amount of a standard of the compound.
  • Tables 1 -9 provide examples of sequences that may be used within embodiments as disclosed in the present application.
  • Table 10 discloses examples of amino acid residues in S9OMT where mutagenesis improves activity of this enzyme.
  • Table 1 Examples of Sequences for Upstream Enzymes
  • aspects of the invention include methods of preparing THP and products therefrom.
  • aspects of the invention include culturing an engineered host cell under conditions in which the one or more host cell modifications (e.g., as described herein) are functionally expressed such that the cell converts starting compounds of interest into THP.
  • methods that include culturing an engineered host cell under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and convert starting compounds of interest into THP.
  • the method is a method of preparing THP that includes culturing an engineered host cell (e.g., as described herein); adding a starting compound to the cell culture; and recovering the THP from the cell culture.
  • Fermentation media may contain suitable carbon substrates.
  • the source of carbon suitable to perform the methods of this disclosure may encompass a wide variety of carbon containing substrates.
  • Suitable substrates may include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof.
  • unpurified mixtures from renewable feedstocks may be used (e.g., cornsteep liquor, sugar beet molasses, barley malt).
  • the carbon substrate may be a one-carbon substrate (e.g., methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol).
  • other carbon containing compounds may be utilized, for example, methylamine, glucosamine, and amino acids.
  • Any convenient methods of culturing engineered host cells may be employed for producing the THP. The particular protocol that is employed may vary, e.g., depending on the engineered host cell, the heterologous coding sequences, the enzymes of interest, etc.
  • the cells may be present in any convenient environment, such as an environment in which the cells are capable of expressing one or more functional heterologous enzymes.
  • the cells are cultured under conditions that are conducive to enzyme expression and with appropriate substrates available to allow production of THP in vivo.
  • the functional enzymes are extracted from the engineered host for production of THP under in vitro conditions.
  • the engineered host cells are placed back into a multicellular host organism.
  • the engineered host cells are in any phase of growth, including, but not limited to, stationary phase and log-growth phase, etc.
  • the cultures themselves may be continuous cultures or they may be batch cultures.
  • Cells may be grown in an appropriate fermentation medium at a temperature between 14-40 e C. Cells may be grown with shaking at any convenient speed (e.g., 200 rpm). Cells may be grown at a suitable pH. Suitable pH ranges for the fermentation may be between pH 5-9. Fermentations may be performed under aerobic, anaerobic, or microaerobic conditions. Any suitable growth medium may be used. Suitable growth media may include, without limitation, common commercially prepared media such as synthetic defined (SD) minimal media or yeast extract peptone dextrose (YEPD) rich media. Any other rich, defined, or synthetic growth media appropriate to the microorganism may be used.
  • SD synthetic defined
  • YEPD yeast extract peptone dextrose
  • Cells may be cultured in a vessel of essentially any size and shape.
  • vessels suitable to perform the methods of this disclosure may include, without limitation, multi-well shake plates, test tubes, flasks (baffled and non-baffled), and bioreactors.
  • the volume of the culture may range from 10 microliters to greater than 10,000 liters.
  • agents to the growth media that are known to modulate metabolism in a manner desirable for the production of alkaloids may be included.
  • cyclic adenosine 2’3’-monophosphate may be added to the growth media to modulate catabolite repression.
  • the host cells that include one or more modifications are cultured under standard or readily optimized conditions, with standard cell culture media and supplements.
  • standard growth media when selective pressure for plasmid maintenance is not required may contain 20 g/L yeast extract, 10 g/L peptone, and 20 g/L dextrose (YPD).
  • Host cells containing plasmids are grown in synthetic complete (SC) media containing 1 .7 g/L yeast nitrogen base, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with the appropriate amino acids required for growth and selection.
  • Alternative carbon sources which may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose.
  • Cells are grown at any convenient temperature (e.g., 30 s C) with shaking at any convenient rate (e.g., 200 rpm) in a vessel, e.g., in test tubes or flasks in volumes ranging from 1-1000 mL, or larger, in the laboratory.
  • Culture volumes may be scaled up for growth in larger fermentation vessels, for example, as part of an industrial process.
  • the industrial fermentation process may be carried out under closed-batch, fed-batch, or continuous chemostat conditions, or any suitable mode of fermentation.
  • the cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for alkaloid production.
  • a batch fermentation is a closed system, in which the composition of the medium is set at the beginning of the fermentation and not altered during the fermentation process.
  • the desired organism(s) are inoculated into the medium at the beginning of the fermentation.
  • the batch fermentation is run with alterations made to the system to control factors such as pH and oxygen concentration (but not carbon).
  • the biomass and metabolite compositions of the system change continuously over the course of the fermentation.
  • Cells typically proceed through a lag phase, then to a log phase (high growth rate), then to a stationary phase (growth rate reduced or halted), and eventually to a death phase (if left untreated).
  • a continuous fermentation is an open system, in which a defined fermentation medium is added continuously to the bioreactor and an equal amount of fermentation media is continuously removed from the vessel for processing.
  • Continuous fermentation systems are generally operated to maintain steady state growth conditions, such that cell loss due to medium being removed must be balanced by the growth rate in the fermentation.
  • Continuous fermentations are generally operated at conditions where cells are at a constant high cell density. Continuous fermentations allow for the modulation of one or more factors that affect target product concentration and/or cell growth.
  • the liquid medium may include, but is not limited to, a rich or synthetic defined medium having an additive component described above.
  • Media components may be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Several media components may be prepared separately and sterilized, and then combined in the fermentation vessel.
  • the culture medium may be buffered to aid in maintaining a constant pH throughout the fermentation.
  • Process parameters including temperature, dissolved oxygen, pH, stirring, aeration rate, and cell density may be monitored or controlled over the course of the fermentation.
  • temperature of a fermentation process may be monitored by a temperature probe immersed in the culture medium.
  • the culture temperature may be controlled at the set point by regulating the jacket temperature. Water may be cooled in an external chiller and then flowed into the bioreactor control tower and circulated to the jacket at the temperature required to maintain the set point temperature in the vessel.
  • a gas flow parameter may be monitored in a fermentation process.
  • gases may be flowed into the medium through a sparger.
  • Gases suitable for the methods of this disclosure may include compressed air, oxygen, and nitrogen. Gas flow may be at a fixed rate or regulated to maintain a dissolved oxygen set point.
  • the pH of a culture medium may also be monitored.
  • the pH may be monitored by a pH probe that is immersed in the culture medium inside the vessel. If pH control is in effect, the pH may be adjusted by acid and base pumps which add each solution to the medium at the required rate.
  • the acid solutions used to control pH may be sulfuric acid or hydrochloric acid.
  • the base solutions used to control pH may be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
  • dissolved oxygen may be monitored in a culture medium by a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is in effect, the oxygen level may be adjusted by increasing or decreasing the stirring speed. The dissolved oxygen level may also be adjusted by increasing or decreasing the gas flow rate.
  • the gas may be compressed air, oxygen, or nitrogen.
  • Stir speed may also be monitored in a fermentation process.
  • the stirrer motor may drive an agitator.
  • the stirrer speed may be set at a consistent rpm throughout the fermentation or may be regulated dynamically to maintain a set dissolved oxygen level.
  • turbidity may be monitored in a fermentation process.
  • cell density may be measured using a turbidity probe.
  • cell density may be measured by taking samples from the bioreactor and analyzing them in a spectrophotometer. Further, samples may be removed from the bioreactor at time intervals through a sterile sampling apparatus. The samples may be analyzed for alkaloids produced by the host cells. The samples may also be analyzed for other metabolites and sugars, the depletion of culture medium components, or the density of cells.
  • a feed stock parameter may be monitored during a fermentation process.
  • feed stocks including sugars and other carbon sources, nutrients, and cofactors that may be added into the fermentation using an external pump.
  • Other components may also be added during the fermentation including, without limitation, antifoam, salts, chelating agents, surfactants, and organic liquids.
  • Any convenient codon optimization techniques for optimizing the expression of heterologous polynucleotides in host cells may be adapted for use in the subject host cells and methods, see e.g., Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety.
  • the subject method may also include adding a starting compound to the cell culture. Any convenient methods of addition may be adapted for use in the subject methods.
  • the cell culture may be supplemented with a sufficient amount of the starting materials of interest (e.g., as described herein), e.g., a mM to pM amount such as between about 1 -5 mM of a starting compound. It is understood that the amount of starting material added, the timing and rate of addition, the form of material added, etc., may vary according to a variety of factors.
  • the starting material may be added neat or pre-dissolved in a suitable solvent (e.g., cell culture media, water, or an organic solvent).
  • the starting material may be added in concentrated form (e.g., 10x over desired concentration) to minimize dilution of the cell culture medium upon addition.
  • the starting material may be added in one or more batches, or by continuous addition over an extended period of time (e.g., hours or days).
  • the subject methods may also include recovering the THP from the cell culture. Any convenient methods of separation and isolation (e.g., chromatography methods or precipitation methods) may be adapted for use in the subject methods to recover the THP from the cell culture. Filtration methods may be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) may be used to separate the THP from other soluble components of the cell culture. In some cases, extraction methods (e.g., liquid extraction, pH based purification, solid phase extraction, affinity chromatography, ion exchange, etc.) may be used to separate the THP from other components of the cell culture.
  • chromatography methods e.g., reverse phase HPLC, size exclusion, normal phase chromatography
  • extraction methods e.g., liquid extraction, pH based purification, solid phase extraction, affinity chromatography, ion exchange, etc.
  • the produced THP may be isolated from the fermentation medium using methods known in the art. A number of recovery steps may be performed immediately after (or in some instances, during) the fermentation for initial recovery of the desired product. Through these steps, the THP may be separated from the cells, cellular debris and waste, and other nutrients, sugars, and organic molecules may remain in the spent culture medium. This process may be used to yield a product enriched with THP.
  • a product stream having THP is formed by providing engineered yeast cells and a feedstock including nutrients and water to a batch reactor.
  • the engineered yeast cells may be subjected to fermentation by incubating the engineered yeast cells for a time period of at least about 5 minutes to produce a solution comprising the THP product and cellular material.
  • at least one separation unit may be used to separate the THP from the cellular material to provide the product stream comprising the THP product.
  • the product stream may include the THP as well as additional components, such as a clarified yeast culture medium. Different methods may be used to remove cells from a bioreactor medium that include THP. In examples, cells may be removed by sedimentation over time.
  • This process of sedimentation may be accelerated by chilling or by the addition of fining agents such as silica.
  • the spent culture medium may then be siphoned from the top of the reactor or the cells may be decanted from the base of the reactor.
  • cells may be removed by filtration through a filter, a membrane, or other porous material. Cells may also be removed by centrifugation, for example, by continuous flow centrifugation or by using a continuous extractor.
  • the cells may be permeabilized or lysed and the cell debris may be removed by any of the methods described above.
  • Agents used to permeabilize the cells may include, without limitation, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate).
  • Methods to lyse the cells may include the addition of surfactants such as sodium dodecyl sulfate, or mechanical disruption by bead milling or sonication.
  • THP may be extracted from the clarified spent culture medium through liquid-liquid extraction by the addition of an organic liquid that is immiscible with the aqueous culture medium.
  • an organic liquid that is immiscible with the aqueous culture medium.
  • suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methylisobutyl ketone, methyl oleate, toluene, oleyl alcohol, ethyl butyrate.
  • the organic liquid may be added to as little as 10% or as much as 100% of the volume of aqueous medium.
  • the organic liquid may be added at the start of the fermentation or at any time during the fermentation. This process of extractive fermentation may increase the yield of THP from the host cells by continuously removing THP to the organic phase.
  • Agitation may cause the organic phase to form an emulsion with the aqueous culture medium.
  • Methods to encourage the separation of the two phases into distinct layers may include, without limitation, the addition of a demulsifier or a nucleating agent, or an adjustment of the pH.
  • the emulsion may also be centrifuged to separate the two phases, for example, by continuous conical plate centrifugation.
  • the organic phase may be isolated from the aqueous culture medium so that it may be physically removed after extraction.
  • the solvent may be encapsulated in a membrane.
  • THP may be extracted from a fermentation medium using adsorption methods.
  • THP may be extracted from clarified spent culture medium by the addition of a resin such as Amberlite® XAD4 or another agent that removes THP by adsorption.
  • the THP may then be released from the resin using an organic solvent.
  • suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone.
  • THP may also be extracted from a fermentation medium using filtration. Under certain conditions, the THP may form a crystalline-like precipitate in the bioreactor. This precipitate may be removed directly by filtration through a filter, membrane, or other porous material. The precipitate may also be collected by centrifugation and/or decantation.
  • the extraction methods described above may be carried out either in situ (in the bioreactor) or ex situ (e.g., in an external loop through which media flows out of the bioreactor and contacts the extraction agent, then is recirculated back into the vessel).
  • the extraction methods may be performed after the fermentation is terminated using the clarified medium removed from the bioreactor vessel.
  • the subject methods may also include recovering the THP from the cell culture. Any convenient methods of separation and isolation (e.g., organic solvent extraction under basic condition, solid phase extraction, chromatography methods, or precipitation methods) may be adapted for use in the subject methods to recover the THP from the cell culture. Filtration methods may be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) are used to separate the THP from other soluble components of the cell culture.
  • Any convenient methods of separation and isolation e.g., organic solvent extraction under basic condition, solid phase extraction, chromatography methods, or precipitation methods
  • Filtration methods may be used to separate soluble from insoluble fractions of the cell culture.
  • liquid chromatography methods e.g., reverse phase HPLC, size exclusion, normal phase chromatography
  • Inserting DNA into host cells may be achieved using any convenient methods.
  • the methods are used to insert the heterologous coding sequences into the host cells such that the host cells functionally express the enzymes and convert starting compounds of interest into THP or precursors thereof.
  • the cell includes one or more promoters for the one or more of the heterologous coding sequences (e.g., as described herein). Any convenient promoters may be utilized in the subject host cells and methods.
  • the promoters driving expression of the heterologous coding sequences may be constitutive promoters or inducible promoters, provided that the promoters can be active in the host cells.
  • the heterologous coding sequences may be expressed from their native promoters, or nonnative promoters may be used. Such promoters may be low to high strength in the host in which they are used.
  • the cell includes one or more strong promoters. Promoters may be regulated or constitutive.
  • promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium are used.
  • Promoters of interest include but are not limited to, promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding fructose bisphosphate aldolase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase), the ADH1 promoter of baker's yeast, the phosphate-starvation induced promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from B.
  • promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding fructose bisphosphate aldolase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase), the ADH1 promoter of bake
  • yeast inducible promoters such as Gall -10, Gall , GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH1), translation-elongation factor-1 -a promoter (TEF), cytochrome c-oxidase promoter (CYC1 ), MRP7 promoter, GAL1 , HXT7, PGK1 , TPI1 , PYK1 , TEF1 , etc.
  • GPD glyceraldehyde 3-phosphate dehydrogenase promoter
  • ADH1 alcohol dehydrogenase promoter
  • TEZ1 translation-elongation factor-1 -a promoter
  • CYC1 cytochrome c-oxidase promoter
  • MRP7 promoter GAL1 , HXT7, PGK1
  • Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE).
  • GRE glucorticoid responsive element
  • TRE thyroid hormone responsive element
  • Additional vectors containing constitutive or inducible promoters such as a factor, alcohol oxidase, and PGH may be used.
  • any promoter/enhancer combination as per the Eukaryotic Promoter Data Base EPDB
  • Any convenient appropriate promoters may be selected for the host cell, e.g., E. coli. One can also use promoter selection to optimize transcript, and hence, enzyme levels to maximize production while minimizing energy resources.
  • the cell includes one or more strong promoters selected from HXT7, ADH1 , PGK1 , TPI1 , PYK1 , and TEF1 .
  • the cell includes one or more heterologous coding sequences that encode CYP82Y1 or a CYP82Y1 mutant and includes a HXT7 promoter.
  • the cell produces 1 -hydroxy-N-methylcanadine.
  • the cell produces 1-hydroxycanadine.
  • the cell includes one or more heterologous coding sequences that encode CYP82X2 or a CYP82X2 mutant and includes a HXT7 promoter.
  • the cell produces 1 ,13- dihydroxy-N-methylcanadine.
  • the cell includes one or more heterologous coding sequences that encode CYP82X2 or a CYP82X2 mutant and includes one or more promoters selected from PGK1 and GPD.
  • the cell produces N- methyl-ophiocarpine.
  • the cell includes one or more heterologous coding sequences that encode CYP82X1 or a CYP82X1 mutant and includes a HXT7 promoter.
  • the cell produces 4’-0-desmethyl-3-0-acetylpapaveroxine.
  • Vectors of interest include vectors for use in yeast and other cells.
  • Yeast vectors can be broken up into 4 general categories: integrative vectors (Yip), autonomously replicating high copy-number vectors (YEp), autonomously replicating low copy-number vectors (YCp) and vectors for cloning large fragments (YACs).
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via any convenient transformation or transfection techniques.
  • Subsequent purification steps may involve treating the post-fermentation solution enriched with THP using methods known in the art to recover individual product species of interest to high purity.
  • THP extracted in an organic phase may be transferred to an aqueous solution.
  • the organic solvent may be evaporated by heat and/or vacuum, and the resulting powder may be dissolved in an aqueous solution of suitable pH.
  • the THP may be extracted from the organic phase by addition of an aqueous solution at a suitable pH that promotes extraction of the THP into the aqueous phase. The aqueous phase may then be removed by decantation, centrifugation, or another method.
  • the THP-containing solution may be further treated to remove metals, for example, by treating with a suitable chelating agent.
  • the THP-containing solution may be further treated to remove other impurities, such as proteins and DNA, by precipitation.
  • the THP-containing solution is treated with an appropriate precipitation agent such as ethanol, methanol, acetone, or isopropanol.
  • DNA and protein may be removed by dialysis or by other methods of size exclusion that separate the smaller alkaloids from contaminating biological macromolecules.
  • the solution containing THP may be extracted to high purity by continuous cross-flow filtration using methods known in the art.
  • the solution contains a mixture of THP and other products, e.g., noscapinoid products, it may be subjected to acid-base treatment to yield THP using methods known in the art. In this process, the pH of the aqueous solution is adjusted to precipitate THP.
  • the THP may be purified in a single step by liquid chromatography.
  • Inserting DNA into host cells may be achieved using any convenient methods.
  • the methods are used to insert the heterologous coding sequences into the engineered host cells such that the host cells functionally express the enzymes and convert starting compounds of interest into THP.
  • the promoters driving expression of the heterologous coding sequences may be constitutive promoters or inducible promoters, provided that the promoters are active in the engineered host cells.
  • the heterologous coding sequences may be expressed from their native promoters, or non-native promoters may be used. Such promoters may be low to high strength in the host in which they are used. Promoters may be regulated or constitutive. In certain embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. Promoters of interest include but are not limited to, promoters of glycolytic genes such as the promoter of the B.
  • subtilis tsr gene (encoding the promoter region of the fructose bisphosphate aldolase gene) or the promoter from yeast S. cerevisiae gene coding for glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, the phosphate-starvation induced promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from B.
  • GPD glyceraldehyde 3-phosphate dehydrogenase
  • ADH1 promoter of baker's yeast the phosphate-starvation induced promoters
  • PHO5 promoter of yeast the alkaline phosphatase promoter from B.
  • yeast inducible promoters such as Gall -10, Gall , GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1 -a promoter (TEF), cytochrome c-oxidase promoter (CYC1 ), MRP7 promoter, etc.
  • GPD glyceraldehyde 3-phosphate dehydrogenase promoter
  • ADH alcohol dehydrogenase promoter
  • TEZ translation-elongation factor-1 -a promoter
  • CYC1 cytochrome c-oxidase promoter
  • MRP7 promoter etc.
  • Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE).
  • GRE glucorticoid responsive element
  • TRE thyroid hormone responsive element
  • Additional vectors containing constitutive or inducible promoters such as a factor, alcohol oxidase, and PGH may be used.
  • any promoter/enhancer combination as per the Eukaryotic Promoter Data Base EPDB
  • Any convenient appropriate promoters may be selected for the host cell, e.g., E coli. One may also use promoter selection to optimize transcript, and hence, enzyme levels to maximize production while minimizing energy resources.
  • Vectors of interest include vectors for use in yeast and other cells.
  • the types of yeast vectors may be broken up into 4 general categories: integrative vectors (Yip), autonomously replicating high copy-number vectors (YEp or 2p plasmids), autonomously replicating low copy-number vectors (YCp or centromeric plasmids) and vectors for cloning large fragments (YACs).
  • Vector DNA is introduced into prokaryotic or eukaryotic cells via any convenient transformation or transfection techniques. DNA of another source (e.g.
  • PCR-generated double stranded DNA product may be used to engineer the yeast by integration into the genome.
  • Any single transformation event may include one or several nucleic acids (vectors, double stranded or single stranded DNA fragments) to genetically modify the host cell.
  • aspects of the invention further include producing a product from the THP produced by host cells of the invention.
  • Products of interest may vary, and include any desired product that may be produced from THP.
  • methods of the invention produce Active Pharmaceutical Ingredients (APIs) from THP produced by a THP-producing cells of the invention.
  • APIs may vary, where examples of such APIs include atracurium, cisatracurium and papaverine.
  • the methods include producing papaverine from THP.
  • the methods may include oxidizing the THP product, e.g., produced by THP- producing cells of the invention (such as described above) to produce papaverine. Oxidizing the THP to produce papaverine may be accomplished using any convenient protocol. In some instances, a chemical oxidizer may be employed to produce papaverine from THP.
  • Chemical oxidizers of interest include, but are not limited to: hydrogen peroxide, potassium persulfate, fremy’s salt, azodicarboxamide, periodic acid, chloramine T trihydrate, peracetic acid, cumene hydroperoxide, dess-martin periodinane, luperox, TEMPO, 3- chloroperbenzoic acid, ammonium persulfate, sodium hypochlorite, cobalt (II) phthalocyanine, cb22’bpdcruthenium hydrate, and the like.
  • oxidation may be carried out under appropriate conditions, e.g., a pH ranging from 6 to 11 , such as 7 to 10.
  • an enzymatic oxidizer may be employed to produce papaverine from THP, where enzymatic oxidizers include dihydrobenzophenanthridine oxidase (DBOX), tetrahydropapaverine oxidase (TPOX), and the like. While the chemical oxidation method of producing papaverine from THP is described herein in terms of THP produced by THP-producing cells of the invention, it is not so limited, as the method may be employed with any THP starting material.
  • the methods include producing atracurium from THP. Any convenient protocol for producing atracurium from THP may be employed, such as but not limited to the protocols described in U.S. Patent No. 5,684,154; 8,354,537; and 8,461 ,338, the disclosures of which are herein incorporated by reference. In some instances, the methods include producing cisatracurium from THP. Any convenient protocol for producing cisatracurium from THP may be employed, such as but not limited to the protocols described in U.S. Patent No. 8,293,912, the disclosure of which is herein incorporated by reference.
  • compositions e.g., that include active agents produced from THP, e.g., as described above.
  • a pharmaceutical formulation may include an API (e.g., as described above) or a pharmaceutically acceptable salt thereof, and one or mere of pharmaceutically acceptable carriers or excipients.
  • a pharmaceutical formulation includes an amount of an API effective to treat a disease; and an excipient.
  • the pharmaceutical formulation may be formulated for administration by any suitable means.
  • the composition is formulated for administration orally, intradermally, intramuscularly, parenterally, intravenously, intra-arterially, intracranially, subcutaneously, intraorbitally, intraventricularly, intraspinally, intraperitoneally, or intranasally.
  • the pharmaceutical formulations or compositions can be formulated into various dosage forms, including tablets, powders, fine granules, granules, dry syrups, capsules, liquid compositions, etc.
  • the pharmaceutical formulation is a capsule or tablet.
  • the pharmaceutic formulation is a parenteral formulation.
  • the pharmaceutical formulation is an intraperitoneal formulation.
  • Additives and diluents normally utilized in the pharmaceutical arts can optionally be added to the pharmaceutical formulation. These include thickening, granulating, dispersing, flavoring, sweetening, coloring, and stabilizing agents, including pH stabilizers, other excipients, anti-oxidants (e.g., tocopherol, BHA, BHT, TBHQ, tocopherol acetate, ascorbyl palmitate, ascorbic acid propyl gallate, and the like), preservatives (e.g., parabens), and the like.
  • thickening granulating, dispersing, flavoring, sweetening, coloring, and stabilizing agents, including pH stabilizers, other excipients, anti-oxidants (e.g., tocopherol, BHA, BHT, TBHQ, tocopherol acetate, ascorbyl palmitate, ascorbic acid propyl gallate, and the like), preservatives (e.g., parabens), and the like.
  • Exemplary preservatives include, but are not limited to, benzylalcohol, ethylalcohol, benzalkonium chloride, phenol, chlorobutanol, and the like.
  • Some useful antioxidants provide oxygen or peroxide inhibiting agents for the formulation and include, but are not limited to, butylated hydroxytoluene, butylhydroxyanisole, propyl gallate, ascorbic acid palmitate, a-tocopherol, and the like.
  • Thickening agents such as lecithin, hydroxypropylcellulose, aluminum stearate, and the like, may improve the texture of the formulation.
  • a container for holding the formulation may be configured to hold any suitable amount or volume of the formulation or composition.
  • the size of the container may depend on the volume formulation to be held in the container.
  • the container may be configured to hold an amount of composition ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to
  • the container is configured to hold an amount of composition ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In certain instances, the container is configured to hold a volume (e.g., a volume of a composition) ranging from 0.1 ml to 200 ml.
  • a volume e.g., a volume of a composition
  • the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml.
  • a volume e.g., a volume of a liquid
  • the shape of the container may also vary.
  • the container may be configured in a shape that is compatible with the assay and/or the method or other devices used to perform the assay.
  • the container may be configured in a shape of typical laboratory equipment used to perform the assay or in a shape that is compatible with other devices used to perform the assay.
  • the container is a liquid container.
  • the liquid container is a vial or a test tube.
  • the liquid container is a vial.
  • the liquid container is a test tube.
  • the container is a blister pack.
  • the container can be compatible with the formulation or composition held therein.
  • suitable materials for the containers include, but are not limited to, glass and plastic.
  • the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREXTM), fused quartz glass, fused silica glass, and the like.
  • suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.
  • plastics such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.
  • the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container.
  • the seal of the container may also substantially prevent other substances from entering the container.
  • the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container.
  • the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container.
  • the seal is made of a resilient material to provide a barrier (e.g., a water-tight and/or air-tight seal) for retaining a sample in the container.
  • a barrier e.g., a water-tight and/or air-tight seal
  • Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container.
  • Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof.
  • the seal is a septum pierceable by a needle, syringe, or cannula.
  • the seal may also provide convenient access to a sample in the container, as well as a protective barrier that overlies the opening of the container.
  • the seal is a removable seal, such as a threaded or snap-on cap or other suitable sealing element that can be applied to the opening of the container. For instance, a threaded cap can be screwed over the opening before or after a sample has been added to the container.
  • the engineered host cells and methods of the invention find use in a variety of applications.
  • Applications of interest include, but are not limited to: research applications and therapeutic applications.
  • Methods of the invention find use in a variety of different applications including any convenient application where the production of THP is desired.
  • the subject engineered host cells and methods find use in a variety of therapeutic applications.
  • Therapeutic applications of interest include those applications in which the preparation of pharmaceutical products that include an API derivative of THP.
  • the engineered host cells described herein produce THP.
  • the subject host cells may be utilized to produce API end products from the THP, such as atracurium, cisatracurium and papaverine. As such, the subject host cells find use in the supply of therapeutically active products, such as atracurium, cisatracurium and papaverine.
  • the engineered host cells and methods find use in the production of commercial scale amounts of THP.
  • the host cells and methods are utilized in a fermentation facility that would include bioreactors (fermenters) of e.g., 5,000- 200,000 liter capacity allowing for rapid production of THP for therapeutic products.
  • bioreactors e.g., 5,000- 200,000 liter capacity allowing for rapid production of THP for therapeutic products.
  • Such applications may include the industrial-scale production of THP from fermentable carbon sources such as cellulose, starch, and free sugars.
  • the subject engineered host cells and methods find use in a variety of research applications.
  • the subject host cells and methods may be used to analyze the effects of a variety of enzymes on the biosynthetic pathways.
  • the engineered host cells may be engineered to produce products that find use in testing for bioactivity of interest in as yet unproven therapeutic functions.
  • the engineering of host cells to include a variety of heterologous coding sequences that encode for a variety of enzymes elucidates the high yielding biosynthetic pathways towards desired products.
  • research applications include the production of products for therapeutic molecules of interest that may then be further chemically modified or derivatized to desired products or for screening for increased therapeutic activities of interest.
  • host cell strains are used to screen for enzyme activities that are of interest in such pathways, which may lead to enzyme discovery via conversion of metabolites produced in these strains.
  • the subject host cells and methods may be used to as a production platform for plant specialized metabolites.
  • the subject engineered host cells and methods may be used as a production platform for plant specialized metabolites.
  • the subject host cells and methods may be used as a platform for drug library development as well as plant enzyme discovery.
  • the subject engineered host cells and methods may find use in the development of natural product based drug libraries by taking yeast strains producing interesting scaffold molecules, such as protopine, and further functionalizing the compound structure through combinatorial biosynthesis or by chemical means. By producing drug libraries in this way, any potential drug hits are already associated with a production host that is amenable to large-scale culture and production.
  • these subject engineered host cells and methods may find use in plant enzyme discovery.
  • the subject host cells provide a clean background of defined metabolites to express plant EST libraries to identify new enzyme activities.
  • the subject host cells and methods provide expression methods and culture conditions for the functional expression and increased activity of plant enzymes in yeast.
  • kits and systems may include one or more components employed in methods of the invention, e.g., engineered host cells, starting compounds, heterologous coding sequences, vectors, culture medium, etc., as described herein.
  • the subject kit includes an engineered host cell (e.g., as described herein), and one or more components selected from the following: starting compounds, a heterologous coding sequence and/or a vector including the same, vectors, growth feedstock, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bi-directional promoters, an internal ribosome entry site (IRES), etc.), and a culture medium.
  • an engineered host cell e.g., as described herein
  • components suitable for use in expression systems e.g., cells, cloning vectors, multiple cloning sites (MCS), bi-directional promoters, an internal ribosome entry site (IRES), etc.
  • a culture medium e.g
  • kits e.g., host cells including one or more modifications, starting compounds, culture medium, etc.
  • a variety of components suitable for use in making and using heterologous coding sequences, cloning vectors and expression systems may find use in the subject kits.
  • Kits may also include tubes, buffers, etc., and instructions for use.
  • the various reagent components of the kits may be present in separate containers, or some or all of them may be pre-combined into a reagent mixture in a single container, as desired.
  • systems for producing THP may include engineered host cells including one or more modifications (e.g., as described herein), starting compounds, culture medium, a fermenter and fermentation equipment, e.g., an apparatus suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, and the like.
  • engineered host cells including one or more modifications (e.g., as described herein), starting compounds, culture medium, a fermenter and fermentation equipment, e.g., an apparatus suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, and the like.
  • the system includes components for the large scale fermentation of engineered host cells, and the monitoring and purification of THP produced by the fermented host cells.
  • one or more starting compounds e.g., as described herein
  • the host cells produce a THP (e.g., as described herein).
  • the system includes processes for monitoring and or analyzing THP produced by the subject host cells.
  • a LC-MS analysis system as described herein, a chromatography system, or any convenient system where the sample may be analyzed and compared to a standard, e.g., as described herein.
  • the fermentation medium may be monitored at any convenient times before and during fermentation by sampling and analysis. When the conversion of starting compounds to THP is complete, the fermentation may be halted and purification of the THP may be done.
  • the subject system includes a purification component suitable for purifying THP from the host cell medium into which it is produced.
  • the purification component may include any convenient means that may be used to purify the THP produced by fermentation, including but not limited to, silica chromatography, reverse-phase chromatography, ion exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction, and pH extraction methods.
  • the subject system provides for the production and isolation of THP fermentation products of interest following the input of one or more starting compounds to the system.
  • Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles.
  • THP is a precursor in the production of the drugs atracurium and cisatracurium(l).
  • Papaverine is used as an anti-spasmodic during vascular surgery(2).
  • metabolic engineering advances have enabled the production of other natural products through heterologous expression of pathway enzymes in yeast(3).
  • Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products.
  • the biosynthesis of THP and papaverine have not been achieved to date, in part, because multiple pathway enzymes have not been elucidated(4).
  • THP is achieved through the heterologous expression of two novel enzyme variants with activity on non-native substrates.
  • protein engineering we developed a variant of /V-methylcoclaurine hydroxylase with activity on coclaurine enabling de novo norreticuline biosynthesis.
  • scoulerine 9-O-methyltransferase capable of O-methylating 1 -benzylisoquinoline alkaloids at the 3’ position enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multi-drug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 pg/L.
  • THP tetrahydropapaverine
  • MDR yeast multi-drug resistance
  • Difco yeast nitrogen base without amino acids and ammonium sulfate (YNB), Bacto peptone, Bacto yeast extract, Luria Broth (LB), LB agar, dextrose, and galactose were obtained from BD (Becton, Dickinson and Company). Adenine hemisulfate, Kanamycin monosulfate, and ampicillin were obtained from Sigma Chemicals.
  • G418 (sc-29065A) used for yeast integration selections was obtained from Santa Cruz Biotech. Amino acid dropout media was obtained from Takara Bio (product #630400 - #630431 ). Frozen-EZ transformation kit for yeast was obtained from Zymo Research. E.
  • coli were selected on LB agar plates with 50 mg/L kanamycin, 75 mg/L carbenicillin, and grown in LB liquid media with the appropriate antibiotic.
  • Yeast 10x drop out (DO) supplement was prepared from Takara as a synthetic complete supplement with desired dropout component omitted.
  • S. cerevisiae strains were selected on YNB-DO (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% dextrose, and 1x DO) agar or on YPD-G418 (1% yeast extract, 2% peptone, and 2% dextrose, 250 p/mL Geneticin). Yeast were grown in selective YNB-DO.
  • (S)-Reticuline and (S)-tetrahydropapaverine (THP) were purchased from Toronto Research Chemicals.
  • (S)-Norreticuline was purchased from Alfa Chemistry.
  • Papaverine hydrochloride, S-adenosylmethionine (SAM), hydrogen peroxide (30% w/w), and potassium persulfate were purchased from Sigma-Aldrich.
  • Tris base was purchased from Fisher Scientific International, Inc.
  • 5N hydrochloric acid was purchased from VWR International.
  • BL21 (DE3) Escherichia coli cells were purchased from Invitrogen.
  • Nickel-nitrilotriacetic acid (Ni-NTA) resin was purchased from Fisher Scientific International, Inc.
  • Amicon® 30 kD cutoff spin filters were purchased from EMD Millipore.
  • E. coli strain TOP10 (Life Technologies) was used for cloning and amplification of plasmids. Plasmids were recovered using Econospin columns (Epoch Life Sciences) according to manufacturer’s instructions. Oligonucleotides were synthesized by the Stanford Protein and Nucleic Acid Facility. PCR was carried out using Q5 DNA Polymerase (New England Biolabs) unless otherwise specified, and all restriction enzymes, T4 DNA ligase, and deoxynucleotides were purchased from New England Biolabs.
  • Heterologous gene sequences were cloned from previously published plasmids, obtained from Addgene, or synthesized by Twist Bioscience. Cloning was exclusively performed using Gibson assembly(54) followed by transformation into E. coli or by gaprepair directly into yeast, or direct genomic integration.
  • gRNA integration plasmids were constructed from addgene plasmid pCAS, which was a gift from Jamie Cate (Addgene plasmid # 60847)(55).
  • pCAS was modified by Gibson assembly to create SpCas9 expression vector pCS3410, which was digested with Pad followed by Gibson assembly of each gRNA fragment. All gRNA sequences used in this work are listed in Supplementary Table 3.
  • Error-prone libraries of the EcNMCH and T/S9OMT were generated by PCR amplification using Taq polymerase and TriLink Biotechnologies mutagenic dNTPs (Item #2748 and #2746) according to the manufacturer's instructions. Three separate PCR reactions were performed at 20 cycles with varying concentrations of equimolar nucleotide analogs according to manufacturer instructions. Amplicons from mutagenic PCR generally were at least 75 bp long and included up to 10 base pairs upstream and downstream of the indicated region when necessary to meet that length, products from each mutagenic PCR were amplified further using Taq polymerase.
  • the amplified DNA was incorporated into the full TfS9OMT gene using overlap-extension PCR and incorporated into plasmids using Gibson Assembly.
  • T/S9OMT NNK libraries were generated with primers containing NNK-randomized regions at the desired codon. These primers were used to amplify two fragments: The front of the gene up to the NNK codon with 15 additional base pairs of overhang, and the back of the gene from 15 base pairs before the NNK codon to the end of the gene. These two amplicons were amplified and Gibson overhangs were attached using overlap-extension PCR.
  • the DNA purification columns, Zymoclean gel DNA recovery kit, and yeast genomic DNA prep kit (D2002) were all obtained from Zymo Research. All routine sequencing was performed using Quintara Biosciences.
  • strain CSY1171 28, 38 was used as the base for any novel strains reported in this work. All yeast plasmid transformations were performed using EZ transformation kit from Zymo Research according to manufacturer’s instructions, unless otherwise specified. Scar-free yeast integrations were carried out as previously described(55), by co-transformation of gRNA/Cas9 expression plasmid targeting the edit site, along with repair DNA to be integrated genomically. Briefly, expression cassettes or gene modifications were amplified using primers with 15-40 bp overhangs for gap repair in yeast. Error-prone libraries of the NMCH and TfS9OMT were generated as previously described before direct transformation for genomically integrated libraries, with guides designed as previously described(35).
  • Genomically integrated NNK libraries were generated using overlapping 70 bp region with the relevant codon NNK randomized in both primers. Each assembly was flanked by an integration homology region of 30-60 base pairs. gRNAs were generated at a target region such that integration of the desired modification would result in disruption of the gRNA binding site. Typically, 500 ng of purified PCR product was introduced with 300 ng of gRNA plasmid using the Zymo EZ Kit for transformation.
  • Variants combined using DNA shuffling were amplified using PCR and pooled in an equimolar mixture with a final DNA concentration of 200 ng/pL.
  • a DNAse I (Zymo Research - E1010) solution was prepared with 167 mM Tris-HCI buffer, 83.3 mM manganese chloride, and 1 .67 U/mL DNase I.
  • 30 ⁇ L DNAse I solution was added to 70 ⁇ L of the equimolar amplicon mixture and the reactions were incubated for 60 seconds at 15°C before being quenched with 6 pL of 500 mM EDTA.
  • the resulting DNA fragments were separated using an agarose gel.
  • DNA fragments between 50 bp and 150 bp in length were cut from the gel and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). DNA fragments were reassembled using the following cycling procedure: 1x: 96°C for 2 min; 35x: 95°C for 15s -> 65°C for 30s -> 62°C for 30s -> 59°C for 30s -> 56°C for 30s -> 53°C for 30s 50°C for 30s 47°C for 30s 44°C for 30s 41 °C for 30s 72°C for 1 min; 1x: 72°C for 3 minutes. A second PCR reaction with terminal primers was used to lift full length sequences and attach overhangs for Gibson Assembly.
  • yeast peptone (YP) base (10 g yeast extract, 20 g peptone, and 80mg adenine hemisulfate for 1 L media
  • yeast nitrogen base (YNB) (1 .7 g yeast nitrogen base, 5 g ammonium sulfate for 1 L media, pH to 5.8 using NaOH).
  • concentration of the carbon source was 2% (w/v) in all media with a single carbon source present. Dextrose was the carbon source unless otherwise noted.
  • the media contained 2% (w/v) dextrose and the concentration of the secondary carbon source was also 2% (w/v).
  • Synthetic complete (SC) media was prepared by combining seven parts YNB, one part 10x amino acid supplement mix, one part water, and one part dextrose.
  • Synthetic defined (SD) media was prepared similarly using 10x amino acid supplement mixes lacking either uracil or tryptophan depending on the auxotrophic marker on the plasmid being used.
  • THP Chemically synthesized THP was used in initial experiments to determine favorable reaction conditions. Final concentrations were 500 nM THP, 75 mM Tris-HCI buffer (pH as indicated), and oxidizing agent concentrations were as indicated in a total reaction volume of 200 ⁇ L. Reactions were incubated at the indicated temperature with shaking at 600 r.p.m. using an Eppendorf Thermomixer F1 .5. Reactions were quenched by adding an equal volume of 1 M sodium thiosulfate and stored at 4°C before analysis via LC-MS/MS. Reactions with biosynthetically-produced THP contained three components: spent yeast media containing THP, Tris-HCI buffer, and hydrogen peroxide.
  • the sum of the volume of the first two components was always 190 ⁇ L at the ratio indicated (e.g. 40:150 media-to- buffer ratio means 40 ⁇ L media and 150 ⁇ L buffer were used). 10 ⁇ L of concentrated hydrogen peroxide solution were then added for a total reaction volume of 200 ⁇ L. These reactions were similarly incubated, quenched, and stored at 4°C prior to analysis via LC- MS/MS.
  • ChemDraw 18.0 was used for illustrating chemical structures. Pymol was used for visualizing protein structures. RaptorX was used to construct protein homology model(33). SwissDock was used for substrate docking simulations(34, 59). MassHunter Workstation (Agilent) was used to collect and analyze LC-MS/MS data. ChemStation (Agilent) and DataAnalysis (Bruker) were used to collect and analyse, respectively, chiral LC-MS data. Microsoft Excel was used to perform statistical analyses and prepare graphs. Inkscape was used to create all figures shown in this work.
  • a reconstructed biosynthetic pathway for reticuline serves as the starting point for a de novo reconstructed THP pathway
  • de novo (S)-reticuline-producing yeast platform strain As the starting point for a de novo THP biosynthetic pathway(27, 28, 32).
  • This reticuline- producing platform strain incorporates five modules with a total of 17 heterologously expressed enzymes to produce reticuline. Each of these modules was integrated into distinct chromosomal regions in a wild-type, haploid Cen.PK2-1 D.
  • Module I encodes the expression of four yeast enzymes to improve the production of the metabolites tyrosine and 4-hydroxyphenylacetaldehyde (4-HPAA). Copies of 3-deoxy-D- arabino-2-heptulosonic acid 7-phosphate synthase (Aro4p Q166K ) and chorismate mutase (Aro7p T2261 ), which were modified to decrease feedback inhibition from tyrosine, and transketolase (TKL1p) and phenylpyruvate decarboxylase (Arol Op) are overexpressed.
  • TKL1p transketolase
  • Arol Op phenylpyruvate decarboxylase
  • Module II encodes expression of four Rattus norvegicus enzymes to synthesize and recycle tetrahydrobiopterin, a cofactor for tyrosine hydroxylase (TyrH); sepiapterin reductase (RnSepR) and 6-pyruvoyl tetrahydrobiopterin synthase (RnPTPS) increase tetrahydrobiopterin production, while quinonoid dihydropteridine reductase (RnQDHPR) and pterin carbinolamine dehydratase (RnPCD) improve cofactor recycling.
  • Module III encodes expression of four enzymes that increase (S)-norcoclaurine titers; R.
  • norvegicus dihydrofolate reductase improves tetrahydrobiopterin (BH 4 ) salvage, mutant (R37E, R38E, and W166Y) R.
  • norvegicus tyrosine hydroxylase produces L- DOPA with improved resistance to negative feedback
  • DOPA decarboxylase from Pseudomonas putida PpDODC
  • PpDODC Pseudomonas putida
  • C/NCS Coptis japonica
  • Module IV encodes the expression of five plant proteins that convert norcoclaurine to reticuline; Papaver somniferum norcoclaurine 6-O-methyltransferase (Ps60MT), coclaurine-A/-methyltransferase (PsCNMT), cytochrome P450 reductase (PsCPR), and 4’-O-methyltransferase (Ps4’0MT) and Eschscholzia californica N- methylcoclaurine hydroxylase (EcNMCH).
  • Module V encodes the overexpression of three proteins that were previously identified as bottlenecks in the pathway (RnTyrH WR , Ps4’0MT, CjNCS), resulting in strain CSY1060(27, 28, 32).
  • Reticuline production in the platform strains were assayed as a benchmark for flux through the native benzylisoquinoline pathway in yeast.
  • the strains were grown for three days at 30°C in synthetic complete (SC) media.
  • Reticuline concentrations in the growth media were assayed by liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS).
  • the base platform strain CSY1060 produces 19.1 pg/L reticuline ( ⁇ 2.23 pg/L). We further incorporated two previously reported modifications to increase reticuline production(28).
  • strain CSY1171 which produces 1.14 mg/L reticuline ( ⁇ 47.3 pg/L).
  • the platform strain CSY1171 was used as the starting point for constructing the THP-producing yeast strains.
  • Error-prone PCR was used to amplify -100 base pair (bp) DNA fragments encoding the peptide chain of interest and overhangs for homologous recombination using conditions generating, on average, one to two mutations per fragment.
  • the fragments were transformed into CSY1172 along with a plasmid encoding CRISPR-Cas9 and a gRNA targeting the corresponding chain.
  • the CRISPR gRNA targets double-stranded breaks in the DNA of each chain, and when the breaks are repaired by the error-containing PCR fragments via homologous recombination, further cutting is inhibited by mutations in the region targeted by the CRISPR guide RNA.
  • norreticuline In order to produce THP via the NH biosynthetic route, norreticuline must be O- methylated at the 7 and 3’ positions on the 1 -BIA scaffold (Fig. 1 A).
  • the enzyme responsible for O-methylating the 3’ position of the 1 - BIA scaffold in planta had not been reported at the time of this study.
  • a number of plant OMTs have been extensively characterized that perform similar reactions, and thus we took an enzyme engineering approach leveraging known plant OMTs as a starting point to complete the reconstructed THP pathway.
  • TfS9OMT was included in our 3’- O-methylation screen because of the structural similarity between norreticuline and the enzyme’s native substrate, scoulerine (Fig. 3A). Scoulerine and norreticuline are similar molecules except for the presence of a carbon bridge between the isoquinoline nitrogen and the 2' benzyl moiety in scoulerine. Screens for 3’OMT activity were performed in the norlaudanine-producing strain, CSY1 174. The strain was grown for three days in synthetic defined (SD) media with the appropriate dropout for plasmid-based expression, and THP and norcodamine were assayed using LC- MS/MS.
  • SD synthetic defined
  • THP was produced at a concentration of 372 ng/L ( ⁇ 52 ng/L) in CSY1174 with a high-copy plasmid encoding TfS9OMT CS , whereas it was not detected in negative control samples with a similar plasmid encoding green fluorescent protein (GFP) (Fig. 3B).
  • GFP green fluorescent protein
  • TfS9OMT activity was improved at the 3' position on the non-native substrates norreticuline and norlaudanine.
  • the previously elucidated crystal structure(38) of the TfS9OMT enzyme was used to identify six contiguous chains of amino acids (amino acid positions — C1 : 10-20, C2: 108-120, C3: 161 -168, C4: 250-256, C5: 281 -283, C6: 293-314) with proximity to the substrate binding pocket of the enzyme (Fig. 3D).
  • Each of the chains was individually targeted for mutagenesis using an error-prone PCR approach.
  • Enzyme variants from 12 colonies with improved THP titers were sequenced, and the sequencing data indicated that mutations at several off-target residues (S47, S77, T83, C98) and on-target residues (A119, F296, L304) are present in enzyme variants exhibiting up to three-fold higher THP titers compared to TfS9OMT DS .
  • NNK libraries were generated and screened at each residue where mutations were shown to improve 3’ OMT activity in the initial site-directed mutagenesis screen.
  • the TfS9OMT NNK libraries were tested by expression from high-copy plasmids in CSY1174. For each NNK library, 92 colonies were screened in order to reach a 95% chance of full library coverage. NNK libraries allowed us to identify novel mutations superior to those found in the initial screen at five of the seven amino acid residues (Table S5).
  • the initial F296S mutation which yielded a three-fold higher THP titer than T/S9OMT DS , was improved to F296L, which produced a five-fold higher THP concentration than TfS9OMT DS (Fig. S3).
  • DNA shuffling to screen for synergistic combinations of the identified amino acid mutations.
  • the site-directed mutagenesis and NNK libraries identified mutations at seven residues that individually improved THP production (S47, T83, S77, C98, A119, F296, L304).
  • a library to test both optimized and unoptimized mutations in combination was generated via DNA shuffling(39) with three input templates: TfS9OMT DS , TfS9OMT V1 (S47F, T83A, C98R), and TfS9OMT V2 (S77W, C98P, A1 19L, F296L, L304Y).
  • TfS9OMT shuffled variants were similarly characterized by expressing the variants from high-copy plasmids transformed into strain CSY1 174. 720 colonies were screened for THP titers and 36% of these colonies produced THP titers higher than TfS9OMT DS . The three top variants increased THP titers more than 10-fold compared to TfS9OMT DS .
  • TfS9OMT sl incorporates A119L and the off- target mutation V281 1 and increases THP titer 12-fold;
  • TfS9OMT S incorporates four on- target mutations, S47P, T83A, C98R, and A119L and increases THP titer 17-fold;
  • TfS9OMT SI incorporates T83A, C98P, A1 19L, and the off-target mutation P208Q and improves THP titer 16-fold (Fig. S4).
  • TfS9OMT OPT contains mutations T83A, C98P, A119L, S160V, V281 I, F296L, and N309T.
  • TfS9OMT DS contains mutations T/S9OMT F29L (the highest performing single mutant), TfS9OMT SI "*, and T/S9OMT OPT by expressing them from a low-copy plasmid in CSY1354 (strain described in next section) grown for three days in SD media with appropriate dropout.
  • TfS9OMT OPT produced 35-fold more THP than TfS9OMT DS (Fig. 3E).
  • TfS9OMT DS and T/S9OMT OPT were purified by Ni-NTA affinity chromatography and their activities examined in vitro.
  • TfS9OMT OPT produced 55-fold more norcodamine than TfS9OMT DS when fed norreticuline as a substrate, indicating that the observed improvements in THP titer are the result of improved catalytic efficiency.
  • TfS9OMT OPT was incorporated into the LYP locus of CSY1 174 resulting in CSY1359, which produces THP at 271 ng/L THP ( ⁇ 24.2 ng/L).
  • engineering a TfS9OMT variant capable of 3’ O-methylating the 1- benzylisoquinoline scaffold for de novo THP production included site-directed mutagenesis, individual NNK optimization, and DNA shuffling (Fig. 3C), resulting in a variant harboring seven mutations that supported a 35-fold increase in THP production from yeast.
  • MDR multi-drug resistance
  • the MDRs were knocked out individually and in combination in the norlaudanine-producing strain CSY1174.
  • the engineered TfS9OMT OPT was expressed in each of the resulting knockout strains from a low-copy plasmid.
  • the strains were grown for three days in SD media with the appropriate dropout, and THP levels were assayed using LC-MS/MS.
  • PDR5 and SNQ2 knockouts improved the overall production of THP compared to equivalent strains with the transporters expressed.
  • metabolites were analyzed as we predicted they may be targets for yeast MDR transporters due to their size and unique scaffold when compared to native yeast metabolites.
  • concentration measured in the media with CSY1354 was higher than with CSY1174 at every timepoint, except for the 12- and 18-hour timepoints for coclaurine.
  • the increase in metabolite titers resulting from the removal of SNQ2 and PDR5 varied significantly (Table S6).
  • coclaurine concentration in CSY1354 was 1.4-fold higher than CSY1174, whereas the concentration of THP in CSY1354 was 10.1 -fold higher. Since overall pathway flux is increased, we predicted that metabolites with smaller increases in extracellular concentration, like coclaurine, may be exported less efficiently with SNQ2 and PDR5 knocked out.
  • the ratio of intracellular concentration to extracellular concentration was higher in CSY1354 than it was for CSY1174 (Fig. 4B).
  • the concentration of coclaurine intracellularly was 9.1 -fold higher than in the media, whereas in CSY1174 the intracellular coclaurine concentration was only 1 .8-fold higher than in the media.
  • the intracellular accumulation of coclaurine resulting from SNQ2 and PDR5 knockouts suggests that these MDR transporters may play a role in exporting coclaurine in CSY1174.
  • the decreased rate of coclaurine export in CSY1354 may play a role in increasing flux through the THP production pathway and improving THP titers.
  • TfS9OMT OPT As the introduction of a second copy of TfS9OMT OPT more than doubled THP production, we examined whether increasing copies of this bottleneck enzyme via plasmidbased expression would lead to additional increases in THP titers.
  • Expression of TfS9OMT OPT from a high-copy plasmid in CSY1354 resulted in higher THP titers than any fully genomically integrated strain, with a THP concentration of 68.9 pg/L ( ⁇ 304 ng/L) when grown in SC with appropriate dropout.
  • THP concentration 68.9 pg/L ( ⁇ 304 ng/L) when grown in SC with appropriate dropout.
  • galactose and raffinose As the introduction of a second copy of TfS9OMT OPT more than doubled THP production, we examined whether increasing copies of this bottleneck enzyme via plasmidbased expression would lead to additional increases in THP titers.
  • the final step in papaverine production biosynthesis is the four-electron oxidation of THP to produce papaverine.
  • An enzyme from Papaver somniferum called dihydrobenzophenanthridine oxidase (PsDBOX) has been proposed to facilitate this reaction in planta and has demonstrated THP oxidase activity when tested in vitro.
  • PsDBOX dihydrobenzophenanthridine oxidase
  • reaction specificity defined as product produced divided by substrate consumed (Table S7).
  • the highest yield achieved with potassium persulfate as an oxidizing agent was 7.41% at a temperature of 40°C, a concentration of 30 mM, and a reaction time of six hours (Fig. S21).
  • the highest yield achieved overall was 16.3% at a temperature of 85°C, a hydrogen peroxide concentration of 0.5%, and a time of 60 minutes (Fig. S22).
  • Biosynthesized THP was produced in CSY1354 with TfS9OMT OPT expressed from a high-copy plasmid as previously described.
  • the spent media from this experiment containing 121 pg/L THP was used as the substrate for oxidation reactions.
  • TfS9OMT OPT a variant with improved catalytic efficiency.
  • these improvements to pathway flux increased THP titers in the media 600-fold resulting in 121 pg/L THP production.
  • reaction conditions that allow for the aqueous oxidation of THP to produce papaverine using hydrogen peroxide, demonstrating the first reported semisynthesis of papaverine from biosynthesized THP.
  • MDR transporter knockouts have broad substrate specificity and generally export compounds with drug-like structures that may be harmful to the cell viability(52) .
  • Export by MDR transporters of pathway intermediates from the cytosol to the media results in those metabolites no longer being co-localized with pathway enzymes, thereby decreasing pathway flux.
  • knocking out one or more MDR transporters could increase the intracellular concentration of intermediate pathway metabolites without decreasing product export.
  • EcNMCH Eschscholzia californica M-methyl-coclaurine hydroxylase
  • OMTs Glaucium flavum norreticuline 7-O-methyltransferase
  • TfS9OMT Thalictrum flavum scoulerine 9-0- methyltransferase
  • MDR transporters Two multi-drug resistance (MDR) transporters, SNQ2 and PDR5, were knocked out in the strain to increase flux through the THP production pathway, resulting in a 15-fold increase in THP titers.
  • the highest titers of THP were achieved by expressing the engineered S90MT from a high-copy plasmid with galactose as a carbon source, resulting in 121 pg/L THP ( ⁇ 3.00 pg/L).
  • hydrogen peroxide is capable of oxidizing THP to produce papaverine in a one-step, aqueous oxidation reaction that converts the biosynthesized THP to papaverine with a yield of approximately 15%.
  • yeast cultures were pelleted by centrifugation at 1 ,500g for 10 minutes. 110 ⁇ L of supernatant was removed for analysis. Metabolites were analyzed by LC-MS/MS using an Agilent 1260 Infinity Binary HPLC and an Agilent 6420 Triple Quadrupole mass spectrometer. Chromatography was performed using a Zorbax EclipsePlus C18 column (2.1x50 mm, 1.8 pm; Agilent Technologies) with 0.1% (v/v) formic acid in water as mobile phase solvent A and 0.1% (v/v) formic acid in acetonitrile as solvent B.
  • Method A 0-1.0 min, 0-100% B; 1.0-2.5, 100% b; 1.5 min post-time.
  • Method B 0-4.0 min, 0-100% B; 4.0-6.5 min, 100% B; 2.5 min post-time.
  • Method C 0-2.0 min, 0% B; 2.0-5.0 min, 0-10% B; 5.0-10.0 min, 10-20% B; 10.0-11.0 min, 20-100% B; 3.0 min post-time.
  • Method A was used for all extracellular metabolite measurements.
  • Method B was used for chemical oxidation reactions.
  • Method C was used for analyzing in vitro protein reactions.
  • the LC eluent was directed to the MS operating with electrospray ionization (ESI) in positive mode, source gas temperature 350 °C, gas flow rate 11 I min-1 , and nebulizer pressure 40 psi.
  • Data collection was performed using MassHunter Workstation LC/MS Data Acquisition software (Agilent). Metabolites were identified and quantified by integrated peak area in MassHunter Workstation Quantitative Analysis software (Agilent) using the mass fragment/transition parameters in Supplementary Table 4 and by comparison to standard curves.
  • ESI electrospray ionization
  • Plasmids containing the TfS9OMT DS and TfS9OMT OPT pET28 expression constructs were used to transform E. coli BL21 (DE3) via heat shock. Briefly, 1 ng of plasmid DNA was added to a 50 ⁇ L aliquot of competent cells, the tube was chilled on ice for 15 minutes, then placed in a 42 °C water bath for 35 seconds, then returned to ice for 2 minutes. Seven hundred fifty pL of SOC media were then added and the tube was rotated at 37 °C for 45 minutes before being plated on an LB agar plate containing 50 pg/mL kanamycin (GoldBio).
  • a single colony was then picked and used to inoculate a primary culture of 5 mL of LB media containing 50 pg/mL kanamycin. This primary culture was then used to inoculate a secondary or expression culture of 500 mL of LB medium containing 50 pg/mL kanamycin. This expression culture was grown to an QD600 of 0.6-1.0 and then induced with IPTG at a final concentration of 1 mM. The expression cultures were then grown at 16 °C for 20 hours at 250 r.p.m., after which, the cultures were harvested by centrifugation (10 min at 3,500 r.p.m. in a 50 mL Falcon tube) and stored at -20 °C until lysis and purification.
  • Ni-NTA resin (Fisher Scientific) was equilibrated with at least 5 volumes of Ni-NTA equilibration buffer (described above) and then loaded with the clarified lysate. The loaded resin was then washed with at least 5 volumes of NH 01 NTA wash buffer (50 mM sodium phosphate, 300 mM NaCI, 50 mM imidazole, pH 7.4) and then the bound protein was eluted with 5 volumes of Ni-NTA elution buffer (50 mM sodium phosphate, 300 mM NaCI, 250 mM imidazole, pH 7.4).
  • NH 01 NTA wash buffer 50 mM sodium phosphate, 300 mM NaCI, 50 mM imidazole, pH 7.4
  • Ni-NTA elution buffer 50 mM sodium phosphate, 300 mM NaCI, 250 mM imidazole, pH 7.4
  • the eluted fractions were then combined and concentrated using an Amicon® 30 kDa cutoff spin filter (EMD Millipore) at 5,000 g at 4 °C. Concentrated protein fractions were then buffer exchanged into storage buffer (50 mM potassium phosphate, 100 mM NaCI, 10% glycerol, pH 7.5), split into separate aliquots, and stored at -20 °C until use.
  • storage buffer 50 mM potassium phosphate, 100 mM NaCI, 10% glycerol, pH 7.5
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Abstract

Non-plant cells that produce tetrahydropapaverine (THP) via an engineered THP-biosynthetic pathway are provided. In embodiments of the invention, the engineered THP-biosynthetic pathway is a norreticuline mediated pathway. Also provided are methods of producing THP using the cells, as well as methods of producing papaverine, e.g., via oxidation of THP, as well as other produces from THP, e.g., atracurium and cisatracurium.

Description

TETRAHYDROPAPA VERINE-PRODUCING MICROBES AND METHODS OF MAKING AND USING THE SAME
GOVERNMENT RIGHTS
This invention was made with Government support under contract AT007886 awarded by the National Institutes of Health. The Government has certain rights in the invention.
CROSS-REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Application Serial No. 63/389,206 filed on July 14, 2022, which application is incorporated by reference herein.
INTRODUCTION
Plant secondary metabolism is a rich source of structurally unique and functionally diverse bioactive compounds. These secondary metabolites, called plant natural products (PNPs), serve crucial functions in plant development, communication, and defense(5). In addition, many PNPs exhibit therapeutic activities and have been used as antibiotics, analgesics, antivirals, neurocognitive therapies, and chemotherapeutics(6). Isolation of PNPs from plant biomass requires laborious extraction and purification procedures and the yields are subject to both seasonal and long-term instability due to weather and climate change. Furthermore, because of the significant lead time required for planting and maturation of crops, the supply of plant-derived PNPs is less able to respond to sudden shocks in demand.
1 -Benzylisoquinoline alkaloids (1 -Bl As) are a family of PNPs with diverse structures and significant medicinal value, including the analgesics morphine and codeine, the antimicrobials sanguinarine and berberine, and the cough suppressant noscapine(7). Many widely used medicinal compounds are still produced through extraction of the active ingredient, or its precursors, from commercially grown plant material(8). (S)- Tetrahydropapaverine (THP) and papaverine are 1-BIAs with established clinical significance that are derived from the opium poppy (Papaver somniferum). THP is a direct precursor to papaverine and a precursor in the production of the neuromuscular blocking agents atracurium and cisatracurium(l ). Atracurium and cisatracurium are often administered during anesthesia to facilitate intubation(9). Due to increased incidence of patient intubation during the COVID-19 pandemic, atracurium and cisatracurium have experienced recent global supply shortages(I O). Papaverine is used directly in the clinic as a vasodilator and antispasmodic(11-13). In addition, recent studies have demonstrated potential clinical applications of papaverine due to its anti-cancer(14) and antiviral5 activities. Despite the availability of several chemical syntheses of papaverine dating back to the early 1900s(16-18), shortages of papaverine in the 2010s forced many vascular surgeons to seek replacements for the drug due to the disruption in supply(2).
The natural biosynthetic pathway for papaverine in opium poppy has not been fully elucidated and differing hypotheses have been suggested. Evidence has been presented for the existence of two biosynthetic pathways for papaverine production - the NH route involving norreticuline(19, 20) and the NCH3 route involving reticuline(21 ) (Fig. S1). Despite rigorous study on both potential pathways, key enzymes required for reactions in the pathway remain uncharacterized. For the NH route an enzyme to hydroxylate the 3’ position of the benzylisoquinoline scaffold has not been characterized. For the NCH3 route, an enzyme to A/-demethylate the benzylisoquinoline scaffold has not been described(4). In addition, for both routes no enzyme is known to efficiently O-methylate the 3’ position and while an enzyme has been proposed to carry out the final oxidation step(22), it has not been confirmed as the main in planta driver of papaverine production. While DBOX has been shown to oxidize THP in vitro, the accumulation of the enzyme mainly in plant tissue void of papaverine raises questions as to the role of this enzyme in the native biosynthetic pathway.
Saccharomyces cerevisiae (baker’s yeast) has served as a platform for enzyme characterization, pathway elucidation(23), and de novo metabolite production for various plant natural products(24). S. cerevisiae can be more readily engineered and provides a cleaner secondary metabolite background compared to plants(25) and is able to functionally express eukaryotic cytochromes P450 with higher efficacy than prokaryotic microbes(3, 26). As a result, S. cerevisiae has been used as a platform for the construction of several complex plant natural product pathways, including opioids(27), noscapinoids(23, 28), artemisinin(29), strictosidine(30), and tropane alkaloids(31 ). Significant work on benzylisoquinoline alkaloids has already been accomplished in yeast, including the de novo production of key intermediates of THP and papaverine production(32).
SUMMARY
Non-plant cells that produce tetrahydropapaverine (THP) via an engineered THP- biosynthetic pathway are provided. In embodiments of the invention, the engineered THP- biosynthetic pathway is a norreticuline mediated pathway. Also provided are methods of producing THP using the cells, as well as methods of producing papaverine, e.g., via oxidation of THP, as well as other produces from THP, e.g., atracurium and cisatracurium.
INCORPORATION BY REFERENCE
ALL publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
BRIEF DESCRIPTION OF THE DRAWINGS
The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
Figure 1 : De novo production of THP is achieved following the N- desmethylated route with enzymes from four kingdoms, overexpressed native yeast genes, and multiple engineered variants of plant enzymes. Light gray arrows, native yeast enzymes; dark gray arrows, native yeast enzymes that have been modified to improve activity in the context of this pathway or that are overexpressed with the addition of multiple copies to the strain; purple arrows; mammalian enzymes; light green arrows, wildtype plant enzymes; dark green arrows; plant enzymes that have been modified to improve their activity on a non-native substrate; orange enzymes, bacterial enzymes. Engineered enzymes modified from their wild-type sequence are noted with a superscript identifying the mutation if single mutation or with an abbreviation if multiple mutations. Moieties added by a single reaction in the pathway are highlighted in blue.
Figure 2: Structure-guided semi-targeted mutagenesis helps identify NMCH variant with improved activity on the N-desmethylated substrate coclaurine. A) Comparison between native substrate for EcNMCH, /V-methylcoclaurine, and the non-native substrate in the reconstructed THP biosynthetic pathway, coclaurine. Key differences are highlighted in red. B) Homology model of NMCH with regions adjacent to the binding pocket highlighted. EcNMCH is shown in green; binding pocket adjacent residues targeted for mutagenesis are highlighted in blue; native enzyme substrate, /V-methylcoclaurine, is shown in cyan. The green bar below represents the linearized form of the enzyme with the binding pocket adjacent regions highlighted in blue and identified as regions for targeted mutagenesis. C) Detailed view of N-methylcoclaurine (cyan) docked in the binding pocket of EcNMCH (green). The L203 position is highlighted in blue, demonstrating its proximity to the nitrogen that is differentially methylated between the desired substrate, coclaurine, and the native substrate, A/-methylcoclaurine. D) Relative norreticuline production of different EcNMCH variants in an NNK library including all possible amino acids at the L203 position, tested in CSY1172 with L-DOPA feeding. Wild-type negative controls are shown in blue, and norreticuline titers are normalized to the average of the wild-type being equal to one. Glycine and serine mutants are shown in green; other library members are shown in gray. Figure 3: Multiple protein engineering strategies were applied to generate variants of TfS9OMT with an improved capacity to produce de novo THP. A) Comparison between the native substrate of TfS9OMT, scoulerine, and the non-native substrate in the context of this pathway, norreticuline. Key differences are highlighted in red. B) LC-MS/MS traces for the MRM transition 344
Figure imgf000005_0001
192 used to detect the presence of
THP. CSY1174 was grown with a high-copy plasmid encoding expression of either the DS variant of T/S9OMT (blue) or with GFP as a negative control (gray), and the media was analyzed for the presence of THP. Traces shown show a single sample which was representative of tests in triplicate. C) Schematic of the protein engineering strategy used on TfS9OMT. Targeted mutagenesis over individual chains of the enzyme, DNA shuffling, and targeted NNK libraries were used to construct and identify a variant of the enzyme with substantially improved 3’-0-methylation activity. D) Crystal structure of T/S90MT (green) with the substrate binding pocket adjacent region highlighted in blue and the native substrate, scoulerine, in cyan. The green bar below represents the linearized form of the enzyme with the binding pocket adjacent regions highlighted in blue. E) Relative THP concentration produced by CSY1354 when expressing different TfS9OMT variants: TfS9OMTDS; the best single mutant identified, TfS9OMTF296L; the DNA shuffled variant, TfS9OMTSI"*; the final optimized mutant incorporating additional NNK library selections, TfS9OMTOPT. Error bars represent the standard deviation of triplicate samples.
Figure 4: Multi-drug resistance transporter knockouts affect the transport of pathway intermediates into the media and contribute to higher THP producing strains. A) Relative THP titers produced in CSY1174 and related strains with one to three MDR transporters knocked out. All strains are expressing TfS9OMTOPT from a low-copy plasmid. Relative THP concentration is normalized to the THP concentration in CSY1174. B) Relative intracellular concentrations of pathway metabolites in CSY1174, the base norlaudanine-producing strain, and CSY1354, which is identical to CSY1174 except the MDR transporters SNQ2 and PDR5 are knocked out. Both strains contain a low-copy plasmid expressing TfS9OMTOPT. Intracellular concentration is reported as relative to the extracellular concentration of that metabolite in the same sample. C) THP concentration in the media when CSY1361 is grown at the indicated media conditions. 2x synthetic complete (2x SC) and 4x synthetic complete (4x SC) indicate twice and four times the standard concentration of amino acid supplements when preparing synthetic complete media, respectively. Carbon sources indicated on the x-axis were present at 2% (w/v). Error bars represent the standard deviation of triplicate samples in all panels.
Figure 5: Chemical oxidation of biosynthesized THP enables semi-synthetic papaverine production. A) Yield of papaverine in chemical oxidation reactions with biosynthesized THP. All reactions were carried out with 0.5% hydrogen peroxide at 85°C for one hour. The media-to-buffer ratio of each reaction is indicated on the x-axis. Error bars represent the standard deviation of triplicate samples. B) Yield of papaverine in chemical oxidation reactions with 0.5% hydrogen peroxide at 85°C using chemically synthesized (white circles) and biosynthesized (black squares) THP. Synthesized THP reaction used 500 nM THP and 75 mM Tris-HCI buffer. Biosynthesized THP reaction used spent media from CSY1354 grown with TfS9OMTOPT expressed from a high-copy plasmid. The reactions had a media-to-buffer ratio of 40:150 and had a final Tris-HCI concentration of 75 mM. Error bars represent the standard deviation of triplicate samples in all panels.
Supplementary Figure 1 : N-methylated and N-desmethylated routes of papaverine production. Structures and names of each molecule in the proposed N- methylated (blue background) and N-desmethylated (green background) routes to papaverine production. The nitrogen molecule, whose methylation or lack thereof distinguishes the two paths from one another is highlighted in blue. Truncated enzyme names accompany the arrows between molecules and presently uncharacterized enzymes are highlighted in red. Enzyme names: coclaurine N-methyltransferase (CNMT), Coclaurine hydroxylase (CocH), N-methylcoclaurine hydroxylase (NCMH), 4’-O-methyltransferase (4’OMT), 7-O-methyltransferase (7OMT), 3'-O-methyltransferase (3’OMT), laudanosine demethylase (LdM), and dihydrobenzophenanthridine oxidase (DBOX).
Supplementary Figure 2: Integration areas of pathway intermediates in
CSY1172. Integration area is used as a proxy for concentration. The scaling of the y-axis is logarithmic. 3’-OH-coclaurine is equivalent to 3'-hydroxycoclaurine. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 3: Relative THP concentration produced by NNK library at the F296 position of TfS9OMT. Library variants were expressed from a high-copy plasmid in CSY1174. TfS9OMTDS controls are colored in blue. Variants that sequenced as TfS9OMTF296L are colored in green. Relative THP concentrations are reported with the average of T/S9OMTDS samples being equal to one.
Supplementary Figure 4: Relative THP concentration produced by highest performing variants identified during DNA shuffling. TfS9OMTDS and the indicated variants were characterized via expression from a high-copy plasmid in CSY1174. Relative THP concentrations are reported with the average of T/S9OMTDS samples being equal to one. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 5: Growth curves for CSY1174 and CSY1354 cultured in SD media. Both strains harbor a low-copy plasmid expressing TfS9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples. Supplementary Figure 6: Integration area of norcoclaurine measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a high-copy plasmid expressing T/S9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 7: Integration area of coclaurine measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a low-copy plasmid expressing TfS9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 8: Integration area of 3’-hydroxycoclaurine measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a low-copy plasmid expressing TfS9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 9: Integration area of norreticuline measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a low-copy plasmid expressing TfS9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 10: Integration area of norcodamine measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a low-copy plasmid expressing TfS9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 11 : Integration area of norlaudanine measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a low-copy plasmid expressing 7/S9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 12: Integration area of THP measured in the spent media. Integration area is used as a proxy for concentration. CSY1174 (black diamonds) and CSY1354 (white squares) harbor a low-copy plasmid expressing TfS9OMTOPT. Measurements were taken at approximately six-hour intervals. Triplicate 50 mL cultures were started at 0 hours and 600-1000 μL were collected at each timepoint for analysis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 13: Relative intracellular concentrations of pathway metabolites in CSY1174 and CSY1354. Both strains harbor a low-copy plasmid expressing TfS9OMTOPT. Intracellular concentration is reported as relative to the extracellular concentration of that metabolite in the same sample. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 14: THP titer produced by CSY1361 with different media bases and carbon sources. All carbon sources in the media were present at 2% except when indicated otherwise. Dex/Raf indicates 2% dextrose and 2% raffinose. 2x synthetic complete (2x SC) and 4x synthetic complete (4x SC) indicate twice and four times the standard concentration of amino acid supplements when preparing synthetic complete media. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 15: THP titer produced by CSY1361 with different media bases and carbon sources. All carbon sources were present at 2% unless otherwise indicated (including samples with multiple carbon sources. Dex indicates dextrose, Tre indicates trehalose, Gly indicates glycerol, Sue indicates sucrose, and Gal indicates galactose. 2x synthetic complete (2x SC) and 4x synthetic complete (4x SC) indicate twice and four times the standard concentration of amino acid supplements when preparing synthetic complete media. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 16: Biomass normalized THP titers produced by CSY1361 grown with different media bases and carbon sources. All carbon sources in the media were present at 2%. 4x synthetic complete (4x SC) indicates four times the standard concentration of amino acid supplements when preparing synthetic complete media. Error bars represent the standard deviation of triplicate samples. THP titers are normalized by OD6oo measurements to account for the difference in biomass between samples.
Supplementary Figure 17: THP titer produced by CSY1354 harboring a high- copy plasmid encoding an expression cassette for TfS9OMTOPT grown in different media bases and carbon sources. 2x synthetic complete (2x SC) indicates twice the standard concentration of amino acid supplements were used when preparing synthetic complete media. SC 2x carbon indicates synthetic complete prepared with 4% (w/v) of the indicated carbon source. 2% (w/v) of the carbon source was used in the media for all other samples. Error bars represent the standard deviation of triplicate samples. Supplementary Figure 18: Biomass normalized THP titer produced by CSY1354 harboring a high-copy plasmid encoding an expression cassette for TfS9OMTOPT grown in different media bases and carbon sources. 2x synthetic complete (2x SC) indicates twice the standard concentration of amino acid supplements were used when preparing synthetic complete media. SC 2x carbon indicates synthetic complete prepared with 4% (w/v) of the indicated carbon source. 2% (w/v) of the carbon source was used in the media for all other samples. Error bars represent the standard deviation of triplicate samples. THP titers are normalized by OD6oo measurements to account for the difference in biomass between samples.
Supplementary Figure 19: Overlay of fluorescence microscopy and light microscopy images of CSY1361 expressing fusions of DBOX and GFP from a low- copy plasmid. Large, dark outlines are droplets of media containing yeast, which are smaller and have fainter outlines. The scale bar in each image is 5 μm. A) The C-terminal GFP fusion shows strong expression of GFP throughout the cytosol. B) The N-terminal GFP fusion shows many cells that do not express functional GFP and some cells contain localized bright spots, which may indicate poor protein folding. Images are indicative of triplicate samples analyzed by microscopy for both panels.
Supplementary Figure 20: Yield of papaverine in chemical oxidation reactions. 500 nM chemically synthesized THP was present in each reaction as a substrate. Reaction pH was controlled with 75 mM Tris-HCl. Hydrogen peroxide (blue) concentration was 0.5% (w/v) and potassium persulfate (green) concentration was 30 μM. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 21 : Normalized molar concentration of THP and papaverine in chemical oxidation reactions with 30 mM potassium persulfate at 40°C. Reactions began with 500 nM THP and 75 mM Tris-HCl pH = 9.5. The percent yield of papaverine is indicated above the marker corresponding with the highest production of papaverine. The concentration of THP and papaverine are normalized with 100 being equal to the molar concentration of THP at the start of the reaction. 30 mM potassium persulfate at 40°C for six hours resulted in the highest yield of papaverine (7.41%) for any reaction with potassium persulfate as the oxidizing agent. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 22: Normalized molar concentration of THP and papaverine in chemical oxidation reactions with 0.5% hydrogen peroxide at 85°C. Reactions began with 500 nM THP and 75 mM Tris-HCl pH = 9.5. The percent yield of papaverine is indicated above the marker corresponding with the highest production of papaverine. The concentration of THP and papaverine are normalized with 100 being equal to the molar concentration of THP at the start of the reaction. 0.5% hydrogen peroxide at 85°C for 60 minutes resulted in the highest yield of papaverine for any reaction with hydrogen peroxide as the oxidizing agent. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 23: Normalized molar concentration of THP and papaverine in chemical oxidation reactions with 0.3% hydrogen peroxide at 70°C. Reactions began with 500 nM THP and 75 mM Tris-HCI pH = 9.5. The percent yield of papaverine is indicated above the marker corresponding with the highest production of papaverine. The concentration of THP and papaverine are normalized with 100 being equal to the molar concentration of THP at the start of the reaction. 0.3% hydrogen peroxide at 70°C for three hours resulted in the second highest yield of papaverine and were further tested as reaction conditions for biosynthesized THP. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 24: Yield of papaverine in chemical oxidation reactions with biosynthesized THP. All reactions were carried out with 0.3% hydrogen peroxide at 70°C for three hours. The media-to-buffer ratio of each reaction is indicated on the x-axis. Error bars represent the standard deviation of triplicate samples.
Supplementary Figure 25: Normalized molar concentration of THP and papaverine in chemical oxidation reactions with 0.5% mM hydrogen peroxide at 85°C using biosynthesized THP. THP was produced in CSY1354 harboring a high-copy plasmid expressing TfS9OMTOPT with raffinose as a carbon source. Reactions also used 100 mM Tris-HCI pH = 9.5 and the media-to-buffer ratio was 40:150. The percent yield of papaverine is indicated above the marker corresponding with the highest production of papaverine. The concentration of THP and papaverine are normalized with 100 being equal to the molar concentration of THP at the start of the reaction. These conditions resulted in the highest yield of papaverine using biosynthesized THP. Error bars represent the standard deviation of triplicate samples.
DEFINITIONS
Before describing exemplary embodiments in greater detail, the following definitions are set forth to illustrate and define the meaning and scope of the terms used in the description.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, New York (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, N.Y. (1991 ) provide one of skill with the general meaning of many of the terms used herein. Still, certain terms are defined below for the sake of clarity and ease of reference.
As used herein, the terms “determining,” “measuring,” “assessing,” and “assaying” are used interchangeably and include both quantitative and qualitative determinations.
As used herein, the term “polypeptide” refers to a polymeric form of amino acids of any length, including peptides that range from 2-50 amino acids in length and polypeptides that are greater than 50 amino acids in length. The terms “polypeptide” and “protein” are used interchangeably herein. The term “polypeptide” includes polymers of coded and noncoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones. A polypeptide may be of any convenient length, e.g., 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, 50 or more amino acids, 100 or more amino acids, 300 or more amino acids, such as up to 500 or 1000 or more amino acids. “Peptides” may be 2 or more amino acids, such as 4 or more amino acids, 10 or more amino acids, 20 or more amino acids, such as up to 50 amino acids. In some embodiments, peptides are between 5 and 30 amino acids in length.
As used herein the term “isolated,” refers to an moiety of interest that is at least 60% free, at least 75% free, at least 90% free, at least 95% free, at least 98% free, and even at least 99% free from other components with which the moiety is associated with prior to purification.
As used herein, the term “encoded by” refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of 3 or more amino acids, such as 5 or more, 8 or more, 10 or more, 15 or more, or 20 or more amino acids from a polypeptide encoded by the nucleic acid sequence. Also encompassed by the term are polypeptide sequences that are immunologically identifiable with a polypeptide encoded by the sequence.
A “vector” is capable of transferring gene sequences to target cells. As used herein, the terms, “vector construct,” “expression vector,” and “gene transfer vector,” are used interchangeably to mean any nucleic acid construct capable of directing the expression of a gene of interest and which may transfer gene sequences to target cells, which is accomplished by genomic integration of all or a portion of the vector, or transient or inheritable maintenance of the vector as an extrachromosomal element. Thus, the term includes cloning, and expression vehicles, as well as integrating vectors.
An “expression cassette” includes any nucleic acid construct capable of directing the expression of a gene/coding sequence of interest, which is operably linked to a promoter of the expression cassette. Such cassette is constructed into a “vector,” “vector construct,” “expression vector,” or “gene transfer vector,” in order to transfer the expression cassette into target ceils. Thus, the term includes cloning and expression vehicles, as well as viral vectors.
A “plurality” contains at least 2 members. In certain cases, a plurality may have 10 or more, such as 100 or more, 1000 or more, 10,000 or more, 100,000 or more, 106 or more, 107 or more, 108 or more, or 109 or more members. In any embodiments, a plurality can have 2-20 members.
Numeric ranges are inclusive of the numbers defining the range.
The methods described herein include multiple steps. Each step may be performed after a predetermined amount of time has elapsed between steps, as desired. As such, the time between performing each step may be 1 second or more, 10 seconds or more, 30 seconds or more, 60 seconds or more, 5 minutes or more, 10 minutes or more, 60 minutes or more, and including 5 hours or more. In certain embodiments, each subsequent step is performed immediately after completion of the previous step. In other embodiments, a step may be performed after an incubation or waiting time after completion of the previous step, e.g., a few minutes to an overnight waiting time.
Other definitions of terms may appear throughout the specification.
DETAILED DESCRIPTION
Non-plant cells that produce tetrahydropapaverine (THP) via an engineered THP- biosynthetic pathway are provided. In embodiments of the invention, the engineered THP- biosynthetic pathway is a norreticuline mediated pathway. Also provided are methods of producing THP using the cells, as well as methods of producing papaverine, e.g., via oxidation of THP, as well as other produces from THP, e.g., atracurium and cisatracurium.
Tetrahydropapaverine (THP), along with and papaverine, are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium. Papaverine is used as an anti-spasmodic during vascular surgery. In recent years, metabolic engineering advances have enabled the production of other natural products through heterologous expression of pathway enzymes in yeast. Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products.
The biosynthesis of THP and papaverine have not been achieved to date, in part, because multiple pathway enzymes have not been elucidated. Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through the heterologous expression of two novel enzyme variants with activity on non-native substrates. The first of the two novel enzyme variants is a protein engineered variant of M-methylcoclaurine hydroxylase with activity on coclaurine enabling de novo norreticuline biosynthesis. The second of the two novel enzyme variants is a protein engineered variant of scoulerine 9-O-methyltransferase capable of O-methylating 1 - benzylisoquinoline alkaloids at the 3’ position enabling de novo THP biosynthesis.
Additionally, strain engineering aspects were also developed so as to increase flux through the heterologous pathway for the production of THP. In some embodiments, flux through the heterologous pathway was improved by knocking out yeast multi-drug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 pg/L.
Additionally, embodiments provide for production of papaverine. In particular, some embodiments of provide for papaverine semi-synthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce de novo semi-synthesized papaverine through the oxidation of biosynthesized THP.
Provided herein are methods and engineered host cells for producing de novo microbial biosynthesis of tetrahydropapaverine (THP). Additionally, provided herein are methods for semi-synthesis of papaverine using microbially biosynthesized THP. The biosynthesis of THP demonstrates the ability to use protein homologs and protein engineering to replace the activity of unknown enzymes in heterologous biosynthetic pathways. Further, methods are provided herein for improving THP pathway flux by knocking out two yeast multi-drug resistance (MDR) transporters, which reduces the export of pathway intermediates. MDR knockouts may be applied to increase flux through other heterologous pathways. The strain engineering as disclosed herein provides methods for fermentation-based production of clinically significant molecules THP and papaverine, which have experienced recent supply chain shortages.
Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.
It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.
As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.
THP PRODUCING CELLS
Host cells which produce tetrahydropapaverine (THP) are provided. As such, one aspect of the invention is a host cell that produces THP. THP is described by the structure:
Figure imgf000015_0001
As used herein, the term “THP-producing cell” is meant to include cells that are engineered to produce THP from a starting compound via an engineered synthetic pathway.
Any convenient cells may be utilized in the subject host cells and methods. In some cases, the host cells are non-plant cells. In some instances, the host cells may be characterized as microbial cells. In certain cases, the host cells are insect cells, mammalian cells, bacterial cells, fungal cells or yeast cells. Any convenient type of host cell may be utilized in producing the subject cells producing the subject THP-producing cells, see, e.g., US2008/0176754; WO/2012/039438; WO2013136057; US2017/0253898;
US2018/0163241 and WO 2020/185626, as well as U.S. Patent Nos. 9,534,241 ; 11 ,124,814 and 10,752,903; the disclosures of which are incorporated by reference in their entirety. Host cells of interest include, but are not limited to, bacterial cells, such as Bacillus subtilis, Escherichia coli, Streptomyces, Anabaena, Arthrobacter, Acetobacter, Acetobacterium, Bacillus, Bifidobacterium, Brachybacterium, Brevibacterium, Carnobacterium, Clostridium, Corynebacterium, Enterobacter, Escherichia, Gluconacetobacter, Gluconobacter, Hafnia, Halomonas, Klebsiella, Kocuria, Lactobacillus, Leucononstoc, Macrococcus, Methylomonas, Methylobacter, Methylocella, Methylococcus, Microbacterium, Micrococcus, Microcystis, Moorella, Oenococcus, Pediococcus, Prochlorococcus, Propionibacterium, Proteus, Pseudoalteromonas, Pseudomonas, Psychrobacter, Rhodobacter, Rhodococcus, Rhodopseudomonas, Serratia, Staphylococcus, Streptococcus, Streptomyces, Synechococcus, Synechocystis, Tetragenococcus, Weissella, Zymomonas, and Salmonella typhimuium cells, insect cells such as Drosophila melanogaster S2 and Spodoptera frugiperda Sf9 cells, and yeast cells such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris, Yarrowia lipolytica, Candida albicans, Aspergillus spp., Rhizopus spp., Peniciilium spp., and Trichoderma reesei cells.
In some embodiments, the host cells are yeast cells or E. coli cells. In some cases, the host cell is a yeast cell. Any of the host cells described in e.g., US2008/0176754; WO/2012/039438; WO2013136057; US2017/0253898; US2018/0163241 and WO 2020/185626, as well as U.S. Patent Nos. 9,534,241 ; 11 ,124,814 and 10,752,903; may be adapted for use in the subject cells and methods. In certain embodiments, the yeast cells may be of the species Saccharomyces cerevisiae (S. cerevisiae). In certain embodiments, the yeast cells may be of the species Schizosaccharomyces pombe. In certain embodiments, the yeast cells may be of the species Pichia pastoris. Yeast is of interest as a host cell because cytochrome P450 proteins are able to fold properly into the endoplasmic reticulum membrane so that their activity is maintained. In examples, cytochrome P450 proteins are involved in some biosynthetic pathways of interest. Yeast strains of interest that find use in the invention include, but are not limited to, CEN.PK (Genotype: MATa/a ura3-52/ura3-52 trp 1-289/trp 1-289 leu2-3_ 112/leu2-3_ 112 his3 A 1/his3 A 1 MAL2-8C/MAL2- 8C SUC2/SUC2), S288C, W303, D273-10B, X2180, A364A, £1278B, AB972, SK1 , and FL100. In certain cases, the yeast strain is any of S288C (MATa; SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1 ), BY4741 (MATa; his3A1 ; leu2A0; met15A0; ura3A0), BY4742 (MATa; his3A1 ; leu2A0; lys2A0; ura3A0), BY4743 (MATa/MATa; his3A1/his3A1 ; leu2A0/leu2A0; met15A0/MET15; LYS2/lys2A0; ura3A0/ura3A0), and WAT11 or \N(R), derivatives of the W303-B strain (MATa; ade2-1 ; his3-11 , -15; leu2-3,-112; ura3-1 ; canR; cyr+) which express the Arabidopsis thaliana NADPH-P450 reductase ATR1 and the yeast NADPH-P450 reductase CPR1 , respectively. In another embodiment, the yeast cell is W303alpha (MATa; his3-11 ,15 trp1-1 leu2-3 ura3-1 ade2-1 ). The identity and genotype of additional yeast strains of interest may be found at EUROSCARF (web.uni- frankfurt.de/fb15/mikro/euroscarf/col_index.html).
In some instances, the host cell is a fungal cell. In certain embodiments, the fungal cells may be of the Aspergillus species and strains include Aspergillus niger (ATCC 1015, ATCC 9029, CBS 513.88), Aspergillus oryzae (ATCC 56747, RIB40), Aspergillus terreus (NIH 2624, ATCC 20542) and Aspergillus nidulans (FGSC A4).
The host cells may be engineered to include one or more modifications (such as two or more, three or more, four or more, five or more, or even more modifications) that provide for the production of THP. In some cases, a modification is a genetic modification, such as a mutation, addition, or deletion of a gene or fragment thereof, or transcription regulation of a gene or fragment thereof. As used herein, the term "mutation" refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence or motif. The mutation may be incorporated as a directed mutation to the native gene at the original locus. In some cases, the mutation may be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy on an episomal vector such as a 2p or centromeric plasmid. In certain instances, the substrate inhibited copy of the enzyme is under the native cell transcriptional regulation. In some instances, the substrate inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter. In some examples, the object of one or more modifications may be a native gene. In some examples, the object of one or more modifications may be a non-native gene. In some examples, a non-native gene may be inserted into a host cell. In further examples, a non-native gene may be altered by one or more modifications prior to being inserted into a host cell.
An engineered host cell may overproduce one or more products. By overproduce is meant that the cell has an improved or increased production of a product relative to a control cell (e.g., an unmodified cell). By improved or increased production is meant both the production of some amount of the THP product where the control has no THP product production, as well as an increase of about 10% or more, such as about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 80% or more, about 100% or more, such as 2-fold or more, such as 5-fold or more, including 10-fold or more in situations where the control has some THP product production.
As summarized above, THP-producing cells of embodiments of the invention include an engineered THP-biosynthetic pathway. By engineered THP-biosynthetic pathway is meant a metabolic pathway that produces THP as a product and includes one or more enzymatic activities exogenous to the host cell, i.e., one or more heterologous coding sequences. The one or more exogeneous enzymatic activities may be naturally occurring, e.g., wild type, or synthetic, e.g., mutant. The THP-biosynthetic pathway may vary, where examples of THP-biosynthetic pathways that may be present cells of the invention include those that synthesize THP via the NH route involving norreticuline and the NCH3 route involving reticuline. In some instances, the engineered THP-biosynthetic pathway is a norreticuline mediated pathway (i.e., a pathway that synthesizes THP via the NH route).
Where the engineered THP-biosynthetic pathway is a norreticuline mediated pathway, in some embodiments, the host cell includes one or more heterologous coding sequences encoding at least one enzyme involved in the norreticuline mediated pathway, such as two or more heterologous coding sequences encoding two or more enzymes involved in the norreticuline mediated pathway. In some such embodiments, the non-plant cell includes one or more heterologous coding sequences encoding at least one enzyme involved in conversion of coclaurine to THP in the norreticuline mediated pathway, such as the conversion of coclaurine to 3'-OH Coclaurine. In some such instances, the pathway includes a heterologous coding sequence for a N-methylcoclaurine hydroxylase (NMCH). When present, the heterologous coding sequence for NMCH may be a wild-type or mutant NMCH coding sequence from any convenient source, e.g., Eschscholzia californica, Papaver somniferum, Papaver bracteatum, Coptis japonica, and the like. In some instances, the NMCH coding sequence encodes a mutant NMCH that is capable of accepting the non-native substrate (S)-coclaurine to produce (S)-3’-hydroxy-coclaurine at a higher concentration as compared to the wild-type enzyme, such as 5-fold or higher concentration, 10-fold or higher concentration, 25-fold or higher concentration and in some instances 40-fold or higher concentration. Specific mutant or variant NMCH enzymes that may be employed in embodiments of the invention include, but are not limited to: EcNMCHL203S, PsNMCHL203S, PbNMCHL203S, C/NMCH12035, and the like.
In some instances, the THP-biosynthetic pathway includes at least one enzyme having an activity that is capable of O-methylating norreticuline at the 7' and 3' positions to produce THP, such as first and second O-methylating enzymes capable of O-methylating norreticuline at the 7' and 3' positions, respectively, to produce THP. In some such embodiments, the first O-methylating enzyme is capable of O-methylating norreticuline at the 3' position, such as an O-methyl transferase (OMT). Any convenient OMT may be employed, where examples of suitable OMTs include, but are not limited to scoulerine 9-0- methyltransferases and the like. In some such instances, the pathway includes a heterologous coding sequence for a scoulerine 9-0-methyltransferases (S90MT). When present, the heterologous coding sequence for S90MT may be a wild-type or mutant S90MT coding sequence from any convenient source, e.g., Thalictrum flavum, Papaver somniferum, Papaver bracteatum, Coptis japonica, Coptis chinensis, and the like. In some instances, the S90MT coding sequence encodes a mutant S90MT that is capable of accepting the non-native substrates, e.g., norreticuline to produce norcodamine and/or norlaudanine to produce THP, to ultimately produce THP at a higher concentration as compared to a suitable control, such as 2-fold or higher concentration, e.g., 3-fold or higher concentration. Specific mutant or variant S90MT enzymes that may be employed in embodiments of the invention include, but are not limited to: TfS9OMTOPT, TfS9OMTF296L, T/S9OMTT83A C98P A"9L 77ggQMTT83A C98P.A I I9LV28 I y-^ggQ|^|-|-T83A,C98P,A1 19L,V281 I,F296L 77S9QMTT83A,C98P,A119L,V281 l,F296L,N309T j-^ggQ|\y]-|-T83A,C98P,A119L.S160V, V281 l,F296L,N309T |j^Q
As reviewed above, the THP biosynthetic pathway may include a second O- methylating enzyme, where the second O-methylating enzyme is capable of O-methylating norreticuline at the 7' position. In some instances, the second O-methylating enzyme is a norreticuline 7-O-methyltransferase (N7OMT). In some such instances, the pathway includes a heterologous coding sequence for a N7OMT). When present, the heterologous coding sequence for N70MT may be a wild-type or mutant N7OMT coding sequence from any convenient source, e.g., Glaucium flavum, Papaver somniferum, Papaver armeniacum, Eschscholzia californica, and the like.
In some cases, one or more (such as two or more, three or more, or four or more) additional modifications may be present, where such modifications may be selected from: a substrate inhibition alleviating mutation in a biosynthetic enzyme gene; a product inhibition alleviating mutation in a biosynthetic enzyme gene; a cofactor recovery promoting mechanism; a feedback inhibition alleviating mutation in a biosynthetic enzyme gene; and a transcriptional modulation modification of a biosynthetic enzyme gene; an inactivating mutation in an enzyme gene. Further details regarding such modifications may be found in Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
In some instances, the engineered host cells are cells that include one or more substrate inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell). In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term “substrate inhibition alleviating mutation” refers to a mutation that alleviates a substrate inhibition control mechanism of the cell.
A mutation that alleviates substrate inhibition reduces the inhibition of a regulated enzyme in the cell of interest relative to a control cell and provides for an increased level of the regulated compound or a downstream biosynthetic product thereof. In some cases, by alleviating inhibition of the regulated enzyme is meant that the IC5o of inhibition is increased by 2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or even more. By increased level is meant a level that is 110% or more of that of the regulated compound in a control cell or a downstream product thereof, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more or even more of the regulated compound in the engineered host cell or a downstream product thereof.
A variety of substrate inhibition control mechanisms and biosynthetic enzymes in the engineered host cell that are directed to regulation of levels of THP may be targeted for substrate inhibition alleviation. The engineered host cell may include one or more substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes. The one or more mutations may be located in any convenient biosynthetic enzyme genes where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the engineered host cell may include one or more substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
Any convenient numbers and types of mutations may be utilized to alleviate a substrate inhibition control mechanism. In certain embodiments, the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more substrate inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 substrate inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell.
In some instances, the engineered host cells are cells that include one or more cofactor recovery promoting mechanisms (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell). In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term “cofactor recovery promoting mechanism” refers to a mechanism that promotes a cofactor recovery control mechanism of the cell. In some examples, the one or more cofactors of interest for recovery include but are not limited to S-adenosyl methionine, nicotinamide adenine dinucleotide phosphate, nicotinamide adenine dinucleotide, tetrahydrobiopterin, and flavin adenine dinucleotide.
A variety of cofactor recovery control mechanisms and biosynthetic enzymes in the engineered host cell that are directed to regulation of levels of THP may be targeted for cofactor recovery promotion. The engineered host cell may include one or more cofactor recovery promoting mechanism in one or more biosynthetic enzyme genes. In some examples, the engineered host cell may include one or more cofactor recovery promoting mechanisms in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
Any convenient numbers and types of mechanisms may be utilized to promote a cofactor recovery control mechanism. In certain embodiments, the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more cofactor recovery promoting mechanisms such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 cofactor recovery promoting mechanisms in one or more biosynthetic enzyme genes within the engineered host cell.
In some instances, the engineered host cells are cells that include one or more product inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell). In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term “product inhibition alleviating mutation” refers to a mutation that alleviates a short term and/or long term product inhibition control mechanism of an engineered host cell. Short term product inhibition is a control mechanism of the cell in which there is competitive binding at a cosubstrate binding site. Long term product inhibition is a control mechanism of the cell in which there is irreversible binding of a compound away from a desired pathway.
A mutation that alleviates product inhibition reduces the inhibition of a regulated enzyme in the cell of interest relative to a control cell and provides for an increased level of the regulated compound or a downstream biosynthetic product thereof. In some cases, by alleviating inhibition of the regulated enzyme is meant that the IC5o of inhibition is increased by 2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300-fold or more, 1000-fold or more, or even more. By increased level is meant a level that is 110% or more of that of the regulated compound in a control cell or a downstream product thereof, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, such as at least 3-fold or more, at least 5-fold or more, at least 10-fold or more or even more of the regulated compound in the engineered host cell or a downstream product thereof.
A variety of product inhibition control mechanisms and biosynthetic enzymes in the engineered host cell that are directed to regulation of levels of THP may be targeted for product inhibition alleviation. The engineered host cell may include one or more product inhibition alleviating mutations in one or more biosynthetic enzyme genes. The mutation may be located in any convenient biosynthetic enzyme genes where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the engineered host cell includes one or more product inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
Any convenient numbers and types of mutations may be utilized to alleviate a product inhibition control mechanism. In certain embodiments, the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more product inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 product inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell.
In some instances, the engineered host cells are cells that include one or more feedback inhibition alleviating mutations (such as two or more, three or more, four or more, five or more, or even more) in one or more biosynthetic enzyme genes of the cell. In some cases, the one or more biosynthetic enzyme genes are native to the cell (e.g., is present in an unmodified cell). Additionally or alternatively, in some examples the one or more biosynthetic enzyme genes are non-native to the cell. As used herein, the term “feedback inhibition alleviating mutation” refers to a mutation that alleviates a feedback inhibition control mechanism of an engineered host cell. Feedback inhibition is a control mechanism of the cell in which an enzyme in the synthetic pathway of a regulated compound is inhibited when that compound has accumulated to a certain level, thereby balancing the amount of the compound in the cell. A mutation that alleviates feedback inhibition reduces the inhibition of a regulated enzyme in the engineered host cell relative to a control cell. In this way, the engineered host cell provides for an increased level of the regulated compound or a downstream biosynthetic product thereof. In some cases, by alleviating inhibition of the regulated enzyme is meant that the IC5o of inhibition is increased by 2-fold or more, such as by 3-fold or more, 5-fold or more, 10-fold or more, 30-fold or more, 100-fold or more, 300- fold or more, 1000-fold or more, or even more. By increased level is meant a level that is 110% or more of that of the regulated compound in a control cell or a downstream product thereof, such as 120% or more, 130% or more, 140% or more, 150% or more, 160% or more, 170% or more, 180% or more, 190% or more, or 200% or more, such as at least 3- fold or more, at least 5-fold or more, at least 10-fold or more or even more of the regulated compound in the host cell or a downstream product thereof.
A variety of feedback inhibition control mechanisms and biosynthetic enzymes that are directed to regulation of levels of BIAs of interest may be targeted for alleviation in the host cell. The host cell may include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes native to the cell. The one or more mutations may be located in any convenient biosynthetic enzyme genes where the biosynthetic enzyme is subject to regulatory control. In some embodiments, the engineered host cell may include one or more feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes such as one of those genes described in Table 1 of Published United States Patent Application Publication No. 2017/0253898; the disclosure of which is herein incorporated by reference.
Any convenient numbers and types of mutations may be utilized to alleviate a feedback inhibition control mechanism. As used herein, the term "mutation" refers to a deletion, insertion, or substitution of an amino acid(s) residue or nucleotide(s) residue relative to a reference sequence or motif. The mutation may be incorporated as a directed mutation to the native gene at the original locus. In some cases, the mutation may be incorporated as an additional copy of the gene introduced as a genetic integration at a separate locus, or as an additional copy on an episomal vector such as a 2g or centromeric plasmid. In certain instances, the feedback inhibited copy of the enzyme is under the native cell transcriptional regulation. In some instances, the feedback inhibited copy of the enzyme is introduced with engineered constitutive or dynamic regulation of protein expression by placing it under the control of a synthetic promoter.
In certain embodiments, the engineered host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more feedback inhibition alleviating mutations, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 feedback inhibition alleviating mutations in one or more biosynthetic enzyme genes within the engineered host cell.
The host cells may include one or more transcriptional modulation modifications (such as two or more, three or more, four or more, five or more, or even more modifications) of one or more biosynthetic enzyme genes of the cell. In some examples, the one or more biosynthetic enzyme genes are native to the cell. In some examples, the one or more biosynthetic enzyme genes are non-native to the cell. Any convenient biosynthetic enzyme genes of the cell may be targeted for transcription modulation. By transcription modulation is meant that the expression of a gene of interest in a modified cell is modulated, e.g., increased or decreased, enhanced or repressed, relative to a control cell (e.g., an unmodified cell). In some cases, transcriptional modulation of the gene of interest includes increasing or enhancing expression. By increasing or enhancing expression is meant that the expression level of the gene of interest is increased by 2-fold or more, such as by 5-fold or more and sometimes by 25-, 50-, or 100-fold or more and in certain embodiments 300- fold or more or higher, as compared to a control, i.e., expression in the same cell not modified (e.g., by using any convenient gene expression assay). Alternatively, in cases where expression of the gene of interest in a cell is so low that it is undetectable, the expression level of the gene of interest is considered to be increased if expression is increased to a level that is easily detectable. In certain instances, transcriptional modulation of the gene of interest includes decreasing or repressing expression. By decreasing or repressing expression is meant that the expression level of the gene of interest is decreased by 2-fold or more, such as by 5-fold or more and sometimes by 25-, 50-, or 100- fold or more and in certain embodiments 300-fold or more or higher, as compared to a control. In some cases, expression is decreased to a level that is undetectable.
Modifications of host cell processes of interest that may be adapted for use in the subject host cells are described in U.S. Patent No. 9,5434,241 , the disclosure of which is herein incorporated by reference in its entirety.
In some embodiments, the transcriptional modulation modification may include a substitution of a strong promoter for a native promoter of the one or more biosynthetic enzyme genes or the expression of an additional copy(ies) of the gene or genes under the control of a strong promoter. The promoters driving expression of the genes of interest may be constitutive promoters or inducible promoters, provided that the promoters may be active in the host cells. The genes of interest may be expressed from their native promoters. Additionally or alternatively, the genes of interest may be expressed from non-native promoters. Although not a requirement, such promoters may be medium to high strength in the host in which they are used. Promoters may be regulated or constitutive. In some embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, may be used. There are numerous suitable promoters, examples of which include promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding fructose biphosphate aldolase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase) (Bitter G. A., Meth. Enzymol. 152:673 684 (1987)). Other strong promoters of interest include, but are not limited to, the ADHI promoter of baker's yeast (Ruohonen L., et al, J. Biotechnol. 39:193 203 (1995)), the phosphate-starvation induced promoters such as the PHO5 promoter of yeast (Hinnen, A., et al, in Yeast Genetic Engineering, Barr, P. J., et al. eds, Butterworths (1989), the alkaline phosphatase promoter from B. licheniformis (Lee. J. W. K., et al., J. Gen. Microbiol. 137:1127 1133 (1991 )), GPD1 , and TEF1 . Yeast promoters of interest include, but are not limited to, inducible promoters such as Gall -10, Gall , GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3- phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation f actor- 1 -alpha promoter (TEF), cytochrome c-oxidase promoter (CYC1 ), MRP7 promoter, etc. In some instances, the strong promoter is GPD1 . In certain instances, the strong promoter is TEF1 . Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones are also known and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE), see e.g., those promoters described in U.S. Pat. No. 7,045,290. Vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes of interest. It is understood that any convenient promoters specific to the host cell may be selected, e.g., E. coli. In some cases, promoter selection may be used to optimize transcription, and hence, enzyme levels to maximize production while minimizing energy resources.
The engineered host cells may include one or more inactivating mutations to an enzyme of the cell (such as two or more, three or more, four or more, five or more, or even more). The inclusion of one or more inactivating mutations may modify the flux of a synthetic pathway of an engineered host cell to increase the levels of a BIA of interest or a desirable enzyme or precursor leading to the same. In some examples, the one or more inactivating mutations are to an enzyme native to the cell. Additionally or alternatively, the one or more inactivating mutations are to an enzyme non-native to the cell. As used herein, by “inactivating mutation” is meant one or more mutations to a gene or regulatory DNA sequence of the cell, where the mutation(s) inactivates a biological activity of the protein expressed by that gene of interest. In some cases, the gene is native to the cell. In some instances, the gene encodes an enzyme that is inactivated and is part of or connected to the synthetic pathway of a BIA of interest produced by the host cell. In some instances, an inactivating mutation is located in a regulatory DNA sequence that controls a gene of interest. In certain cases, the inactivating mutation is to a promoter of a gene. Any convenient mutations (e.g., as described herein) may be utilized to inactivate a gene or regulatory DNA sequence of interest. By “inactivated” or “inactivates” is meant that a biological activity of the protein expressed by the mutated gene is reduced by 10% or more, such as by 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 97% or more, or 99% or more, relative to a control protein expressed by a non-mutated control gene. In some cases, the protein is an enzyme and the inactivating mutation reduces the activity of the enzyme.
In some examples, the engineered host cell includes an inactivating mutation in an enzyme native to the cell. Any convenient enzymes may be targeted for inactivation. Enzymes of interest may include, but are not limited to those enzymes, described in Table 1 of Published United States Patent Application Publication No. 2017/0253898 (the disclosure of which is herein incorporated by reference), whose action in the synthetic pathway of the engineered host cell tends to reduce the levels of THP products.
In some instances, the engineered host cells harbor one or more heterologous coding sequences (such as two or more, three or more, four or more, five or more) which encode activity(ies) that enable the engineered host cells to produce THP, e.g., as described herein. As used herein, the term "heterologous coding sequence" is used to indicate any polynucleotide that codes for, or ultimately codes for, a peptide or protein or its equivalent amino acid sequence, e.g., an enzyme, that is not normally present in the host organism and may be expressed in the host cell under proper conditions. As such, "heterologous coding sequences" includes multiple copies of coding sequences that are normally present in the host cell, such that the cell is expressing additional copies of a coding sequence that are not normally present in the cells. The heterologous coding sequences may be RNA or any type thereof, e.g., mRNA, DNA or any type thereof, e.g., cDNA, or a hybrid of RNA/DNA. Coding sequences of interest include, but are not limited to, full-length transcription units that include such features as the coding sequence, introns, promoter regions, 3'-UTRs, and enhancer regions.
The engineered host cells may also be modified to possess one or more genetic alterations to accommodate the heterologous coding sequences. Alterations of the native host genome include, but are not limited to, modifying the genome to reduce or ablate expression of a specific protein that may interfere with the desired pathway. The presence of such native proteins may rapidly convert one of the intermediates or final products of the pathway into a metabolite or other compound that is not usable in the desired pathway. Thus, if the activity of the native enzyme were reduced or altogether absent, the produced intermediates would be more readily available for incorporation into the desired product. In some instances, where ablation of expression of a protein may be of interest, as in proteins involved in the pleiotropic drug response, including, but not limited to, ATP-binding cassette (ABC) transporters, multidrug resistance (MDR) pumps, e.g., PDR5, SNG2, and associated transcription factors.
The host cells may be modified to include a variety of plant proteins that provide for a desirable activity or property. Any convenient plant proteins related to the synthesis of THP or precursor thereof may be utilized in the engineered host cells, such as enzymes, chaperones, co-factors, and the like. In some cases, the host cell includes a plant chaperone protein. The plant chaperone may facilitate the action of an enzyme of interest in the host cell, thereby providing for an improved production of THP or precursor thereof. Plant chaperones of interest include, but are not limited to, binding immunoglobulin protein (BiP), DnaJ protein, glucose regulated protein (GRP) 94, binding protein (BiP), protein disulphide isomerase (PDI), cyclophilin, and calnexin.
Heterologous coding sequences include but are not limited to sequences that encode enzymes, either wild-type or equivalent sequences, that are normally responsible for the production of noscapinoid products in plants. In some cases, the enzymes for which the heterologous sequences code may be any of the enzymes in the 1 -BIA pathway, and may be from any convenient source. The choice and number of enzymes encoded by the heterologous coding sequences for the particular synthetic pathway may be selected based upon the desired product. In certain embodiments, the host cells of the invention may include 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, or even 15 or more heterologous coding sequences, such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, or 15 heterologous coding sequences.
As used herein, the term "heterologous coding sequences" also includes the coding portion of the peptide or enzyme, i.e., the cDNA or mRNA sequence, of the peptide or enzyme, as well as the coding portion of the full-length transcriptional unit, i.e., the gene including introns and exons, as well as "codon optimized" sequences, truncated sequences or other forms of altered sequences that code for the enzyme or code for its equivalent amino acid sequence, provided that the equivalent amino acid sequence produces a functional protein. Such equivalent amino acid sequences may have a deletion of one or more amino acids, with the deletion being N-terminal, C-terminal, or internal. Truncated forms are envisioned as long as they have the catalytic capability indicated herein. Fusions of two or more enzymes are also envisioned to facilitate the transfer of metabolites in the pathway, provided that catalytic activities are maintained.
Operable fragments, mutants, or truncated forms may be identified by modeling and/or screening. In some cases, this is achieved by deletion of, for example, N-terminal, C- terminal, or internal regions of the protein in a step-wise fashion, followed by analysis of the resulting derivative with regard to its activity for the desired reaction compared to the original sequence. If the derivative in question operates in this capacity, it is considered to constitute an equivalent derivative of the enzyme proper. Any convenient enzyme of interest may be mutated or engineered to provide for a desirable biological activity in the engineered host cell. In some cases, the mutant enzyme is engineered to facilitate the correct folding of the enzyme. In certain instances, the mutant enzyme is engineered to increase a desirable activity or property of the enzyme relative to a non-mutated enzyme. In certain instances, the mutant enzyme is engineered to decrease an undesirable activity or property of the enzyme relative to a non-mutated enzyme. In some embodiments, the cell includes one or more heterologous coding sequences that encode one or more mutant enzymes. Aspects of the invention also relate to heterologous coding sequences that code for amino acid sequences that are equivalent to the native amino acid sequences for the various enzymes. An amino acid sequence that is "equivalent" is defined as an amino acid sequence that is not identical to the specific amino acid sequence, but rather contains at least some amino acid changes (deletions, substitutions, inversions, insertions, etc.) that do not essentially affect the biological activity of the protein as compared to a similar activity of the specific amino acid sequence, when used for a desired purpose. Equivalent sequences are also meant to include those which have been engineered and/or evolved to have properties different from the original amino acid sequence. Mutable properties of interest include catalytic activity, substrate specificity, selectivity, stability, solubility, localization, etc. In certain embodiments, an "equivalent" amino acid sequence contains at least 80%-99% identity at the amino acid level to the specific amino acid sequence, in some cases at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% and more in certain cases, at least 95%, 96%, 97%, 98%, and 99% identity, at the amino acid level. In some cases, the amino acid sequence may be identical but the DNA sequence is altered such as to optimize codon usage for the host organism, for example.
In some instances, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and/or production of THP. Embodiments of the invention include increased production of THP in a host cell through simultaneous expression of multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient methods may be utilized including multiple copies of a heterologous coding sequence for an enzyme in the host cell.
In some examples, the engineered host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies. In certain embodiments, the engineered host cell includes multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc. In some cases, the multiple copies of the heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell. For example, the engineered host cell may include multiple copies of one heterologous coding sequence, where each of the copies is derived from a different source organism. As such, each copy may include some variations in explicit sequences based on inter-species differences of the enzyme of interest that is encoded by the heterologous coding sequence.
Unless otherwise noted, the heterologous coding sequences are as reported in GENBANK. A list of enzymes of interest is disclosed herein as well as in Table 1 of Published United States Patent Application Publication No. US20170253898; the disclosure of which is herein incorporate reference. The host cells of the invention may include any combination of the listed enzymes, from any source. Unless otherwise indicated, Accession numbers disclosed herein refer to GenBank. Some accession numbers refer to the Saccharomyces genome database (SGD), which is available on the world-wide web at www.yeastgenome.org.
In some embodiments, the cell is a norreticuline producing cell. In some such instances, the cell is a modified version of cells described in Published United States Patent Application Publication No. US20170253898; the disclosure of which is herein incorporate reference. In such instances, the cells may be modified to produce norreticuline instead of reticuline, e.g., by removing the PsCNMT activity. In some instances, the cell includes a coding sequence for producing a 6-pyruvoyl tetrahydrobiopterin synthase (PTPS), such as Rattus norvegicus PTPS (RnPTPS). In some instances, the cell includes a coding sequence for producing a sepiapterin reductase (SepR), such as RnSepR. In some instances, the cell includes a coding sequence for producing a pterin carbinolamine dehydratase (PCD), such as F?nPCD. In some instances, the cell includes a coding sequence for producing a quinonoid dihydropteridine reductase (QDHPR), such as RnQDHPR. In some instances, the cell includes a coding sequence for producing a dihydrofolate reductase (DHFR), such as RnDHFR. In some instances, the cell includes a coding sequence for producing a dihydrofolate reductase a tyrsosine hydroxylase (TyrH), such as RnTyrH, including a mutant thereof, e.g., F?nTyrHWR. In some instances, the cell includes a coding sequence for producing a norcoclaurine synthase (NOS), such as Coptis japonica (CyNCS). In some instances, the cell includes a coding sequence for producing a DOPA decarboxylase (DODC), such as form Pseudomonas putida (PpDODC). In some instances, the host cell includes a heterologous coding sequence for a CPR enzyme. Any convenient CPR enzymes may be utilized in the subject host cells. In certain instances, the CPR enzyme is an Arabidopsis thaliana P450 Reductase (ATR), e.g., ATR1 , or CPR enzyme from Papaver somniferum (PsCPR). In some instances, the cell includes a coding sequence for producing a norcoclaurine 6-0-methyltransferase (6OMT), such as ps6OMT. In some instances, the cell includes a coding sequence for producing a 4'-0-methyltransferase (4'OMT), such as ps4'OMT. In some instances, the cell includes a coding sequence for producing a coclaurine-N-methyltransferase (CNMT), such as PsCNMT. In some instances, the cell includes a coding sequence for producing a 3-deoxy-D-arabino-2-heptulosonic acid 7- phosphate synthase (ARO4), such as Aro4pQ166K. In some instances, the cell includes a coding sequence for producing a chorismate mutase (ARO7), such as Aro7pT2261. In some instances, the cell includes a coding sequence for producing a phenylpyruvate decarboxylase (ARO10). In some instances, the cell includes a coding sequence for producing a transketolase (TKL1 ). The cell may produce THP using one or more enzymes that provide for derivatization in the cell. In certain embodiments, the cell includes one or more heterologous coding sequences for one or more enzymes selected from a P450, a halogenase, a glycosylase, a methyltransferase, an acetyltransferase, a short-chain dehydrogenase, a carboxylesterase, and a prenyltransferase.
In some embodiments, the host cell (e.g., a yeast strain) is engineered for selective production of THP, or a precursor thereof, by localizing one or more enzymes to a compartment in the cell. Any convenient compartments or structures of a cell may be targeted for localization of an enzyme of interest. In some embodiments, the cell includes an enzyme that is spatially localized to a compartment in the yeast cell, wherein the compartment is selected from mitochondrion, endoplasmic reticulum (ER), golgi, vacuole, nucleus, plasma membrane, peroxisome, and periplasm. In some instances, an enzyme is localized to the yeast endoplasmic reticulum by fusing an ER targeting sequence to the N- terminus of the protein. In certain cases, an enzyme of interest is spatially localized to the outside of the compartment in the yeast cell. In some instances, an enzyme of interest is spatially localized to the inside of the compartment in the yeast cell.
In some cases, an enzyme may be located in the host cell such that the compound produced by this enzyme spontaneously rearranges, or is converted by another enzyme to a desirable metabolite before reaching a localized enzyme that may convert the compound into an undesirable metabolite. The spatial distance between two enzymes may be selected to prevent one of the enzymes from acting directly on a compound to make an undesirable metabolite, and restrict production of undesirable end products (e.g., an undesirable opioid by-product). In certain embodiments, any of the enzymes described herein, either singularly or together with a second enzyme, may be localized to any convenient compartment in the host cell, including but not limited to, an organelle, endoplasmic reticulum, golgi, vacuole, nucleus, plasma membrane, or the periplasm.
In some embodiments, the host cell includes one or more of the enzymes that include a localization tag. Any convenient localization tags may be utilized. In some cases, the localization tag is a peptidic sequence that is attached at the N-terminal and or C- terminal of the enzyme. Any convenient methods may be utilized for attaching a tag to the enzyme. In some cases, the localization tag is derived from an endogenous yeast protein. Such tags may provide routing to a variety of yeast organelles, including but not limited to, the endoplasmic reticulum (ER), mitochondria (MT), plasma membrane (PM), and vacuole (V). In certain instances, the tag includes or is derived from, a transmembrane domain from within the tail-anchored class of proteins. In some embodiments, the localization tag locates the enzyme on the outside of an organelle. In certain embodiments, the localization tag locates the enzyme on the inside of an organelle. In some instances, the expression of each type of enzyme is increased through additional gene copies (i.e., multiple copies), which increases intermediate accumulation and ultimately THP production. Embodiments of the invention include increased THP production in a host cell through simultaneous expression of multiple species variants of a single or multiple enzymes. In some cases, additional gene copies of a single or multiple enzymes are included in the host cell. Any convenient methods may be utilized including multiple copies of a heterologous coding sequence for an enzyme in the host cell.
In some embodiments, the host cell includes multiple copies of a heterologous coding sequence for an enzyme, such as 2 or more, 3 or more, 4 or more, 5 or more, or even 10 or more copies. In certain embodiments, the host cell include multiple copies of heterologous coding sequences for one or more enzymes, such as multiple copies of two or more, three or more, four or more, etc.
In some cases, the multiple copies of the heterologous coding sequence for an enzyme are derived from two or more different source organisms as compared to the host cell. For example, the host cell may include multiple copies of one heterologous coding sequence, where each of the copies is derived from a different source organism. In certain cases, the copies are derived from P. somniferum and E. californica source organisms. As such, each copy may include some variations in explicit sequences based on inter-species differences of the enzyme of interest that is encoded by the heterologous coding sequence. In some instances, the host cell includes multiple heterologous coding sequences that each encode an enzyme and are each derived from a different source organisms as compared to the host cell. In some embodiments, the host cell includes copies of an enzyme derived from two or more different source organisms as compared to the host cell.
The engineered host cell medium may be sampled and monitored for the production of THP. The THP may be observed and measured using any convenient methods.
Methods of interest include, but are not limited to, LC-MS methods (e.g., as described herein) where a sample of interest is analyzed by comparison with a known amount of a standard compound. Additionally, there are other ways that THP may be observed and/or measured. Examples of alternative ways of observing and/or measuring BIAs include GC- MS, UV-vis spectroscopy, NMR, LC-NMR, LC-UV, TLC, capillary electrophoresis, among others. Identity may be confirmed, e.g., by m/z and MS/MS fragmentation patterns, and quantitation or measurement of the compound may be achieved via LG trace peaks of know retention time and/or EIC MS peak analysis by reference to corresponding LC-MS analysis of a known amount of a standard of the compound.
EXAMPLES OF SEQUENCES Tables 1 -9 provide examples of sequences that may be used within embodiments as disclosed in the present application. Table 10 discloses examples of amino acid residues in S9OMT where mutagenesis improves activity of this enzyme. Table 1. Examples of Sequences for Upstream Enzymes
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Table 2. Examples of Sequence Variants for 60MT
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Table 3. Examples of Sequence Variants for NMCH
Figure imgf000041_0002
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Table 4. Examples of Sequence Variants for CPR
Figure imgf000044_0002
Figure imgf000045_0001
Figure imgf000046_0001
Table 5. Examples of Sequence Variants for 4’0MT
Figure imgf000046_0002
Figure imgf000047_0001
Table 6. Examples of Sequence Variants for N70MT
Figure imgf000047_0002
Figure imgf000048_0001
Table 7. Example of Sequence for DBOX
Figure imgf000048_0002
Figure imgf000049_0001
Table 8. Examples of Sequence Variants for S90MT
Figure imgf000049_0002
Figure imgf000050_0001
Figure imgf000051_0001
Table 9. Examples of Sequences for MDR Transporters
Figure imgf000051_0002
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Table 10. Examples of Amino Acid Residues in S9OMT where mutagenesis improves activity
Figure imgf000055_0001
METHODS
Process Steps
As summarized above, aspects of the invention include methods of preparing THP and products therefrom. As such, aspects of the invention include culturing an engineered host cell under conditions in which the one or more host cell modifications (e.g., as described herein) are functionally expressed such that the cell converts starting compounds of interest into THP. Also provided are methods that include culturing an engineered host cell under conditions suitable for protein production such that one or more heterologous coding sequences are functionally expressed and convert starting compounds of interest into THP. In examples, the method is a method of preparing THP that includes culturing an engineered host cell (e.g., as described herein); adding a starting compound to the cell culture; and recovering the THP from the cell culture.
Fermentation media may contain suitable carbon substrates. The source of carbon suitable to perform the methods of this disclosure may encompass a wide variety of carbon containing substrates. Suitable substrates may include, without limitation, monosaccharides (e.g., glucose, fructose, galactose, xylose), oligosaccharides (e.g., lactose, sucrose, raffinose), polysaccharides (e.g., starch, cellulose), or a combination thereof. In some cases, unpurified mixtures from renewable feedstocks may be used (e.g., cornsteep liquor, sugar beet molasses, barley malt). In some cases, the carbon substrate may be a one-carbon substrate (e.g., methanol, carbon dioxide) or a two-carbon substrate (e.g., ethanol). In other cases, other carbon containing compounds may be utilized, for example, methylamine, glucosamine, and amino acids. Any convenient methods of culturing engineered host cells may be employed for producing the THP. The particular protocol that is employed may vary, e.g., depending on the engineered host cell, the heterologous coding sequences, the enzymes of interest, etc. The cells may be present in any convenient environment, such as an environment in which the cells are capable of expressing one or more functional heterologous enzymes. In some embodiments, the cells are cultured under conditions that are conducive to enzyme expression and with appropriate substrates available to allow production of THP in vivo. In some embodiments, the functional enzymes are extracted from the engineered host for production of THP under in vitro conditions. In some instances, the engineered host cells are placed back into a multicellular host organism. The engineered host cells are in any phase of growth, including, but not limited to, stationary phase and log-growth phase, etc. In addition, the cultures themselves may be continuous cultures or they may be batch cultures.
Cells may be grown in an appropriate fermentation medium at a temperature between 14-40eC. Cells may be grown with shaking at any convenient speed (e.g., 200 rpm). Cells may be grown at a suitable pH. Suitable pH ranges for the fermentation may be between pH 5-9. Fermentations may be performed under aerobic, anaerobic, or microaerobic conditions. Any suitable growth medium may be used. Suitable growth media may include, without limitation, common commercially prepared media such as synthetic defined (SD) minimal media or yeast extract peptone dextrose (YEPD) rich media. Any other rich, defined, or synthetic growth media appropriate to the microorganism may be used.
Cells may be cultured in a vessel of essentially any size and shape. Examples of vessels suitable to perform the methods of this disclosure may include, without limitation, multi-well shake plates, test tubes, flasks (baffled and non-baffled), and bioreactors. The volume of the culture may range from 10 microliters to greater than 10,000 liters.
The addition of agents to the growth media that are known to modulate metabolism in a manner desirable for the production of alkaloids may be included. In a non-limiting example, cyclic adenosine 2’3’-monophosphate may be added to the growth media to modulate catabolite repression.
Any convenient cell culture conditions for a particular cell type may be utilized. In certain embodiments, the host cells that include one or more modifications are cultured under standard or readily optimized conditions, with standard cell culture media and supplements. As one example, standard growth media when selective pressure for plasmid maintenance is not required may contain 20 g/L yeast extract, 10 g/L peptone, and 20 g/L dextrose (YPD). Host cells containing plasmids are grown in synthetic complete (SC) media containing 1 .7 g/L yeast nitrogen base, 5 g/L ammonium sulfate, and 20 g/L dextrose supplemented with the appropriate amino acids required for growth and selection. Alternative carbon sources which may be useful for inducible enzyme expression include, but are not limited to, sucrose, raffinose, and galactose. Cells are grown at any convenient temperature (e.g., 30sC) with shaking at any convenient rate (e.g., 200 rpm) in a vessel, e.g., in test tubes or flasks in volumes ranging from 1-1000 mL, or larger, in the laboratory.
Culture volumes may be scaled up for growth in larger fermentation vessels, for example, as part of an industrial process. The industrial fermentation process may be carried out under closed-batch, fed-batch, or continuous chemostat conditions, or any suitable mode of fermentation. In some cases, the cells may be immobilized on a substrate as whole cell catalysts and subjected to fermentation conditions for alkaloid production.
A batch fermentation is a closed system, in which the composition of the medium is set at the beginning of the fermentation and not altered during the fermentation process. The desired organism(s) are inoculated into the medium at the beginning of the fermentation. In some instances, the batch fermentation is run with alterations made to the system to control factors such as pH and oxygen concentration (but not carbon). In this type of fermentation system, the biomass and metabolite compositions of the system change continuously over the course of the fermentation. Cells typically proceed through a lag phase, then to a log phase (high growth rate), then to a stationary phase (growth rate reduced or halted), and eventually to a death phase (if left untreated).
A continuous fermentation is an open system, in which a defined fermentation medium is added continuously to the bioreactor and an equal amount of fermentation media is continuously removed from the vessel for processing. Continuous fermentation systems are generally operated to maintain steady state growth conditions, such that cell loss due to medium being removed must be balanced by the growth rate in the fermentation. Continuous fermentations are generally operated at conditions where cells are at a constant high cell density. Continuous fermentations allow for the modulation of one or more factors that affect target product concentration and/or cell growth.
The liquid medium may include, but is not limited to, a rich or synthetic defined medium having an additive component described above. Media components may be dissolved in water and sterilized by heat, pressure, filtration, radiation, chemicals, or any combination thereof. Several media components may be prepared separately and sterilized, and then combined in the fermentation vessel. The culture medium may be buffered to aid in maintaining a constant pH throughout the fermentation.
Process parameters including temperature, dissolved oxygen, pH, stirring, aeration rate, and cell density may be monitored or controlled over the course of the fermentation. For example, temperature of a fermentation process may be monitored by a temperature probe immersed in the culture medium. The culture temperature may be controlled at the set point by regulating the jacket temperature. Water may be cooled in an external chiller and then flowed into the bioreactor control tower and circulated to the jacket at the temperature required to maintain the set point temperature in the vessel.
Additionally, a gas flow parameter may be monitored in a fermentation process. For example, gases may be flowed into the medium through a sparger. Gases suitable for the methods of this disclosure may include compressed air, oxygen, and nitrogen. Gas flow may be at a fixed rate or regulated to maintain a dissolved oxygen set point.
The pH of a culture medium may also be monitored. In examples, the pH may be monitored by a pH probe that is immersed in the culture medium inside the vessel. If pH control is in effect, the pH may be adjusted by acid and base pumps which add each solution to the medium at the required rate. The acid solutions used to control pH may be sulfuric acid or hydrochloric acid. The base solutions used to control pH may be sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
Further, dissolved oxygen may be monitored in a culture medium by a dissolved oxygen probe immersed in the culture medium. If dissolved oxygen regulation is in effect, the oxygen level may be adjusted by increasing or decreasing the stirring speed. The dissolved oxygen level may also be adjusted by increasing or decreasing the gas flow rate. The gas may be compressed air, oxygen, or nitrogen.
Stir speed may also be monitored in a fermentation process. In examples, the stirrer motor may drive an agitator. The stirrer speed may be set at a consistent rpm throughout the fermentation or may be regulated dynamically to maintain a set dissolved oxygen level.
Additionally, turbidity may be monitored in a fermentation process. In examples, cell density may be measured using a turbidity probe. Alternatively, cell density may be measured by taking samples from the bioreactor and analyzing them in a spectrophotometer. Further, samples may be removed from the bioreactor at time intervals through a sterile sampling apparatus. The samples may be analyzed for alkaloids produced by the host cells. The samples may also be analyzed for other metabolites and sugars, the depletion of culture medium components, or the density of cells.
In another example, a feed stock parameter may be monitored during a fermentation process. In particular, feed stocks including sugars and other carbon sources, nutrients, and cofactors that may be added into the fermentation using an external pump. Other components may also be added during the fermentation including, without limitation, antifoam, salts, chelating agents, surfactants, and organic liquids.
Any convenient codon optimization techniques for optimizing the expression of heterologous polynucleotides in host cells may be adapted for use in the subject host cells and methods, see e.g., Gustafsson, C. et al. (2004) Trends Biotechnol, 22, 346-353, which is incorporated by reference in its entirety.
The subject method may also include adding a starting compound to the cell culture. Any convenient methods of addition may be adapted for use in the subject methods. The cell culture may be supplemented with a sufficient amount of the starting materials of interest (e.g., as described herein), e.g., a mM to pM amount such as between about 1 -5 mM of a starting compound. It is understood that the amount of starting material added, the timing and rate of addition, the form of material added, etc., may vary according to a variety of factors. The starting material may be added neat or pre-dissolved in a suitable solvent (e.g., cell culture media, water, or an organic solvent). The starting material may be added in concentrated form (e.g., 10x over desired concentration) to minimize dilution of the cell culture medium upon addition. The starting material may be added in one or more batches, or by continuous addition over an extended period of time (e.g., hours or days).
Methods for Isolating Products from the Fermentation Medium
The subject methods may also include recovering the THP from the cell culture. Any convenient methods of separation and isolation (e.g., chromatography methods or precipitation methods) may be adapted for use in the subject methods to recover the THP from the cell culture. Filtration methods may be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) may be used to separate the THP from other soluble components of the cell culture. In some cases, extraction methods (e.g., liquid extraction, pH based purification, solid phase extraction, affinity chromatography, ion exchange, etc.) may be used to separate the THP from other components of the cell culture.
The produced THP may be isolated from the fermentation medium using methods known in the art. A number of recovery steps may be performed immediately after (or in some instances, during) the fermentation for initial recovery of the desired product. Through these steps, the THP may be separated from the cells, cellular debris and waste, and other nutrients, sugars, and organic molecules may remain in the spent culture medium. This process may be used to yield a product enriched with THP.
In an example, a product stream having THP is formed by providing engineered yeast cells and a feedstock including nutrients and water to a batch reactor. In particular, the engineered yeast cells may be subjected to fermentation by incubating the engineered yeast cells for a time period of at least about 5 minutes to produce a solution comprising the THP product and cellular material. Once the engineered yeast cells have been subjected to fermentation, at least one separation unit may be used to separate the THP from the cellular material to provide the product stream comprising the THP product. In particular, the product stream may include the THP as well as additional components, such as a clarified yeast culture medium. Different methods may be used to remove cells from a bioreactor medium that include THP. In examples, cells may be removed by sedimentation over time. This process of sedimentation may be accelerated by chilling or by the addition of fining agents such as silica. The spent culture medium may then be siphoned from the top of the reactor or the cells may be decanted from the base of the reactor. Alternatively, cells may be removed by filtration through a filter, a membrane, or other porous material. Cells may also be removed by centrifugation, for example, by continuous flow centrifugation or by using a continuous extractor.
If THP is present inside the cells, the cells may be permeabilized or lysed and the cell debris may be removed by any of the methods described above. Agents used to permeabilize the cells may include, without limitation, organic solvents (e.g., DMSO) or salts (e.g., lithium acetate). Methods to lyse the cells may include the addition of surfactants such as sodium dodecyl sulfate, or mechanical disruption by bead milling or sonication.
THP may be extracted from the clarified spent culture medium through liquid-liquid extraction by the addition of an organic liquid that is immiscible with the aqueous culture medium. In examples, the use of liquid-liquid extraction may be used in addition to other processing steps. Examples of suitable organic liquids include, but are not limited to, isopropyl myristate, ethyl acetate, chloroform, butyl acetate, methylisobutyl ketone, methyl oleate, toluene, oleyl alcohol, ethyl butyrate. The organic liquid may be added to as little as 10% or as much as 100% of the volume of aqueous medium.
In some cases, the organic liquid may be added at the start of the fermentation or at any time during the fermentation. This process of extractive fermentation may increase the yield of THP from the host cells by continuously removing THP to the organic phase.
Agitation may cause the organic phase to form an emulsion with the aqueous culture medium. Methods to encourage the separation of the two phases into distinct layers may include, without limitation, the addition of a demulsifier or a nucleating agent, or an adjustment of the pH. The emulsion may also be centrifuged to separate the two phases, for example, by continuous conical plate centrifugation.
Alternatively, the organic phase may be isolated from the aqueous culture medium so that it may be physically removed after extraction. For example, the solvent may be encapsulated in a membrane.
In examples, THP may be extracted from a fermentation medium using adsorption methods. In examples, THP may be extracted from clarified spent culture medium by the addition of a resin such as Amberlite® XAD4 or another agent that removes THP by adsorption. The THP may then be released from the resin using an organic solvent. Examples of suitable organic solvents include, but are not limited to, methanol, ethanol, ethyl acetate, or acetone. THP may also be extracted from a fermentation medium using filtration. Under certain conditions, the THP may form a crystalline-like precipitate in the bioreactor. This precipitate may be removed directly by filtration through a filter, membrane, or other porous material. The precipitate may also be collected by centrifugation and/or decantation.
The extraction methods described above may be carried out either in situ (in the bioreactor) or ex situ (e.g., in an external loop through which media flows out of the bioreactor and contacts the extraction agent, then is recirculated back into the vessel). Alternatively, the extraction methods may be performed after the fermentation is terminated using the clarified medium removed from the bioreactor vessel.
The subject methods may also include recovering the THP from the cell culture. Any convenient methods of separation and isolation (e.g., organic solvent extraction under basic condition, solid phase extraction, chromatography methods, or precipitation methods) may be adapted for use in the subject methods to recover the THP from the cell culture. Filtration methods may be used to separate soluble from insoluble fractions of the cell culture. In some cases, liquid chromatography methods (e.g., reverse phase HPLC, size exclusion, normal phase chromatography) are used to separate the THP from other soluble components of the cell culture.
Also included are methods of engineering host cells for the purpose of producing THP. Inserting DNA into host cells may be achieved using any convenient methods. The methods are used to insert the heterologous coding sequences into the host cells such that the host cells functionally express the enzymes and convert starting compounds of interest into THP or precursors thereof.
In some embodiments, the cell includes one or more promoters for the one or more of the heterologous coding sequences (e.g., as described herein). Any convenient promoters may be utilized in the subject host cells and methods. The promoters driving expression of the heterologous coding sequences may be constitutive promoters or inducible promoters, provided that the promoters can be active in the host cells. The heterologous coding sequences may be expressed from their native promoters, or nonnative promoters may be used. Such promoters may be low to high strength in the host in which they are used. In some embodiments, the cell includes one or more strong promoters. Promoters may be regulated or constitutive. In certain embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. Promoters of interest include but are not limited to, promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding fructose bisphosphate aldolase) or GAPDH promoter from yeast S. cerevisiae (coding for glyceraldehyde-phosphate dehydrogenase), the ADH1 promoter of baker's yeast, the phosphate-starvation induced promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from B. licheniformis, yeast inducible promoters such as Gall -10, Gall , GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH1), translation-elongation factor-1 -a promoter (TEF), cytochrome c-oxidase promoter (CYC1 ), MRP7 promoter, GAL1 , HXT7, PGK1 , TPI1 , PYK1 , TEF1 , etc. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE). These and other examples are described U.S. Pat. No. 7,045,290, which is incorporated by reference, including the references cited therein. Additional vectors containing constitutive or inducible promoters such as a factor, alcohol oxidase, and PGH may be used. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes. Any convenient appropriate promoters may be selected for the host cell, e.g., E. coli. One can also use promoter selection to optimize transcript, and hence, enzyme levels to maximize production while minimizing energy resources.
In some instances, the cell includes one or more strong promoters selected from HXT7, ADH1 , PGK1 , TPI1 , PYK1 , and TEF1 . In certain embodiments, the cell includes one or more heterologous coding sequences that encode CYP82Y1 or a CYP82Y1 mutant and includes a HXT7 promoter. In certain cases, the cell produces 1 -hydroxy-N-methylcanadine. In certain instances, the cell produces 1-hydroxycanadine. In some embodiments, the cell includes one or more heterologous coding sequences that encode CYP82X2 or a CYP82X2 mutant and includes a HXT7 promoter. In certain instances, the cell produces 1 ,13- dihydroxy-N-methylcanadine. In some instances, the cell includes one or more heterologous coding sequences that encode CYP82X2 or a CYP82X2 mutant and includes one or more promoters selected from PGK1 and GPD. In certain embodiments, the cell produces N- methyl-ophiocarpine. In some instances, the cell includes one or more heterologous coding sequences that encode CYP82X1 or a CYP82X1 mutant and includes a HXT7 promoter. In certain embodiments, the cell produces 4’-0-desmethyl-3-0-acetylpapaveroxine.
Any convenient vectors may be utilized in the subject host cells and methods. Vectors of interest include vectors for use in yeast and other cells. Yeast vectors can be broken up into 4 general categories: integrative vectors (Yip), autonomously replicating high copy-number vectors (YEp), autonomously replicating low copy-number vectors (YCp) and vectors for cloning large fragments (YACs). Vector DNA can be introduced into prokaryotic or eukaryotic cells via any convenient transformation or transfection techniques.
Methods for Purifying Products from Alkaloid-Enriched Solutions Subsequent purification steps may involve treating the post-fermentation solution enriched with THP using methods known in the art to recover individual product species of interest to high purity.
In one example, THP extracted in an organic phase may be transferred to an aqueous solution. In some cases, the organic solvent may be evaporated by heat and/or vacuum, and the resulting powder may be dissolved in an aqueous solution of suitable pH. In a further example, the THP may be extracted from the organic phase by addition of an aqueous solution at a suitable pH that promotes extraction of the THP into the aqueous phase. The aqueous phase may then be removed by decantation, centrifugation, or another method.
The THP-containing solution may be further treated to remove metals, for example, by treating with a suitable chelating agent. The THP-containing solution may be further treated to remove other impurities, such as proteins and DNA, by precipitation. In one example, the THP-containing solution is treated with an appropriate precipitation agent such as ethanol, methanol, acetone, or isopropanol. In an alternative example, DNA and protein may be removed by dialysis or by other methods of size exclusion that separate the smaller alkaloids from contaminating biological macromolecules.
In further examples, the solution containing THP may be extracted to high purity by continuous cross-flow filtration using methods known in the art.
If the solution contains a mixture of THP and other products, e.g., noscapinoid products, it may be subjected to acid-base treatment to yield THP using methods known in the art. In this process, the pH of the aqueous solution is adjusted to precipitate THP.
For high purity, small-scale preparations, the THP may be purified in a single step by liquid chromatography.
Methods of Engineering Host Cells
Also included are methods of engineering host cells for the purpose of producing THP. Inserting DNA into host cells may be achieved using any convenient methods. The methods are used to insert the heterologous coding sequences into the engineered host cells such that the host cells functionally express the enzymes and convert starting compounds of interest into THP.
Any convenient promoters may be utilized in the subject engineered host cells and methods. The promoters driving expression of the heterologous coding sequences may be constitutive promoters or inducible promoters, provided that the promoters are active in the engineered host cells. The heterologous coding sequences may be expressed from their native promoters, or non-native promoters may be used. Such promoters may be low to high strength in the host in which they are used. Promoters may be regulated or constitutive. In certain embodiments, promoters that are not glucose repressed, or repressed only mildly by the presence of glucose in the culture medium, are used. Promoters of interest include but are not limited to, promoters of glycolytic genes such as the promoter of the B. subtilis tsr gene (encoding the promoter region of the fructose bisphosphate aldolase gene) or the promoter from yeast S. cerevisiae gene coding for glyceraldehyde 3-phosphate dehydrogenase (GPD, GAPDH, or TDH3), the ADH1 promoter of baker's yeast, the phosphate-starvation induced promoters such as the PHO5 promoter of yeast, the alkaline phosphatase promoter from B. licheniformis, yeast inducible promoters such as Gall -10, Gall , GalL, GalS, repressible promoter Met25, tetO, and constitutive promoters such as glyceraldehyde 3-phosphate dehydrogenase promoter (GPD), alcohol dehydrogenase promoter (ADH), translation-elongation factor-1 -a promoter (TEF), cytochrome c-oxidase promoter (CYC1 ), MRP7 promoter, etc. Autonomously replicating yeast expression vectors containing promoters inducible by hormones such as glucocorticoids, steroids, and thyroid hormones may also be used and include, but are not limited to, the glucorticoid responsive element (GRE) and thyroid hormone responsive element (TRE). These and other examples are described U.S. Pat. No. 7,045,290, which is incorporated by reference, including the references cited therein. Additional vectors containing constitutive or inducible promoters such as a factor, alcohol oxidase, and PGH may be used. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of genes. Any convenient appropriate promoters may be selected for the host cell, e.g., E coli. One may also use promoter selection to optimize transcript, and hence, enzyme levels to maximize production while minimizing energy resources.
Any convenient vectors may be utilized in the subject engineered host cells and methods. Vectors of interest include vectors for use in yeast and other cells. The types of yeast vectors may be broken up into 4 general categories: integrative vectors (Yip), autonomously replicating high copy-number vectors (YEp or 2p plasmids), autonomously replicating low copy-number vectors (YCp or centromeric plasmids) and vectors for cloning large fragments (YACs). Vector DNA is introduced into prokaryotic or eukaryotic cells via any convenient transformation or transfection techniques. DNA of another source (e.g. PCR-generated double stranded DNA product, or synthesized double stranded or single stranded oligonucleotides) may be used to engineer the yeast by integration into the genome. Any single transformation event may include one or several nucleic acids (vectors, double stranded or single stranded DNA fragments) to genetically modify the host cell.
Production of Products from THP
Aspects of the invention further include producing a product from the THP produced by host cells of the invention. Products of interest may vary, and include any desired product that may be produced from THP. In some instances, methods of the invention produce Active Pharmaceutical Ingredients (APIs) from THP produced by a THP-producing cells of the invention. Such APIs may vary, where examples of such APIs include atracurium, cisatracurium and papaverine.
In some instances, the methods include producing papaverine from THP. In such instances, the methods may include oxidizing the THP product, e.g., produced by THP- producing cells of the invention (such as described above) to produce papaverine. Oxidizing the THP to produce papaverine may be accomplished using any convenient protocol. In some instances, a chemical oxidizer may be employed to produce papaverine from THP. Chemical oxidizers of interest include, but are not limited to: hydrogen peroxide, potassium persulfate, fremy’s salt, azodicarboxamide, periodic acid, chloramine T trihydrate, peracetic acid, cumene hydroperoxide, dess-martin periodinane, luperox, TEMPO, 3- chloroperbenzoic acid, ammonium persulfate, sodium hypochlorite, cobalt (II) phthalocyanine, cb22’bpdcruthenium hydrate, and the like. In such instances, oxidation may be carried out under appropriate conditions, e.g., a pH ranging from 6 to 11 , such as 7 to 10. Instead of employing a chemical oxidizer, where desired an enzymatic oxidizer may be employed to produce papaverine from THP, where enzymatic oxidizers include dihydrobenzophenanthridine oxidase (DBOX), tetrahydropapaverine oxidase (TPOX), and the like. While the chemical oxidation method of producing papaverine from THP is described herein in terms of THP produced by THP-producing cells of the invention, it is not so limited, as the method may be employed with any THP starting material.
In some instances, the methods include producing atracurium from THP. Any convenient protocol for producing atracurium from THP may be employed, such as but not limited to the protocols described in U.S. Patent No. 5,684,154; 8,354,537; and 8,461 ,338, the disclosures of which are herein incorporated by reference. In some instances, the methods include producing cisatracurium from THP. Any convenient protocol for producing cisatracurium from THP may be employed, such as but not limited to the protocols described in U.S. Patent No. 8,293,912, the disclosure of which is herein incorporated by reference.
PHARMACEUTICAL FORMULATIONS
Aspects of the invention further include pharmaceutical compositions, e.g., that include active agents produced from THP, e.g., as described above.
A pharmaceutical formulation may include an API (e.g., as described above) or a pharmaceutically acceptable salt thereof, and one or mere of pharmaceutically acceptable carriers or excipients. In some instances, a pharmaceutical formulation includes an amount of an API effective to treat a disease; and an excipient. The pharmaceutical formulation may be formulated for administration by any suitable means. In certain embodiments, the composition is formulated for administration orally, intradermally, intramuscularly, parenterally, intravenously, intra-arterially, intracranially, subcutaneously, intraorbitally, intraventricularly, intraspinally, intraperitoneally, or intranasally.
The pharmaceutical formulations or compositions can be formulated into various dosage forms, including tablets, powders, fine granules, granules, dry syrups, capsules, liquid compositions, etc. In some instances, the pharmaceutical formulation is a capsule or tablet. In some instances, the pharmaceutic formulation is a parenteral formulation. In some instances, the pharmaceutical formulation is an intraperitoneal formulation.
Additives and diluents normally utilized in the pharmaceutical arts can optionally be added to the pharmaceutical formulation. These include thickening, granulating, dispersing, flavoring, sweetening, coloring, and stabilizing agents, including pH stabilizers, other excipients, anti-oxidants (e.g., tocopherol, BHA, BHT, TBHQ, tocopherol acetate, ascorbyl palmitate, ascorbic acid propyl gallate, and the like), preservatives (e.g., parabens), and the like. Exemplary preservatives include, but are not limited to, benzylalcohol, ethylalcohol, benzalkonium chloride, phenol, chlorobutanol, and the like. Some useful antioxidants provide oxygen or peroxide inhibiting agents for the formulation and include, but are not limited to, butylated hydroxytoluene, butylhydroxyanisole, propyl gallate, ascorbic acid palmitate, a-tocopherol, and the like. Thickening agents, such as lecithin, hydroxypropylcellulose, aluminum stearate, and the like, may improve the texture of the formulation.
A container for holding the formulation may be configured to hold any suitable amount or volume of the formulation or composition. In some cases, the size of the container may depend on the volume formulation to be held in the container. In certain embodiments, the container may be configured to hold an amount of composition ranging from 0.1 mg to 1000 mg, such as from 0.1 mg to 900 mg, such as from 0.1 mg to 800 mg, such as from 0.1 mg to 700 mg, such as from 0.1 mg to 600 mg, such as from 0.1 mg to 500 mg, such as from 0.1 mg to 400 mg, or 0.1 mg to 300 mg, or 0.1 mg to 200 mg, or 0.1 mg to 100 mg, 0.1 mg to 90 mg, or 0.1 mg to 80 mg, or 0.1 mg to 70 mg, or 0.1 mg to 60 mg, or 0.1 mg to 50 mg, or 0.1 mg to 40 mg, or 0.1 mg to 30 mg, or 0.1 mg to 25 mg, or 0.1 mg to 20 mg, or 0.1 mg to 15 mg, or 0.1 mg to 10 mg, or 0.1 mg to 5 mg, or 0.1 mg to 1 mg, or 0.1 mg to 0.5 mg. In certain embodiments, the container is configured to hold an amount of composition ranging from 0.1 g to 10 g, or 0.1 g to 5 g, or 0.1 g to 1 g, or 0.1 g to 0.5 g. In certain instances, the container is configured to hold a volume (e.g., a volume of a composition) ranging from 0.1 ml to 200 ml. For instance, the container may be configured to hold a volume (e.g., a volume of a liquid) ranging from 0.1 ml to 1000 ml, such as from 0.1 ml to 900 ml, or 0.1 ml to 800 ml, or 0.1 ml to 700 ml, or 0.1 ml to 600 ml, or 0.1 ml to 500 ml, or 0.1 ml to 400 ml, or 0.1 ml to 300 ml, or 0.1 ml to 200 ml, or 0.1 ml to 100 ml, or 0.1 ml to 50 ml, or 0.1 ml to 25 ml, or 0.1 ml to 10 ml, or 0.1 ml to 5 ml, or 0.1 ml to 1 ml, or 0.1 ml to 0.5 ml.
The shape of the container may also vary. In certain cases, the container may be configured in a shape that is compatible with the assay and/or the method or other devices used to perform the assay. For instance, the container may be configured in a shape of typical laboratory equipment used to perform the assay or in a shape that is compatible with other devices used to perform the assay. In some instances, the container is a liquid container. In some embodiments, the liquid container is a vial or a test tube. In certain cases, the liquid container is a vial. In certain cases, the liquid container is a test tube. In some instances, the container is a blister pack.
As described above, embodiments of the container can be compatible with the formulation or composition held therein. Examples of suitable materials for the containers include, but are not limited to, glass and plastic. For example, the container may be composed of glass, such as, but not limited to, silicate glass, borosilicate glass, sodium borosilicate glass (e.g., PYREX™), fused quartz glass, fused silica glass, and the like. Other examples of suitable materials for the containers include plastics, such as, but not limited to, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), perfluoroethers (PFE), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polyethylene terephthalate (PET), polyethylene (PE), polyetheretherketone (PEEK), and the like.
In some embodiments, the container may be sealed. That is, the container may include a seal that substantially prevents the contents of the container from exiting the container. The seal of the container may also substantially prevent other substances from entering the container. For example, the seal may be a water-tight seal that substantially prevents liquids from entering or exiting the container, or may be an air-tight seal that substantially prevents gases from entering or exiting the container. In some instances, the seal is a removable or breakable seal, such that the contents of the container may be exposed to the surrounding environment when so desired, e.g., if it is desired to remove a portion of the contents of the container. In some instances, the seal is made of a resilient material to provide a barrier (e.g., a water-tight and/or air-tight seal) for retaining a sample in the container. Particular types of seals include, but are not limited to, films, such as polymer films, caps, etc., depending on the type of container. Suitable materials for the seal include, for example, rubber or polymer seals, such as, but not limited to, silicone rubber, natural rubber, styrene butadiene rubber, ethylene-propylene copolymers, polychloroprene, polyacrylate, polybutadiene, polyurethane, styrene butadiene, and the like, and combinations thereof. For example, in certain embodiments, the seal is a septum pierceable by a needle, syringe, or cannula. The seal may also provide convenient access to a sample in the container, as well as a protective barrier that overlies the opening of the container. In some instances, the seal is a removable seal, such as a threaded or snap-on cap or other suitable sealing element that can be applied to the opening of the container. For instance, a threaded cap can be screwed over the opening before or after a sample has been added to the container.
UTILITY
The engineered host cells and methods of the invention, e.g., as described above, find use in a variety of applications. Applications of interest include, but are not limited to: research applications and therapeutic applications. Methods of the invention find use in a variety of different applications including any convenient application where the production of THP is desired.
The subject engineered host cells and methods find use in a variety of therapeutic applications. Therapeutic applications of interest include those applications in which the preparation of pharmaceutical products that include an API derivative of THP. The engineered host cells described herein produce THP. The subject host cells may be utilized to produce API end products from the THP, such as atracurium, cisatracurium and papaverine. As such, the subject host cells find use in the supply of therapeutically active products, such as atracurium, cisatracurium and papaverine.
In some instances, the engineered host cells and methods find use in the production of commercial scale amounts of THP. In certain cases, the host cells and methods are utilized in a fermentation facility that would include bioreactors (fermenters) of e.g., 5,000- 200,000 liter capacity allowing for rapid production of THP for therapeutic products. Such applications may include the industrial-scale production of THP from fermentable carbon sources such as cellulose, starch, and free sugars.
The subject engineered host cells and methods find use in a variety of research applications. The subject host cells and methods may be used to analyze the effects of a variety of enzymes on the biosynthetic pathways. In addition, the engineered host cells may be engineered to produce products that find use in testing for bioactivity of interest in as yet unproven therapeutic functions. In some cases, the engineering of host cells to include a variety of heterologous coding sequences that encode for a variety of enzymes elucidates the high yielding biosynthetic pathways towards desired products. In certain cases, research applications include the production of products for therapeutic molecules of interest that may then be further chemically modified or derivatized to desired products or for screening for increased therapeutic activities of interest. In some instances, host cell strains are used to screen for enzyme activities that are of interest in such pathways, which may lead to enzyme discovery via conversion of metabolites produced in these strains. The subject host cells and methods may be used to as a production platform for plant specialized metabolites.
The subject engineered host cells and methods may be used as a production platform for plant specialized metabolites. The subject host cells and methods may be used as a platform for drug library development as well as plant enzyme discovery. For example, the subject engineered host cells and methods may find use in the development of natural product based drug libraries by taking yeast strains producing interesting scaffold molecules, such as protopine, and further functionalizing the compound structure through combinatorial biosynthesis or by chemical means. By producing drug libraries in this way, any potential drug hits are already associated with a production host that is amenable to large-scale culture and production. As another example, these subject engineered host cells and methods may find use in plant enzyme discovery. The subject host cells provide a clean background of defined metabolites to express plant EST libraries to identify new enzyme activities. The subject host cells and methods provide expression methods and culture conditions for the functional expression and increased activity of plant enzymes in yeast.
KITS AND SYSTEMS
Aspects of the invention further include kits and systems, where the kits and systems may include one or more components employed in methods of the invention, e.g., engineered host cells, starting compounds, heterologous coding sequences, vectors, culture medium, etc., as described herein. In some embodiments, the subject kit includes an engineered host cell (e.g., as described herein), and one or more components selected from the following: starting compounds, a heterologous coding sequence and/or a vector including the same, vectors, growth feedstock, components suitable for use in expression systems (e.g., cells, cloning vectors, multiple cloning sites (MCS), bi-directional promoters, an internal ribosome entry site (IRES), etc.), and a culture medium.
Any of the components described herein may be provided in the kits, e.g., host cells including one or more modifications, starting compounds, culture medium, etc. A variety of components suitable for use in making and using heterologous coding sequences, cloning vectors and expression systems may find use in the subject kits. Kits may also include tubes, buffers, etc., and instructions for use. The various reagent components of the kits may be present in separate containers, or some or all of them may be pre-combined into a reagent mixture in a single container, as desired.
Also provided are systems for producing THP, where the systems may include engineered host cells including one or more modifications (e.g., as described herein), starting compounds, culture medium, a fermenter and fermentation equipment, e.g., an apparatus suitable for maintaining growth conditions for the host cells, sampling and monitoring equipment and components, and the like. A variety of components suitable for use in large scale fermentation of yeast cells may find use in the subject systems.
In some cases, the system includes components for the large scale fermentation of engineered host cells, and the monitoring and purification of THP produced by the fermented host cells. In certain embodiments, one or more starting compounds (e.g., as described herein) are added to the system, under conditions by which the engineered host cells in the fermenter produce THP. In some instances, the host cells produce a THP (e.g., as described herein).
In some cases, the system includes processes for monitoring and or analyzing THP produced by the subject host cells. For example, a LC-MS analysis system as described herein, a chromatography system, or any convenient system where the sample may be analyzed and compared to a standard, e.g., as described herein. The fermentation medium may be monitored at any convenient times before and during fermentation by sampling and analysis. When the conversion of starting compounds to THP is complete, the fermentation may be halted and purification of the THP may be done. As such, in some cases, the subject system includes a purification component suitable for purifying THP from the host cell medium into which it is produced. The purification component may include any convenient means that may be used to purify the THP produced by fermentation, including but not limited to, silica chromatography, reverse-phase chromatography, ion exchange chromatography, HIC chromatography, size exclusion chromatography, liquid extraction, and pH extraction methods. In some cases, the subject system provides for the production and isolation of THP fermentation products of interest following the input of one or more starting compounds to the system.
The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.), but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric. EXPERIMENTAL
I. Biosynthesis of Tetrahydropapaverine and Semi-synthesis of Papaverine in Yeast
A. Introduction
Tetrahydropapaverine (THP) and papaverine are plant natural products with clinically significant roles. THP is a precursor in the production of the drugs atracurium and cisatracurium(l). Papaverine is used as an anti-spasmodic during vascular surgery(2). In recent years, metabolic engineering advances have enabled the production of other natural products through heterologous expression of pathway enzymes in yeast(3). Heterologous biosynthesis of THP and papaverine could play a role in ensuring a stable supply of these clinically significant products. The biosynthesis of THP and papaverine have not been achieved to date, in part, because multiple pathway enzymes have not been elucidated(4). Here, we describe the development of an engineered yeast strain for de novo biosynthesis of THP. The production of THP is achieved through the heterologous expression of two novel enzyme variants with activity on non-native substrates. Through protein engineering, we developed a variant of /V-methylcoclaurine hydroxylase with activity on coclaurine enabling de novo norreticuline biosynthesis. Similarly, we developed a variant of scoulerine 9-O-methyltransferase capable of O-methylating 1 -benzylisoquinoline alkaloids at the 3’ position enabling de novo THP biosynthesis. Flux through the heterologous pathway was improved by knocking out yeast multi-drug resistance transporters and optimization of media conditions. Overall, strain engineering increased the concentration of biosynthesized THP 600-fold to 121 pg/L. Finally, we demonstrate a strategy for papaverine semi-synthesis using hydrogen peroxide as an oxidizing agent. Through optimizing pH, temperature, reaction time, and oxidizing agent concentration, we demonstrated the ability to produce de novo semi-synthesized papaverine through the oxidation of biosynthesized THP.
We report the first de novo microbial biosynthesis of tetrahydropapaverine (THP) and semi-synthesis of papaverine using microbially biosynthesized THP. We used protein engineering to develop variants of two enzymes with improved non-native activity on pathway intermediates. The biosynthesis of THP demonstrates the ability to use protein homologs and protein engineering to replace the activity of unknown enzymes in heterologous biosynthetic pathways. We improved pathway flux by knocking out two yeast multi-drug resistance (MDR) transporters, which reduces the export of pathway intermediates. MDR knockouts may be applied to increase flux through other heterologous pathways. The strain engineering in this work provides a demonstration for fermentationbased production of clinically significant molecules THP and papaverine, which have experienced recent supply chain shortages. B. Materials and Methods
1 . Media, chemicals, and materials
Difco yeast nitrogen base without amino acids and ammonium sulfate (YNB), Bacto peptone, Bacto yeast extract, Luria Broth (LB), LB agar, dextrose, and galactose were obtained from BD (Becton, Dickinson and Company). Adenine hemisulfate, Kanamycin monosulfate, and ampicillin were obtained from Sigma Chemicals. G418 (sc-29065A) used for yeast integration selections was obtained from Santa Cruz Biotech. Amino acid dropout media was obtained from Takara Bio (product #630400 - #630431 ). Frozen-EZ transformation kit for yeast was obtained from Zymo Research. E. coli were selected on LB agar plates with 50 mg/L kanamycin, 75 mg/L carbenicillin, and grown in LB liquid media with the appropriate antibiotic. Yeast 10x drop out (DO) supplement was prepared from Takara as a synthetic complete supplement with desired dropout component omitted. S. cerevisiae strains were selected on YNB-DO (0.17% yeast nitrogen base, 0.5% ammonium sulfate, 2% dextrose, and 1x DO) agar or on YPD-G418 (1% yeast extract, 2% peptone, and 2% dextrose, 250 p/mL Geneticin). Yeast were grown in selective YNB-DO.
(S)-Reticuline and (S)-tetrahydropapaverine (THP) were purchased from Toronto Research Chemicals. (S)-Norreticuline was purchased from Alfa Chemistry. Papaverine hydrochloride, S-adenosylmethionine (SAM), hydrogen peroxide (30% w/w), and potassium persulfate were purchased from Sigma-Aldrich. Tris base was purchased from Fisher Scientific International, Inc. 5N hydrochloric acid was purchased from VWR International. BL21 (DE3) Escherichia coli cells were purchased from Invitrogen. Nickel-nitrilotriacetic acid (Ni-NTA) resin was purchased from Fisher Scientific International, Inc. Amicon® 30 kD cutoff spin filters were purchased from EMD Millipore.
2. Plasmid construction
Strains and plasmids used and constructed in this work are described in Supplementary Tables 1 , 2 and 3. E. coli strain TOP10 (Life Technologies) was used for cloning and amplification of plasmids. Plasmids were recovered using Econospin columns (Epoch Life Sciences) according to manufacturer’s instructions. Oligonucleotides were synthesized by the Stanford Protein and Nucleic Acid Facility. PCR was carried out using Q5 DNA Polymerase (New England Biolabs) unless otherwise specified, and all restriction enzymes, T4 DNA ligase, and deoxynucleotides were purchased from New England Biolabs. Heterologous gene sequences were cloned from previously published plasmids, obtained from Addgene, or synthesized by Twist Bioscience. Cloning was exclusively performed using Gibson assembly(54) followed by transformation into E. coli or by gaprepair directly into yeast, or direct genomic integration. gRNA integration plasmids were constructed from addgene plasmid pCAS, which was a gift from Jamie Cate (Addgene plasmid # 60847)(55). pCAS was modified by Gibson assembly to create SpCas9 expression vector pCS3410, which was digested with Pad followed by Gibson assembly of each gRNA fragment. All gRNA sequences used in this work are listed in Supplementary Table 3.
Error-prone libraries of the EcNMCH and T/S9OMT were generated by PCR amplification using Taq polymerase and TriLink Biotechnologies mutagenic dNTPs (Item #2748 and #2746) according to the manufacturer's instructions. Three separate PCR reactions were performed at 20 cycles with varying concentrations of equimolar nucleotide analogs according to manufacturer instructions. Amplicons from mutagenic PCR generally were at least 75 bp long and included up to 10 base pairs upstream and downstream of the indicated region when necessary to meet that length, products from each mutagenic PCR were amplified further using Taq polymerase. For plasmid-based expression, the amplified DNA was incorporated into the full TfS9OMT gene using overlap-extension PCR and incorporated into plasmids using Gibson Assembly. T/S9OMT NNK libraries were generated with primers containing NNK-randomized regions at the desired codon. These primers were used to amplify two fragments: The front of the gene up to the NNK codon with 15 additional base pairs of overhang, and the back of the gene from 15 base pairs before the NNK codon to the end of the gene. These two amplicons were amplified and Gibson overhangs were attached using overlap-extension PCR. The DNA purification columns, Zymoclean gel DNA recovery kit, and yeast genomic DNA prep kit (D2002) were all obtained from Zymo Research. All routine sequencing was performed using Quintara Biosciences.
3. Yeast Strain Construction
The previously reported strain CSY1171 (28, 38) was used as the base for any novel strains reported in this work. All yeast plasmid transformations were performed using EZ transformation kit from Zymo Research according to manufacturer’s instructions, unless otherwise specified. Scar-free yeast integrations were carried out as previously described(55), by co-transformation of gRNA/Cas9 expression plasmid targeting the edit site, along with repair DNA to be integrated genomically. Briefly, expression cassettes or gene modifications were amplified using primers with 15-40 bp overhangs for gap repair in yeast. Error-prone libraries of the NMCH and TfS9OMT were generated as previously described before direct transformation for genomically integrated libraries, with guides designed as previously described(35). Genomically integrated NNK libraries were generated using overlapping 70 bp region with the relevant codon NNK randomized in both primers. Each assembly was flanked by an integration homology region of 30-60 base pairs. gRNAs were generated at a target region such that integration of the desired modification would result in disruption of the gRNA binding site. Typically, 500 ng of purified PCR product was introduced with 300 ng of gRNA plasmid using the Zymo EZ Kit for transformation.
4. DNA Shuffling
Variants combined using DNA shuffling were amplified using PCR and pooled in an equimolar mixture with a final DNA concentration of 200 ng/pL. Separately, a DNAse I (Zymo Research - E1010) solution was prepared with 167 mM Tris-HCI buffer, 83.3 mM manganese chloride, and 1 .67 U/mL DNase I. 30 μL DNAse I solution was added to 70 μL of the equimolar amplicon mixture and the reactions were incubated for 60 seconds at 15°C before being quenched with 6 pL of 500 mM EDTA. The resulting DNA fragments were separated using an agarose gel. Fragments between 50 bp and 150 bp in length were cut from the gel and purified using the Zymoclean Gel DNA Recovery Kit (Zymo Research). DNA fragments were reassembled using the following cycling procedure: 1x: 96°C for 2 min; 35x: 95°C for 15s -> 65°C for 30s -> 62°C for 30s -> 59°C for 30s -> 56°C for 30s -> 53°C for 30s 50°C for 30s 47°C for 30s 44°C for 30s 41 °C for 30s 72°C for 1 min; 1x: 72°C for 3 minutes. A second PCR reaction with terminal primers was used to lift full length sequences and attach overhangs for Gibson Assembly.
5. Growth conditions for metabolite production
For this study, all strains were grown for three days at 30°C unless otherwise noted, shaking at either 250 r.p.m. (for 1 mL cultures or larger) or 460 r.p.m. (for 300 μL cultures). Yeast were grown in yeast peptone (YP) base (10 g yeast extract, 20 g peptone, and 80mg adenine hemisulfate for 1 L media) or yeast nitrogen base (YNB) (1 .7 g yeast nitrogen base, 5 g ammonium sulfate for 1 L media, pH to 5.8 using NaOH). The concentration of the carbon source was 2% (w/v) in all media with a single carbon source present. Dextrose was the carbon source unless otherwise noted. For samples with multiple carbon sources, the media contained 2% (w/v) dextrose and the concentration of the secondary carbon source was also 2% (w/v). Synthetic complete (SC) media was prepared by combining seven parts YNB, one part 10x amino acid supplement mix, one part water, and one part dextrose. Synthetic defined (SD) media was prepared similarly using 10x amino acid supplement mixes lacking either uracil or tryptophan depending on the auxotrophic marker on the plasmid being used.
6. Intracellular metabolite extraction
For intracellular metabolite analysis, 50 mL cultures were grown for three days at 30°C before being centrifuged at 3,500g for 10 minutes at 4°C. 1 mL was collected for extracellular metabolite analysis. The cells were then washed twice by resuspending in 5 mL water, centrifuging at 3,500g for 10 minutes at 4°C and removing the supernatant. A sample of the wash after centrifugation was collected each time. The cells were resuspended in 1 mL 50% methanol and 250 μL 0.5 mm glass beads (USA Scientific #7400- 2405) were added. Samples were then vortexed at maximum speed for two hours with an ambient temperature of 4°C (Scientific Industries Vortex-Genie 2, SI-0236; TurboMix attachment, SI-0564) before being centrifuged at 20,000g for 90 minutes. The supernatant was collected for analysis by LC-MS/MS. To compare intracellular and extracellular concentrations, we calculated the total intracellular volume of cells in a 50 mL culture using the average volume of a yeast cell(57), number of yeast cells per OD600 unit(58), and the OD600 measurement of the culture:
Figure imgf000075_0001
Our estimate for intracellular volume of 105 RL indicates that our procedure of extracting lysing 50 mL of cells in a volume of 1 mL results in a 1 :9.5 dilution of the intracellular metabolites. We multiplied intracellular metabolite concentrations by 9.5 to more accurately compare them to the metabolite concentrations measured in the media.
7. Chemical oxidation reactions
Chemically synthesized THP was used in initial experiments to determine favorable reaction conditions. Final concentrations were 500 nM THP, 75 mM Tris-HCI buffer (pH as indicated), and oxidizing agent concentrations were as indicated in a total reaction volume of 200 μL. Reactions were incubated at the indicated temperature with shaking at 600 r.p.m. using an Eppendorf Thermomixer F1 .5. Reactions were quenched by adding an equal volume of 1 M sodium thiosulfate and stored at 4°C before analysis via LC-MS/MS. Reactions with biosynthetically-produced THP contained three components: spent yeast media containing THP, Tris-HCI buffer, and hydrogen peroxide. The sum of the volume of the first two components was always 190 μL at the ratio indicated (e.g. 40:150 media-to- buffer ratio means 40 μL media and 150 μL buffer were used). 10 μL of concentrated hydrogen peroxide solution were then added for a total reaction volume of 200 μL. These reactions were similarly incubated, quenched, and stored at 4°C prior to analysis via LC- MS/MS.
8. Software
ChemDraw 18.0 was used for illustrating chemical structures. Pymol was used for visualizing protein structures. RaptorX was used to construct protein homology model(33). SwissDock was used for substrate docking simulations(34, 59). MassHunter Workstation (Agilent) was used to collect and analyze LC-MS/MS data. ChemStation (Agilent) and DataAnalysis (Bruker) were used to collect and analyse, respectively, chiral LC-MS data. Microsoft Excel was used to perform statistical analyses and prepare graphs. Inkscape was used to create all figures shown in this work.
C. Results
1. A reconstructed biosynthetic pathway for reticuline serves as the starting point for a de novo reconstructed THP pathway
We used a previously reported de novo (S)-reticuline-producing yeast platform strain as the starting point for a de novo THP biosynthetic pathway(27, 28, 32). This reticuline- producing platform strain incorporates five modules with a total of 17 heterologously expressed enzymes to produce reticuline. Each of these modules was integrated into distinct chromosomal regions in a wild-type, haploid Cen.PK2-1 D.
Module I encodes the expression of four yeast enzymes to improve the production of the metabolites tyrosine and 4-hydroxyphenylacetaldehyde (4-HPAA). Copies of 3-deoxy-D- arabino-2-heptulosonic acid 7-phosphate synthase (Aro4pQ166K) and chorismate mutase (Aro7pT2261), which were modified to decrease feedback inhibition from tyrosine, and transketolase (TKL1p) and phenylpyruvate decarboxylase (Arol Op) are overexpressed. Module II encodes expression of four Rattus norvegicus enzymes to synthesize and recycle tetrahydrobiopterin, a cofactor for tyrosine hydroxylase (TyrH); sepiapterin reductase (RnSepR) and 6-pyruvoyl tetrahydrobiopterin synthase (RnPTPS) increase tetrahydrobiopterin production, while quinonoid dihydropteridine reductase (RnQDHPR) and pterin carbinolamine dehydratase (RnPCD) improve cofactor recycling. Module III encodes expression of four enzymes that increase (S)-norcoclaurine titers; R. norvegicus dihydrofolate reductase (PnDHFR) improves tetrahydrobiopterin (BH4) salvage, mutant (R37E, R38E, and W166Y) R. norvegicus tyrosine hydroxylase (RnTyrHWR) produces L- DOPA with improved resistance to negative feedback, DOPA decarboxylase from Pseudomonas putida (PpDODC) produces dopamine, and norcoclaurine synthase from Coptis japonica (C/NCS) catalyzes the reaction between dopamine and 4-HPAA to produce norcoclaurine. Module IV encodes the expression of five plant proteins that convert norcoclaurine to reticuline; Papaver somniferum norcoclaurine 6-O-methyltransferase (Ps60MT), coclaurine-A/-methyltransferase (PsCNMT), cytochrome P450 reductase (PsCPR), and 4’-O-methyltransferase (Ps4’0MT) and Eschscholzia californica N- methylcoclaurine hydroxylase (EcNMCH). Lastly, Module V encodes the overexpression of three proteins that were previously identified as bottlenecks in the pathway (RnTyrHWR, Ps4’0MT, CjNCS), resulting in strain CSY1060(27, 28, 32).
Reticuline production in the platform strains were assayed as a benchmark for flux through the native benzylisoquinoline pathway in yeast. The strains were grown for three days at 30°C in synthetic complete (SC) media. Reticuline concentrations in the growth media were assayed by liquid chromatography coupled with tandem mass spectroscopy (LC-MS/MS). The base platform strain CSY1060 produces 19.1 pg/L reticuline (±2.23 pg/L). We further incorporated two previously reported modifications to increase reticuline production(28). Specifically, a variant of NCS with the first 24 amino acids truncated (C/NCS,runc) and a yeast codon-optimized variant of RnTyrHWR were replaced in the appropriate modules, resulting in strain CSY1171 , which produces 1.14 mg/L reticuline (±47.3 pg/L). The platform strain CSY1171 was used as the starting point for constructing the THP-producing yeast strains.
2. An engineered FcNMCH variant with higher activity on the non-native substrate coclaurine increases norreticuline titer
In order to construct the NH biosynthetic route in yeast to THP and papaverine, we first constructed a strain to produce norreticuline. The reticuline-producing platform strain, CSY1171 , produces the intermediate norcoclaurine, which is subsequently O-methylated and M-methylated by Ps6OMT and PsCNMT, respectively, to produce N-methylcoclaurine. In order to produce only N-demethylated products, the coding region for PsCNMT was deleted from strain CSY1171 yielding CSY1172. With the removal of PsCNMT from the biosynthetic pathway, norreticuline becomes the final pathway product (Fig. S1 ).
We confirmed norreticuline production in the base strain CSY1172. Strains were grown for three days at 30°C in SC media and norreticuline concentrations in the growth media were assayed by LC-MS/MS. The results demonstrate that CSY1172 produces norreticuline at a concentration of 5.85 pg/L (±1 .04 pg/L), and reticuline concentration drops below the limit of detection of our assay. The concentration of norreticuline produced by CSY1172 is 200-fold lower than the concentration of reticuline produced by CSY1171 (the reticuline-producing platform strain) on a molar basis. We hypothesized that the lower titer of norreticuline results from a lower activity of EcNMCH on its non-native substrate coclaurine in the reconstructed norreticuline pathway(19, 28, 32). Since Ps4’OMT has been shown to modify both /V-methylated and N-demethylated substrates(19), we predicted that EcNMCH activity would be limiting norreticuline production in CSY1172. This hypothesis was supported by relative accumulation of pathway intermediates in the norreticuline- producing strain CSY1172. The concentration of the substrate of EcNMCH, coclaurine, was estimated to be one to two orders of magnitude higher than the concentration of the product, 3’OH-coclaurine. By comparison, the concentration of the substrate of Ps4'OMT, 3’OH-coclaurine, was estimated to be an order of magnitude lower than the concentration of its product, norreticuline (Fig. S2).
We focused on improving EcNMCH activity on coclaurine to increase norreticuline production in the coclaurine-producing yeast strain. Because of the structural similarity between coclaurine and /V-methylcoclaurine (Fig. 2A), site-directed, random mutagenesis was used to improve the catalytic activity of the enzyme on the non-native substrate coclaurine. A homology model of EcNMCH was developed using RaptorX(33), and the substrate binding pocket for the native substrate, /V-methylcoclaurine, was predicted using SwissDock(34). Six contiguous chains of amino acids (amino acid positions — C1 : 94-109, C2: 202-205, C3: 271-294, 04: 344-356, 05: 416-434, 06: 465-468) were identified as being in proximity to the substrate binding pocket (Fig. 2B). We hypothesized that the amino acids in these six regions may play roles in substrate binding and orientation and selected them as targets for mutagenesis to improve the binding of the non-native substrate, coclaurine. Each of the chains was individually targeted for mutagenesis using a combination of error-prone PCR and CRISPR-targeted(35) integration. Error-prone PCR was used to amplify -100 base pair (bp) DNA fragments encoding the peptide chain of interest and overhangs for homologous recombination using conditions generating, on average, one to two mutations per fragment. The fragments were transformed into CSY1172 along with a plasmid encoding CRISPR-Cas9 and a gRNA targeting the corresponding chain. The CRISPR gRNA targets double-stranded breaks in the DNA of each chain, and when the breaks are repaired by the error-containing PCR fragments via homologous recombination, further cutting is inhibited by mutations in the region targeted by the CRISPR guide RNA. The transformation therefore results in colonies with genomically- integrated mutations in the desired chains. Colonies were picked and grown for three days at 30°C in SC media supplemented with 2 mM L-DOPA and 10 mM ascorbic acid to increase pathway flux. The spent media was subsequently assayed for norreticuline production by LC-MS/MS. After screening 1 ,000 colonies, a variant of EcNMCH that increased norreticuline production 10-fold was identified. Subsequent sequencing of this top variant identified a mutation at the L203 position.
We used site-saturation mutagenesis to further improve the activity of the EcNMCH enzyme on the nonnative substrate, coclaurine. An NNK library representing every possible amino acid substitution at the L203 position was generated, integrated, and screened. In the resulting library, 42 of 93 colonies screened resulted in higher than wild-type norreticuline production indicating that several amino acid substitutions at this position are able to improve the activity on the non-native substrate. The L203S variant exhibited the largest improvement to norreticuline production at approximately 40-fold over the wild-type variant with L-DOPA feeding (Fig. 2D). The previously generated homology model with docked substrate shows proximity between the wild-type leucine at this position and the /V-methyl group of the native substrate (Fig. 2C). The improved potential for hydrogen bonding between a serine at this position and the unmethylated nitrogen in the non-native substrate provides a possible mechanistic explanation for this improvement in activity. An expression cassette encoding the engineered variant EcNMCH12035 was integrated into strain CSY1 172 replacing the wild-type EcNMCH in the strain resulting in CSY1 173, which produces 72.0 μg/L norreticuline (±1 1 .3 pg/L). Overall, we developed an engineered EcNMCH variant that increased norreticuline production 12-fold over the wild-type enzyme in engineered yeast grown in SC without L-DOPA feeding.
3. An engineered TfS9OMT variant with higher activity on non-native substrates norreticuline and norlaudanine increases THP titer
In order to produce THP via the NH biosynthetic route, norreticuline must be O- methylated at the 7 and 3’ positions on the 1 -BIA scaffold (Fig. 1 A). An enzyme that can carry out O-methylation at the 7 position of the 1 -Bl A scaffold, GfN7OMT, has been previously reported(36). The enzyme responsible for O-methylating the 3’ position of the 1 - BIA scaffold in planta had not been reported at the time of this study. A number of plant OMTs have been extensively characterized that perform similar reactions, and thus we took an enzyme engineering approach leveraging known plant OMTs as a starting point to complete the reconstructed THP pathway.
We first examined whether incorporation of the G/N7OMT enzyme into our norreticuline biosynthetic pathway would yield a yeast strain capable of producing (S)- norlaudanine via O-methylation of the 7 position of norreticuline. An expression cassette encoding G/N7OMT was integrated into the URA3 locus of the norreticuline-producing strain CSY1 173 resulting in strain CSY1 174. Norlaudanine production was assayed by LC- MS/MS after three days of growth at 30°C in SC media. The results demonstrate that strain CSY1 174 produced norlaudanine at an estimated titer of 20 pig/L, indicating that G/N7OMT catalyzes the conversion of norreticuline to norlaudanine in the engineered yeast strain.
To complete the reconstructed THP biosynthetic pathway, we next focused on the O-methylation of the 1 -BIA scaffold at the 3’ position. We screened a variety of plant OMTs, including some reported to perform 3’-0-methylation in vitro or in vivo in other organisms(37), and none were able to efficiently O-methylate the 3’ position in the context of a norlaudanine-producing yeast strain. The only enzyme we screened with detectable 3’- O-methylation activity was the DS isoform of TfS9OMT, which produced low titers of (S)- norcodamine and THP. TfS9OMT was included in our 3’- O-methylation screen because of the structural similarity between norreticuline and the enzyme’s native substrate, scoulerine (Fig. 3A). Scoulerine and norreticuline are similar molecules except for the presence of a carbon bridge between the isoquinoline nitrogen and the 2' benzyl moiety in scoulerine. Screens for 3’OMT activity were performed in the norlaudanine-producing strain, CSY1 174. The strain was grown for three days in synthetic defined (SD) media with the appropriate dropout for plasmid-based expression, and THP and norcodamine were assayed using LC- MS/MS. THP was produced at a concentration of 372 ng/L (± 52 ng/L) in CSY1174 with a high-copy plasmid encoding TfS9OMTCS, whereas it was not detected in negative control samples with a similar plasmid encoding green fluorescent protein (GFP) (Fig. 3B). The results indicate that TfS9OMTDS can exhibit the desired 3’ OMT activity for THP production, but this activity is low on the non-native substrates in the reconstructed THP pathway.
We next focused on improving TfS9OMT activity at the 3' position on the non-native substrates norreticuline and norlaudanine. We took a site-directed, random mutagenesis approach with TfS9OMT similar to the approach taken with EcNMCH. The previously elucidated crystal structure(38) of the TfS9OMT enzyme was used to identify six contiguous chains of amino acids (amino acid positions — C1 : 10-20, C2: 108-120, C3: 161 -168, C4: 250-256, C5: 281 -283, C6: 293-314) with proximity to the substrate binding pocket of the enzyme (Fig. 3D). Each of the chains was individually targeted for mutagenesis using an error-prone PCR approach. Because of the low signal of THP detection at the titers produced by genomically integrated TfS9OMT, variants generated by error-prone PCR were expressed from a high-copy plasmid in CSY1174, in contrast to the EcNMCH engineering experiments. Colonies were picked and grown for three days at 30°C in SD media with the appropriate dropout and subsequently assayed for THP production. 1000 colonies were screened for THP titers and 10% of colonies screened exhibited at least a 50% increase in THP titer over a control strain expressing the TfS9OMTDS. Enzyme variants from 12 colonies with improved THP titers were sequenced, and the sequencing data indicated that mutations at several off-target residues (S47, S77, T83, C98) and on-target residues (A119, F296, L304) are present in enzyme variants exhibiting up to three-fold higher THP titers compared to TfS9OMTDS.
In order to identify the optimal amino acid at each identified residue location, NNK libraries were generated and screened at each residue where mutations were shown to improve 3’ OMT activity in the initial site-directed mutagenesis screen. The TfS9OMT NNK libraries were tested by expression from high-copy plasmids in CSY1174. For each NNK library, 92 colonies were screened in order to reach a 95% chance of full library coverage. NNK libraries allowed us to identify novel mutations superior to those found in the initial screen at five of the seven amino acid residues (Table S5). For example, the initial F296S mutation, which yielded a three-fold higher THP titer than T/S9OMTDS, was improved to F296L, which produced a five-fold higher THP concentration than TfS9OMTDS (Fig. S3).
To further improve the 3' OMT activity , we applied DNA shuffling to screen for synergistic combinations of the identified amino acid mutations. The site-directed mutagenesis and NNK libraries identified mutations at seven residues that individually improved THP production (S47, T83, S77, C98, A119, F296, L304). A library to test both optimized and unoptimized mutations in combination was generated via DNA shuffling(39) with three input templates: TfS9OMTDS, TfS9OMTV1 (S47F, T83A, C98R), and TfS9OMTV2 (S77W, C98P, A1 19L, F296L, L304Y). Fully reassembled TfS9OMT shuffled variants were similarly characterized by expressing the variants from high-copy plasmids transformed into strain CSY1 174. 720 colonies were screened for THP titers and 36% of these colonies produced THP titers higher than TfS9OMTDS. The three top variants increased THP titers more than 10-fold compared to TfS9OMTDS. TfS9OMTsl incorporates A119L and the off- target mutation V281 1 and increases THP titer 12-fold; TfS9OMTS" incorporates four on- target mutations, S47P, T83A, C98R, and A119L and increases THP titer 17-fold; and TfS9OMTSI" incorporates T83A, C98P, A1 19L, and the off-target mutation P208Q and improves THP titer 16-fold (Fig. S4).
We achieved additional improvements of 3’ OMT activity in the engineered TfS9OMT variant with additional NNK optimization. In previous experiments, libraries were generated using TfS9OMTDS as a template, but with the availability of improved variants, we used a pool of TfS9OMTsl, TfS9OMTS", TfS9OMTSI", and TfS9OMTsl"* (a version of T/S9OMT with the off-target P208Q reverted to its wild-type identity) as templates for further improvements. We generated and tested NNK libraries at V281 , F296, N309, and S160, using the highest performing variant as the template for each subsequent library. These mutagenesis experiments screened colonies by expression from a low-copy plasmid in CSY1 174 to better match the protein titers that can be achieved through genomic integration. This iterative optimization resulted in TfS9OMTOPT, which contains mutations T83A, C98P, A119L, S160V, V281 I, F296L, and N309T. We compared the TfS9OMTDS, T/S9OMTF29L (the highest performing single mutant), TfS9OMTSI"*, and T/S9OMTOPT by expressing them from a low-copy plasmid in CSY1354 (strain described in next section) grown for three days in SD media with appropriate dropout. Under these conditions, TfS9OMTOPT produced 35-fold more THP than TfS9OMTDS (Fig. 3E). To confirm that the improved THP titer was the result of improved enzyme function, TfS9OMTDS and T/S9OMTOPT were purified by Ni-NTA affinity chromatography and their activities examined in vitro. TfS9OMTOPT produced 55-fold more norcodamine than TfS9OMTDS when fed norreticuline as a substrate, indicating that the observed improvements in THP titer are the result of improved catalytic efficiency. TfS9OMTOPT was incorporated into the LYP locus of CSY1 174 resulting in CSY1359, which produces THP at 271 ng/L THP (±24.2 ng/L). Overall, engineering a TfS9OMT variant capable of 3’ O-methylating the 1- benzylisoquinoline scaffold for de novo THP production included site-directed mutagenesis, individual NNK optimization, and DNA shuffling (Fig. 3C), resulting in a variant harboring seven mutations that supported a 35-fold increase in THP production from yeast.
During the preparation of this manuscript, two enzymes were reported in separate studies to methylate the 3'-hydroxyl group of norreticuline. In one study a putative 3’0MT from P. somniferum (Ps3’0MT) associated with papaverine production was identif ied(40) via virus induced gene silencing. In vitro characterization experiments demonstrated methylation of a structural homolog, dihydroxyacetophenone, but 3’OMT activity on norreticuline and norlaudanine was not demonstrated. In a second study, an engineered variant of an OMT from Glaucium flavum (G/0MT1 , referred to elsewhere as GFLOMT1 (36)) was demonstrated to exhibit 3’0MT activity on the 1 -BIA scaffold(41). The authors of this study reported a variant of G/0MT1 with the ability to methylate four different positions on the 1 -BIA scaffold, including the 3’ position, to produce THP from fed norlaudanosaline in E. coli. We examined the 3’0MT activity of both enzymes in the context of our engineered pathway by expressing them from a low-copy plasmid in the norreticuline- producing strain, CSY1173, and in the norlaudanine-producing strain, CSY1174. Under these conditions, norcodamine and THP, the expected products of 3’-G-methylation, were not detected in the media at levels above background.
4. Knocking out multi-drug resistance transporters improves pathway flux
Despite the improvements to TfS9OMT activity in the THP biosynthetic pathway, the incorporation of two engineered enzymes (EcNMCHL203S, TfS9OMTOPT) modifying nonnative substrates in the pathway results in a strain that exhibits relatively low THP titers compared to the titer of reticuline in CSY1171 . We sought to further improve the titer of THP produced de novo by making complementary strain modifications.
We first examined whether the deletion of multi-drug resistance (MDR) transporters can lead to increased pathway flux. Biosynthetic intermediates in our pathway must remain within cells in order to be accepted and functionalized by pathway enzymes, so premature export of pathway intermediates may be limiting our titer of THP. We hypothesized that removal of specific MDRs may result in higher intracellular concentrations of pathway intermediates resulting in increased pathway flux. Four ATP-utilizing MDRs were selected as knockout targets: PDR5, SNQ2, Y0R1 , and YCF1 (42). Proteins were knocked out by introducing a premature stop codon within the first 50 amino acids of the protein using a CRISPR-based approach. The MDRs were knocked out individually and in combination in the norlaudanine-producing strain CSY1174. The engineered TfS9OMTOPT was expressed in each of the resulting knockout strains from a low-copy plasmid. The strains were grown for three days in SD media with the appropriate dropout, and THP levels were assayed using LC-MS/MS. PDR5 and SNQ2 knockouts improved the overall production of THP compared to equivalent strains with the transporters expressed. The strain harboring the double knockout of SNQ2 and PDR5, CSY1354, exhibited a 15-fold increase in THP titer (Fig. 4A). The strain resulting from the triple knockout of SNQ2, PDR5, and YCF1 (CSY1358) produced the highest THP titer increase at 17-fold higher than CSY1174. We chose to proceed with the double knockout, CSY1354, because additional strain modification can negatively impact strain health(43) and because the YCF1 knockout was not shown to be statistically significant in a head-to-head comparison between CSY1354 and CSY1358 using a t-test (p-value = 0.119).
We next examined the potential mechanism by which CSY1354 results in higher THP titers compared to CSY1174. Based on our initial hypothesis, we expected that export of one or more THP pathway intermediates would be diminished in CSY1354. To better understand the production and export of pathway intermediates in CSY1174 and CSY1354, we expressed TfS9OMTOPT from a low-copy plasmid and we measured the optical density (Fig. S5) and concentrations of norcoclaurine, coclaurine, 3’-hydroxycoclaurine, norreticuline, norlaudanine, norcodamine, and THP at six-hour intervals (Figs. S6-S12). These metabolites were analyzed as we predicted they may be targets for yeast MDR transporters due to their size and unique scaffold when compared to native yeast metabolites. For each metabolite, the concentration measured in the media with CSY1354 was higher than with CSY1174 at every timepoint, except for the 12- and 18-hour timepoints for coclaurine. The increase in metabolite titers resulting from the removal of SNQ2 and PDR5 varied significantly (Table S6). For example, at the 54-hour timepoint, coclaurine concentration in CSY1354 was 1.4-fold higher than CSY1174, whereas the concentration of THP in CSY1354 was 10.1 -fold higher. Since overall pathway flux is increased, we predicted that metabolites with smaller increases in extracellular concentration, like coclaurine, may be exported less efficiently with SNQ2 and PDR5 knocked out.
To further understand the impact of MDR transporter knockouts on pathway metabolite transport, we grew cultures of CSY1354 and CSY1174 with T/S9OMTOPT expressed from a low-copy plasmid for 66 hours and compared intracellular and extracellular metabolite concentrations. The LC-MS/MS integration areas from extracted intracellular metabolites were multiplied by a corrective factor in attempt to compare metabolite concentrations intracellularly and extracellularly (see Methods). For the furthest downstream metabolites (3’-hydroxycoclaurine, norreticuline, norcodamine, norlaudanine, THP), intracellular concentrations were too low to accurately quantify (Fig. S13). For the upstream metabolites (norcoclaurine, coclaurine), the ratio of intracellular concentration to extracellular concentration was higher in CSY1354 than it was for CSY1174 (Fig. 4B). In CSY1354, the concentration of coclaurine intracellularly was 9.1 -fold higher than in the media, whereas in CSY1174 the intracellular coclaurine concentration was only 1 .8-fold higher than in the media. The intracellular accumulation of coclaurine resulting from SNQ2 and PDR5 knockouts suggests that these MDR transporters may play a role in exporting coclaurine in CSY1174. The decreased rate of coclaurine export in CSY1354 may play a role in increasing flux through the THP production pathway and improving THP titers.
5. Optimization of carbon source and TfS9OMTOPT expression results in increased
THP production in yeast
We next sought to increase the titer of THP produced via optimization of growth media and TfS9OMTOPT expression in the engineered yeast strain. We began by integrating a copy of TfS9OMTOPT into the LYP locus of CSY1354, the norlaudanine producing strain with SNQ2 and PDR5 knocked out, to generate CSY1360, which produces 4.38 pg/L THP (±0.25 pg/L). Adding a second copy of TfS9OMTOPI at the TRP locus of CSY1360 yielded CSY1361 , which produces more than double the amount of THP at a concentration of 10.1 pg/L (±0.21 pg/L). Based on the results of previous studies on the biosynthesis of 1 - BIAs(28, 44), we grew CSY1361 with multiple media bases and carbon sources to improve pathway flux. For media bases, we used yeast nitrogen base to prepare a standard synthetic complete media and more amino acid rich versions of synthetic complete with two and four times the standard amino acid concentrations (2xSC and 4xSC, respectively). We also prepared media using a yeast peptone (YP) base for a richer nutrient media option. For carbon sources, we tested the standard carbon source, dextrose, along with galactose, sucrose, trehalose, glycerol, raffinose. Carbon sources were tested individually and in combination with dextrose. Each media was tested at a concentration of 2% (w/v) in the final preparation of the media unless otherwise noted. Overall, the highest titer of THP was observed with the conditions of YP with raffinose as a carbon source, which produced 36.0 pg/L THP (±2.20 pg/L) (Figs. 4C, S14-S16).
As the introduction of a second copy of TfS9OMTOPT more than doubled THP production, we examined whether increasing copies of this bottleneck enzyme via plasmidbased expression would lead to additional increases in THP titers. Expression of TfS9OMTOPT from a high-copy plasmid in CSY1354 resulted in higher THP titers than any fully genomically integrated strain, with a THP concentration of 68.9 pg/L (± 304 ng/L) when grown in SC with appropriate dropout. To maximize the production of THP with a high-copy plasmid, we tested galactose and raffinose as alternates to the carbon source dextrose. Galactose and raffinose were chosen based on the results of the carbon source optimization using CSY1361 , where they consistently yielded the best results compared to other carbon sources regardless of media base. SC with both galactose and raffinose resulted in more than 2-fold higher THP concentration compared to SC with dextrose (Fig. S17, S18). In this context, expression of TfS9OMTOPT from a high-copy plasmid in CSY1354 with raffinose as a carbon source yielded the highest THP titer of any conditions tested at 121 pg/L (± 3.00 ug/L). Taken together, the combination of optimizing MDR transporter knockouts, T/S9OMT engineering, TfS9OMT expression, and carbon source improved the THP titer 600-fold compared to our lowest THP-producing strain (i.e., low-copy plasmid expression of TfS9OMTDS in CSY1174).
6. One-step chemical oxidation of biosynthesized THP yields semi-synthetic papaverine
The final step in papaverine production biosynthesis is the four-electron oxidation of THP to produce papaverine. To accomplish this final step, we first explored enzymatic oxidation of THP to produce papaverine. An enzyme from Papaver somniferum called dihydrobenzophenanthridine oxidase (PsDBOX) has been proposed to facilitate this reaction in planta and has demonstrated THP oxidase activity when tested in vitro. We grew the THP-producing strain CSY1361 harboring a low-copy plasmid encoding a PsDBOX expression cassette in SD media with appropriate dropouts for three days. Despite the presence of the substrate, THP, and PsDBOX, papaverine was not detected in the growth media. We made several attempts to establish PsDBOX activity in this context including feeding high concentrations of THP, localizing PsDBOX to subcellular compartments, improving culture oxygenation by growth in baffled flasks, and tethering PsDBOX to GFP to confirm protein expression via fluorescence microscopy (Fig. S19). Despite significant troubleshooting efforts, PsDBOX activity on THP to produce papaverine in yeast strain CSY1361 was not observed.
In the absence of viable enzymatic oxidation, we explored chemical oxidation of THP. Initial screens, which included numerous oxidizing agents, demonstrated that both hydrogen peroxide and potassium persulfate were able to oxidize THP to produce papaverine in an aqueous setting, and we selected these two oxidizing agents for further testing. To improve reaction yields, pH, oxidizing agent concentration, reaction time, and temperature were adjusted to improve reaction yield. First, we tested the effect of pH on the oxidation of THP by hydrogen peroxide and potassium persulfate by varying the pH of Tris- HCI buffer. Oxidation reactions were performed at 42°C for three hours before being quenched with an equal volume of 1 M sodium thiosulfate. For both hydrogen peroxide and potassium persulfate, the highest yields were achieved at a pH of 9.5 (Fig. S20). Tris-HCI buffer with a pH of 9.5 was used as the base for all subsequent reactions. We next examined reaction yield in the context of reaction time, reaction temperature, and oxidizing agent concentration. These variables were adjusted in combination because of their potential effect on one another. For hydrogen peroxide, we measured the reaction yield at nine conditions with variable temperatures (55°C, 70°C, 85°C) and concentrations (0.1%, 0.3%, 0.5%, 0.75%, 1.0%). For potassium persulfate, we measured reaction yield in six conditions at three temperatures (30°C, 40°C, 55°C) and two concentrations (15 mM, 30 mM). To determine optimal conditions, we also tracked reaction specificity, defined as product produced divided by substrate consumed (Table S7). The highest yield achieved with potassium persulfate as an oxidizing agent was 7.41% at a temperature of 40°C, a concentration of 30 mM, and a reaction time of six hours (Fig. S21). The highest yield achieved overall was 16.3% at a temperature of 85°C, a hydrogen peroxide concentration of 0.5%, and a time of 60 minutes (Fig. S22).
We next used these THP oxidation conditions with biosynthesized THP to produce de novo semi-synthesized papaverine. Biosynthesized THP was produced in CSY1354 with TfS9OMTOPT expressed from a high-copy plasmid as previously described. The spent media from this experiment containing 121 pg/L THP was used as the substrate for oxidation reactions. We first tested how the ratio of substrate-containing media to Tris-HCI buffer would affect the yield and specificity of the reactions. We tested six different ratios of media-to-buffer (40:150, 60:130, 80:110, 100:90, 120:70, 140:50) under the two sets of conditions that produced the highest yield in the previously described oxidation experiments using synthesized THP — 60 minutes with 0.5% hydrogen peroxide at 85°C, and three hours with 0.3% hydrogen peroxide at 70°C (Fig. S23). In both sets of reaction conditions, we found that the yield of papaverine was highest when the media-to-buffer ratio was lowest (Figs. 5A, S24). At the lowest media-to-buffer ratio, the yield of reactions with biosynthesized THP in the presence of media was similar to the yield of reactions with pure, chemically synthesized THP (Fig. 5B). Using biosynthesized THP, the yield of papaverine was 15.1% (Fig. S25) — versus 16.3% with synthesized THP. Overall, we were able to use synthesized substrate to find a set of aqueous reaction conditions capable of oxidizing THP to produce papaverine and validated that these same reaction conditions can be used on biosynthesized THP produced in the media of our yeast strains.
D. Discussion
In this work, we demonstrate the de novo microbial biosynthesis of THP and semisynthetic production of papaverine. We utilized a reticuline-producing platform yeast strain that produces 1 .14 mg/L reticuline. We modified the strain to produce norreticuline by removing PsCNMT and engineering a variant of EcNMCH with improved activity on the nonnative substrate coclaurine. In the absence of a known 1 -BIA 3’-O-methyltransferase, we identified that TfS9OMT has 3’-O-methyltransferase activity on this scaffold. We used a combination of site-directed, random mutagenesis, DNA shuffling, and site-saturation mutagenesis to identify TfS9OMTOPT, a variant with improved catalytic efficiency. We improved the overall flux through the pathway by knocking out two MDR transporters, with results indicating that these transporters reduce the transport of the intermediate coclaurine out of the cell. THP production was further improved by increasing 7/S9OMTOPT copy number and altering the carbon-source of the media. Overall, these improvements to pathway flux increased THP titers in the media 600-fold resulting in 121 pg/L THP production. We also identified reaction conditions that allow for the aqueous oxidation of THP to produce papaverine using hydrogen peroxide, demonstrating the first reported semisynthesis of papaverine from biosynthesized THP.
Despite extensive prior research, the enzymes for in planta papaverine production pathway have remained elusive(4, 45). While evidence for the NCH3 route towards papaverine production has been presented(21), most recent studies have presented data and evidence favoring the NH route(4, 19, 20). Along the NH route, there are three major challenges for microbial production of papaverine: coclaurine hydroxylation, norreticuline or norlaudanine 3’-0-methylation, and THP oxidation. Earlier studies of EcNMCH in which coclaurine was tested as a potential substrate reported no activity(46). Studies of potential norreticuline 3’OMTs have either lacked in vitro tests to confirm activity(40), or 3’-O- methylation has been a low-conversion side reaction compared to the enzyme’s main activity(37). We tested both the proposed Ps3'OMT and a G/OMT1 mutant(41) that has been shown to O-methylate at several positions of the 1 -BIA scaffold, including the 3’ position, and observed no activity in the context of our constructed THP pathway in yeast. While the putative Ps3'OMT activity was not verified on the pathway substrates, the difference in observed activity could result from a number of factors, including differences in protein folding conditions, intracellular pH (6.8 for yeast(47, 48) vs. 7.7 for E. coli(49)), or other pathway and cellular contexts. Finally, earlier in vitro characterization studies of DBOX demonstrated that the conversion of THP to papaverine was 7% of the conversion of dihydrosanguinarine to sanguinarine(22). The relatively low activity of PsDBOX on THP taken together with the localization of PsDBOX to plant roots and the accumulation of papaverine in the aerial organs implies either that another enzyme is involved in papaverine synthesis in planta or that papaverine is transported from the roots, where PsDBOX is present, to the aerial organs.
This work does not evaluate or address the in planta synthesis route for papaverine. Instead, we sought to construct a biosynthetic route to THP and papaverine despite existing gaps in knowledge. The three main tools we used to address these gaps were protein engineering to alter the substrate specificity of enzymes, yeast MDR transporter knockouts to increase the flux through the pathway, and the addition of a chemical oxidation reaction in aqueous conditions to replace the final enzymatic oxidation step. Many enzymes essential to the production of key plant natural products have yet to be elucidated — as for 3’-hydroxylation and O-methylation in this pathway — or cannot be functionally expressed in a heterologous microbial production platform — as we found to be the case for PsDBOX. Similar gaps in knowledge currently face heterologous microbial production systems for important plant natural product drugs like paclitaxel(50) and vinca alkaloids(51). The discovery of new plant enzymes has accelerated as genome and transcriptome sequencing becomes less expensive and more robust(3). Additionally, protein engineering strategies such as truncations to improve activity in a heterologous host and modifications to prevent product inhibition have been shown to significantly boost production of plant natural products in microbial hosts(3). The production of THP in yeast with two enzymes engineered for altered substrate specificity demonstrates the utility of applying protein engineering on known enzymes that act on related secondary metabolites to replace the activity of unknown pathway enzymes.
In our work, we also show significant increases to THP production as a result of MDR transporter knockouts. These host strain modifications may have broad applications in optimizing flux through other complex plant natural product pathways reconstructed in yeast. Yeast MDR transporters have broad substrate specificity and generally export compounds with drug-like structures that may be harmful to the cell viability(52) . Export by MDR transporters of pathway intermediates from the cytosol to the media, results in those metabolites no longer being co-localized with pathway enzymes, thereby decreasing pathway flux. We hypothesized that knocking out one or more MDR transporters could increase the intracellular concentration of intermediate pathway metabolites without decreasing product export. Metabolite analyses showing elevated intracellular coclaurine concentrations and a 15-fold increase in THP titer when SNQ2 and PDR5 are knocked out aligned with that prediction. SNQ2 and PDR5 disruption could improve flux through pathways that include coclaurine, and the concept of knocking out MDR transporters in combination could be applicable to intermediates in other heterologous plant natural product pathways in yeast.
Our work on this biosynthetic pathway began, in part, because of the supply shortages of clinically significant products. Papaverine, cisatracurium, and atracurium, pharmaceuticals produced from THP, have important medicinal uses, but the supply of these drugs has been interrupted over the past decade. Our work to enable the microbial biosynthesis of THP and semi-synthesis of papaverine provides an initial proof-of-concept of a microbial fermentation route that can be further advanced to improve the supply chain for these vital drugs. We expect that further functional genomic discovery efforts may elucidate additional enzymes capable of performing the final oxidation step in papaverine production. Protein engineering of PsDBOX, or another functionally similar oxidase, could enable fully biosynthetic papaverine production. In the absence of an enzyme capable of carrying out the final step, the chemical oxidation reaction that we describe can be improved upon. Further investigation to identify improved oxidizing agents or buffer conditions could improve yields and conditions for commercial production. Finally, incorporating the advances of this work with other efforts to improve 1 -BIA titers(53) , as well as fermentation process development, could enable efficient production of THP via yeast fermentation and ultimately improve the supply chain stability of several important pharmaceutical compounds.
E. Findings
Here, we report the biosynthetic production of THP and the semi-synthetic production of papaverine via a reconstructed NH route in yeast. We built upon a previously reported platform yeast strain designed to produce reticuline(27, 32). The strain was modified by removing the enzyme responsible for M-methylation of the 1 -BIA scaffold, Papaver somniferum coclaurine M-methyltransferase (PsCNMT). We then engineered a novel variant of Eschscholzia californica M-methyl-coclaurine hydroxylase (EcNMCH), capable of accepting the non-native substrate (S)-coclaurine to produce (S)-3'-hydroxy- coclaurine at a 40-fold higher concentration compared to the wild-type enzyme. These modifications resulted in a platform yeast strain that produced (S)-norreticuline de novo instead of reticuline. Two additional O-methyltransferases (OMTs), Glaucium flavum norreticuline 7-O-methyltransferase (G/N70MT) and Thalictrum flavum scoulerine 9-0- methyltransferase (TfS9OMT), were added to the strain to O-methylate norreticuline at the 7 and 3’ positions resulting in de novo yeast biosynthesis of THP. We engineered a variant of S90MT with seven amino acid mutations, which resulted in a 35-fold increase in THP titers compared to the original S90MT isoform. Two multi-drug resistance (MDR) transporters, SNQ2 and PDR5, were knocked out in the strain to increase flux through the THP production pathway, resulting in a 15-fold increase in THP titers. The highest titers of THP were achieved by expressing the engineered S90MT from a high-copy plasmid with galactose as a carbon source, resulting in 121 pg/L THP (± 3.00 pg/L). Finally, we demonstrated that hydrogen peroxide is capable of oxidizing THP to produce papaverine in a one-step, aqueous oxidation reaction that converts the biosynthesized THP to papaverine with a yield of approximately 15%.
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II. Supplementary Methods and Materials
A. Metabolite analysis by LC-MS/MS
For all extracellular metabolite measurements, yeast cultures were pelleted by centrifugation at 1 ,500g for 10 minutes. 110 μL of supernatant was removed for analysis. Metabolites were analyzed by LC-MS/MS using an Agilent 1260 Infinity Binary HPLC and an Agilent 6420 Triple Quadrupole mass spectrometer. Chromatography was performed using a Zorbax EclipsePlus C18 column (2.1x50 mm, 1.8 pm; Agilent Technologies) with 0.1% (v/v) formic acid in water as mobile phase solvent A and 0.1% (v/v) formic acid in acetonitrile as solvent B. The column was operated with a constant flow rate of 0.4 mL/min at 40°C with a sample injection volume of 5 pL. Chromatographic separation was achieved using one of three possible methods. Method A: 0-1.0 min, 0-100% B; 1.0-2.5, 100% b; 1.5 min post-time. Method B: 0-4.0 min, 0-100% B; 4.0-6.5 min, 100% B; 2.5 min post-time. Method C: 0-2.0 min, 0% B; 2.0-5.0 min, 0-10% B; 5.0-10.0 min, 10-20% B; 10.0-11.0 min, 20-100% B; 3.0 min post-time. Method A was used for all extracellular metabolite measurements. Method B was used for chemical oxidation reactions. Method C was used for analyzing in vitro protein reactions. The LC eluent was directed to the MS operating with electrospray ionization (ESI) in positive mode, source gas temperature 350 °C, gas flow rate 11 I min-1 , and nebulizer pressure 40 psi. Data collection was performed using MassHunter Workstation LC/MS Data Acquisition software (Agilent). Metabolites were identified and quantified by integrated peak area in MassHunter Workstation Quantitative Analysis software (Agilent) using the mass fragment/transition parameters in Supplementary Table 4 and by comparison to standard curves. The identity of metabolites for which chemical standards are available (reticuline, norreticuline, THP, and papaverine) were confirmed by comparing retention times and product ion spectra. The identity of other metabolites was confirmed by comparison to known MRM transitions(56). Concentrations were determined by comparing integration areas of samples to a standard curve generated using known concentrations. The concentration of norlaudanine (for which a chemical standard was unavailable) was estimated using standard curves for the structurally similar molecules norreticuline and THP, and averaging the results.
B. Protein purification
Plasmids containing the TfS9OMTDS and TfS9OMTOPT pET28 expression constructs were used to transform E. coli BL21 (DE3) via heat shock. Briefly, 1 ng of plasmid DNA was added to a 50 μL aliquot of competent cells, the tube was chilled on ice for 15 minutes, then placed in a 42 °C water bath for 35 seconds, then returned to ice for 2 minutes. Seven hundred fifty pL of SOC media were then added and the tube was rotated at 37 °C for 45 minutes before being plated on an LB agar plate containing 50 pg/mL kanamycin (GoldBio). A single colony was then picked and used to inoculate a primary culture of 5 mL of LB media containing 50 pg/mL kanamycin. This primary culture was then used to inoculate a secondary or expression culture of 500 mL of LB medium containing 50 pg/mL kanamycin. This expression culture was grown to an QD600 of 0.6-1.0 and then induced with IPTG at a final concentration of 1 mM. The expression cultures were then grown at 16 °C for 20 hours at 250 r.p.m., after which, the cultures were harvested by centrifugation (10 min at 3,500 r.p.m. in a 50 mL Falcon tube) and stored at -20 °C until lysis and purification.
C. Cell lysis and protein purification
Frozen pellets were thawed and resuspended in 25 mL of Ni-NTA 95 equilibration buffer (50 mM sodium phosphate, 300 mM NaCI, 10 mM imidazole, pH 7.4) and lysed by sonication while kept on ice (Branson Sonifier 450, 0.5” horn, 50% duty cycle, 4 x 1 min with 2 min rests). Lysed cultures were then clarified by centrifugation (45 min at 35,000 RCF at 4 °C) and the clarified lysate was purified by Ni-NTA affinity chromatography. Briefly, 1 mL of Ni-NTA resin (Fisher Scientific) was equilibrated with at least 5 volumes of Ni-NTA equilibration buffer (described above) and then loaded with the clarified lysate. The loaded resin was then washed with at least 5 volumes of NH 01 NTA wash buffer (50 mM sodium phosphate, 300 mM NaCI, 50 mM imidazole, pH 7.4) and then the bound protein was eluted with 5 volumes of Ni-NTA elution buffer (50 mM sodium phosphate, 300 mM NaCI, 250 mM imidazole, pH 7.4). The eluted fractions were then combined and concentrated using an Amicon® 30 kDa cutoff spin filter (EMD Millipore) at 5,000 g at 4 °C. Concentrated protein fractions were then buffer exchanged into storage buffer (50 mM potassium phosphate, 100 mM NaCI, 10% glycerol, pH 7.5), split into separate aliquots, and stored at -20 °C until use.
D. In vitro protein assays
Analytical reactions were carried out at the 100 μL scale in triplicate. To either a 1 .5 mL Eppendorf tube were added 1 .5 nmol norreticuline as a substrate (final concentration of 30 pM), 1.5 nmol S-adenosylmethionine (SAM, final concentration of 100 pM), and 15 pmol purified TfS9OMT enzyme (0.3 pM final concentration) in 50 mM potassium phosphate, pH 6.8. The reactions were shaken at 600 r.p.m. at 30 °C for 2 hours before being quenched with an equal volume of ACN, spun down at 20,000 g for 10 minutes at 4°C, and were stored at 4°C until analysis by LC-MS/MS. III. Supplementary Tables
Figure imgf000095_0001
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Figure imgf000098_0003
Figure imgf000099_0001
Figure imgf000099_0002
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Figure imgf000099_0004
Figure imgf000100_0001
References
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In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims.
It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.”
In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.
As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1 -3 articles refers to groups having 1 , 2, or 3 articles. Similarly, a group having 1 -5 articles refers to groups having 1 , 2, 3, 4, or 5 articles, and so forth.
Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.
Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e. , any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.
The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

What is claimed is:
1. A non-plant cell that produces tetrahydropapaverine (THP) via an engineered THP- biosynthetic pathway.
2. The non-plant cell according to Claim 1 , wherein the engineered THP-biosynthetic pathway is a norreticuline mediated pathway.
3. The non-plant cell according to Claim 2, wherein the non-plant cell comprises one or more heterologous coding sequences encoding at least one enzyme involved in the norreticuline mediated pathway.
4. The non-plant cell according to Claim 3, wherein the non-plant cell comprises one or more heterologous coding sequences encoding at least one enzyme involved in conversion of coclaurine to THP in the norreticuline mediated pathway.
5. The non-plant cell according to Claim 4, wherein the at least one enzyme comprises a N-methylcoclaurine hydroxylase (NMCH).
6. The non-plant cell according to Claim 5, wherein the NMCH comprises a mutant NMCH.
7. The non-plant cell according to any of Claims 4 - 6, wherein the at least one enzyme comprises an activity that is capable of O-methylating norreticuline at the 7' and 3' positions to produce THP.
8. The non-plant cell according to Claim 7, wherein the at least one enzyme comprises first and second O-methylating enzymes.
9. The non-plant cell according to Claim 8, wherein the first O-methylating enzyme is capable of O-methylating norreticuline at the 3' position.
10. The non-plant cell according to Claim 9, wherein the first O-methylating enzyme comprises a scoulerine 9-O-methyltransferase (S9OMT).
11. The non-plant cell according to Claim 10, wherein the S90MT comprises a mutant S9OMT.
12. The non-plant cell according to any of Claims 8 - 11 , wherein the second O- methylating enzyme is capable of O-methylating norreticuline at the 7' position.
13. The non-plant cell according to Claim 12, wherein the second O-methylating enzyme comprises a norreticuline 7-O-methyltransferase (N7OMT).
14. The non-plant cell according to any of Claims 4 - 13, wherein the at least one enzyme involved in conversion of coclaurine to THP in the norreticuline mediated pathway is chromosomally integrated into the norreticuline-producing cell.
15. The non-plant cell according to any of the preceding claims, wherein non-plant cell is a norreticuline producing cell that comprises coding sequences for producing PTPS, SepR, PCD, QDHPR, DHFR, TyrH, NCS, DODC, CPR, 60MT, 4’0MT, CNMT, ARO4, ARO7, ARO10, and TKL1.
16. The non-plant cell according to Claim 15, wherein each coding sequence is chromosomally integrated into the norreticuline-producing cell.
17. The non-plant cell according to any of the preceding claims, wherein the non-plant cell comprises a deletion of one or more endogenous multi-drug resistance (MDR) transports.
18. The non-plant cell according to Claim 11 , wherein the non-plant cell comprises a deletion of two or more endogenous multi-drug resistance (MDR) transports.
19. The non-plant cell according to any of the preceding claims, wherein the non-plant cell comprises a microbial cell.
20. The non-plant ell according to Claim 19, wherein the microbial cell comprises a yeast cell.
21. The non-plant cell of Claim 1 , further comprising a protein engineered variant of N- methylcoclaurine hydroxylase.
22. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
23. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
34.
24. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
35.
25. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
36.
26. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
37.
27. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
38.
28. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
39.
29. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO.
40.
30. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 41.
31. The non-plant cell of Claim 21 , wherein the protein engineered variant of N- methylcoclaurine hydroxylase comprises an amino acid sequence substantially similar identical to a SEQ ID NO. selected from SEQ ID NOs. 33-41 .
32. The non-plant cell of any of Claims 21 - 31 , wherein the protein engineered variant of N-methylcoclaurine hydroxylase has an activity on coclaurine that enables de novo norreticuline biosynthesis.
33. The non-plant cell of Claim 1 , further comprising a protein engineered variant of scoulerine 9-O-methyltransferase.
34. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 56.
35. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 57.
36. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 58.
37. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 59.
38. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 60.
39. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 61 .
40. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 62.
41 . The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 63.
42. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 64.
43. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprising an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO. 65.
44. The non-plant cell of Claim 33, wherein the protein engineered variant of scoulerine 9-O-methyltransferase comprises an amino acid sequence substantially similar identical to a SEQ ID NO. selected from SEQ ID NOs. 56-65.
45. The non-plant cell of any of Claims 33 - 44, wherein the protein engineered variant of scoulerine 9-O-methyltransferase has an activity on 1 -benzylisoquinoline alkaloids of O- methylation at the 3’ position.
46. The non-plant cell of Claim 45, wherein the protein engineered variant of scoulerine 9-O-methyltransferase enables de novo biosynthesis of THP.
47. The non-plant cell according to any of Claims 21 -46, wherein the non-plant cell comprises a microbial cell.
48. The non-plant cell according to Claim 47, wherein the microbial cell comprises a yeast cell.
49. The non-plant cell according to any of Claims 1 - 19 and 21 - 47, wherein the non- plant cell comprises a bacterial cell.
50. A method for producing THP, the method comprising
(a) culturing a cell of any of Claims 1 - 49 under conditions suitable for protein production;
(b) adding a starting compound to the cell culture; and
(c) recovering THP from the culture.
51 . The method according to Claim 50, wherein the cells are cultured in a fed-batch or batch fermentation.
52. The method according to any of Claims 50 - 51 , wherein the starting compound added to the cell culture is a sugar or a substrate which contains one or more sugars, or which is converted to one or more sugars during microbial fermentation.
53. The method according to any of Claims 50 - 52, wherein the starting compound added to the cell culture is an amino acid or a mixture comprising one or more amino acids, or a substrate which is converted to one or more amino acids during microbial fermentation.
54. The method according to any of Claims 50 - 53, wherein the THP is recovered via a process comprising a liquid-liquid extraction, chromatography separation, distillation, or recrystallization.
55. The method according to any of Claims 50 - 54, wherein the method further comprises producing a product from the recovered THP.
56. The method according to Claim 55, wherein the product is an API.
57. The method according to Claim 56, wherein the API is selected from the group consisting of atracurium, cisatracurium and papaverine.
58. The method according to Claim 57, wherein the API selected from the group consisting of atracurium and cisatracurium.
59. The method according to Claim 58, wherein the API is papaverine.
60. The method according to Claim 59, wherein the method comprises oxidizing the THP product to produce papaverine.
61 . The method according to Claim 60, wherein the method comprises chemically oxidizing THP with a chemical oxidizer to produce papaverine.
62. The method according to Claim 61 , wherein the chemical oxidizer is selected from the group consisting of hydrogen peroxide and potassium persulfate.
63. The method according to Claim 62, wherein the chemical oxidizer is hydrogen peroxide.
64. The method according to any of Claims 61 - 63, wherein the chemically oxidizing is carried at a pH ranging from 7 to 10.
65. A method of producing papaverine, the method comprising chemically oxidizing THP with a chemical oxidizer to produce papaverine.
66. The method according to Claim 65, wherein the chemical oxidizer is selected from the group consisting of hydrogen peroxide and potassium persulfate.
67. The method according to Claim 66, wherein the chemical oxidizer is hydrogen peroxide.
68. The method according to any of Claims 65 - 67, wherein the chemically oxidizing is carried at a pH ranging from 7 to 10.
69. The method according to any of Claims 65 - 68, wherein the THP is biosynthetically produced by a non-plant cell according to any of Claims 1 - 49.
70. A mutant M-methylcoclaurine hydroxylase (NMCH) or nucleic acid coding sequence therefore.
71. The mutant according to Claim 70, wherein the mutant comprises higher activity on the non-native substrate coclaurine relative to wild type NMCH.
72. A mutant scoulerine 9-O-methyltransferase (S9OMT) or coding sequence therefore.
73. The mutant according to Claim 72, wherein the mutant comprises higher activity on non-native substrates norreticuline and norlaudanine relative to wild-type S9OMT.
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