EP4680720A1 - Optimized production of branch point compounds and derivatives using alternative isopentenyl diphosphate-supplying pathways - Google Patents

Optimized production of branch point compounds and derivatives using alternative isopentenyl diphosphate-supplying pathways

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Publication number
EP4680720A1
EP4680720A1 EP24710767.5A EP24710767A EP4680720A1 EP 4680720 A1 EP4680720 A1 EP 4680720A1 EP 24710767 A EP24710767 A EP 24710767A EP 4680720 A1 EP4680720 A1 EP 4680720A1
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EP
European Patent Office
Prior art keywords
seq
synthase
polypeptide
phosphate
mevalonate
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP24710767.5A
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German (de)
French (fr)
Inventor
Sotirios KAMPRANIS
Victor Forman
Oskar OLOFSSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Evodiabio Aps
Original Assignee
Evodiabio Aps
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Application filed by Evodiabio Aps filed Critical Evodiabio Aps
Publication of EP4680720A1 publication Critical patent/EP4680720A1/en
Pending legal-status Critical Current

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Definitions

  • the present disclosure describes a method for preparing branch point compounds and derivatives using alternative isopentenyl diphosphate-supplying pathways. Further, the present disclosure describes host cells capable of performing said method as well as a fermentation liquid comprising the compounds prepared by the method.
  • Terpenes, terpenoids, derivatives thereof and other prenylated aromatic compounds are widely used e.g. as pharmaceuticals, cosmetics, nutraceuticals, flavors, fragrances and pesticides. Methods for increasing the production of these compounds in natural or engineered cells are abundant in the art.
  • MEP pathway mainly prokaryotes
  • MVA pathway both leading to the formation of DMAPP and IPP
  • GPP is converted either into a wide array of monoterpenes by monoterpene synthases (MTSs) that rearrange the 10-carbons backbone of GPP into various monoterpenes or precursors thereof, or it is further elongated into FPP or GGPP by successive addition of IPP molecules to form sesquiterpenes and diterpenes respectively.
  • MESs monoterpene synthases
  • GPP also serves as the precursor for the synthesis of a number of compounds that contain a terpene moiety, such as cannabinoids, iridoids, monoterpene indole alkaloids, prenylated aromatic compounds, and other meroterpenoids.
  • Yeast is considered a good host for terpene production because of its ease to be engineered, its native mevalonate pathway, and a good capacity to harbor functional cytochromes P450 in its endoplasmic reticulum (ER) membrane for terpene scaffold decoration. It has shown great capacity at producing sesquiterpenes, such as artemisinin and farnesene, at industrial scale. However, the production of monoterpenes has so far been considerably less successful.
  • Compartmentalization is a strategy used by eukaryotic cells to optimize their own metabolism.
  • Organelles such as mitochondria, peroxisomes, and the endoplasmic reticulum (ER) are designed to protect the rest of the cells from toxic compounds, isolate intermediates from competing pathways, shield enzymes from inhibitors, and, overall, provide a more suitable environment for a reaction to occur away from the main bulk of the metabolism.
  • KR20190079575A discloses a recombinant yeast wherein the number of peroxisomes is increased, leading to increased terpenoid production. Also disclosed is insertion of a heterologous geranylgeranyl pyrophosphate synthase.
  • US20130302861A1 discloses terpenoid production in yeast by localizing a terpene synthase to the mitochondria. The exemplification focuses on FPP-derived sesquiterpenes.
  • a host cell comprising, i) a peroxisomally-localized enzyme catalyzing the formation of a branch point compound which in a metabolic pathway can be metabolized by a prioritized metabolic pathway and one or more nonprioritized pathways leading to different metabolites, ii) a peroxisomally-localized enzyme catalyzing the first step of a non-prioritized metabolic pathway for the branch point compound, and iii) a peroxisomally-localized enzyme having kinase activity, and/or genes encoding the peroxisomally-localized enzymes of i), ii) and iii).
  • a cell culture comprising the host cell or yeast cell defined herein, and a growth medium.
  • a method for producing a compound selected from the group consisting of: a monoterpenoid, a sesquiterpenoid, a diterpenoid, a cannabinoid, a monoterpene indole alkaloid, and a prenylated aromatic compound comprising the steps of: a) providing a host cell or a yeast cell as defined herein; b) fermenting the host cell or yeast cell in a substrate supporting growth of the cell; c) when required, providing the cell with a substrate to be prenylated, for example wherein the substrate to be prenylated is selected from: olivetolic acid, olivetolic acid derivatives, naringenin, genistein, resveratrol or p-coumaric acid, and d) recovering the compound from the fermentation broth.
  • Figure 1 shows an evaluation of the performance of an isoprenoid alcohol-type pathway in the yeast peroxisome for terpenoid production.
  • Linalool production in the form of linalool titer i.e., mg of linalool produced per L of culture
  • OD Optical Density
  • Strain xEVO838 serves as the control.
  • Strains xEVO820 and xEVO823 contain the isoprenoid alcohol-type pathway constructed with two different IPK enzymes.
  • Strain xEVO820 contains a peroxisomally-localized variant of IPK from M.
  • Figure 2 shows an evaluation of the performance of an Aeropyrum-type pathway (Pathway 3) in the yeast peroxisome for terpenoid production. Linalool production in the form of linalool titer (i.e., mg of linalool produced per L of culture) per unit of Optical Density (OD) of the culture was evaluated by GC-FID using commercially available linalool (Sigma-Aldrich, USA) as a standard. Strain xEVO838 serves as the control.
  • Figure 3 shows an evaluation of the performance of a Thermoplasma-type pathway (Pathway 2) in the yeast peroxisome for terpenoid production.
  • Linalool production in the form of linalool titer i.e., mg of linalool produced per L of culture
  • OD Optical Density
  • Strain xEVO838 serves as the control.
  • Strains xEVO810 and xEVO813 contain the Thermoplasma-type pathway (Pathway 2) constructed with two different IPK enzymes.
  • heterologous or recombinant or “genetically modified” and their grammatical equivalents as used herein interchangeably refers to entities "derived from a different species or cell".
  • a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell.
  • heterologous also covers one or more endogenous genes under a non-native or altered promoter, or located in a locus other than the native locus.
  • control sequence refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide.
  • a control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide.
  • Control sequences include, but are not limited to leader sequences, polyadenylation sequence, pro-peptide coding sequence, promoter sequences, signal peptide coding sequence, translation terminator (stop) sequences and transcription terminator (stop) sequences.
  • To be operational control sequences usually must include promoter sequences, transcriptional and translational stop signals.
  • Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.
  • host cell refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present disclosure.
  • Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • cell culture refers to a culture medium comprising a plurality of host cells of the disclosure.
  • a cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains.
  • the culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.
  • a recombinant host e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium
  • additional components e.g.,
  • a host cell comprising, i) a peroxisomally-localized enzyme catalyzing the formation of a branch point compound which in a metabolic pathway can be metabolized by a prioritized metabolic pathway and one or more non- prioritized pathways leading to different metabolites, ii) a peroxisomally-localized enzyme catalyzing the first step of a non-prioritized metabolic pathway for the branch point compound, and iii) a peroxisomally-localized enzyme having kinase activity, and/or genes encoding the peroxisomally-localized enzymes of i), ii) and iii).
  • the present disclosure provides peroxisomal localization by inserting a peroxisomal localization signal in the genes encoding the peroxisomally-localized enzymes.
  • the peroxisomally-localized enzyme of i), ii), and/or iii) are heterologous to the host cell.
  • the host cell comprises or expresses or is configured to express one or more sequences selected from the group consisting of: SEQ. ID NO: 1 to SEQ ID NO: 98.
  • the host cell is a yeast cell.
  • the yeast cell belongs to a genus selected from the group consisting of: Saccharomyces, Pichia, Candida, Ogatea, Yarrowia, Kluyveromyces, Rhodotorula, Rhodosporidium, Cryptococcus, Schizosaccharomyces, Trichosporon and Lipomyces.
  • the yeast cell is of a species selected from the group consisting of: Saccharomyces spp., Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Ogataea polymorpha, Kluyveromyces marxianus, Kluyveromyces lactis, Candida albicans, Candida boidinii, Schizosaccharomyces pombe, Scheffersomyces stipidis, and Dekkera bruxellensis.
  • GPP Geranyl diphopsphate
  • NPP neryl diphosphate
  • the branch point compound is geranyl diphopsphate (GPP). In some embodiments, the branch point compound is neryl diphosphate (NPP).
  • the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a GPP synthase or an NPP synthase
  • the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
  • the NPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 48, for example wherein the polypeptide has the sequence of SEQ ID NO: 48.
  • the GPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 45, for example wherein the polypeptide has the sequence of SEQ ID NO: 45.
  • the terpene synthase is selected from the group consisting of: (+)- limonene synthase, (-)-limonene synthase, 1,8-cineole synthase, sabinene synthase, camphene synthase, geraniol synthase, linalool synthase, myrcene synthase, bornyl diphosphate synthase, alphaterpineol synthase, tricyclene synthase, alpha-thujene synthase, alpha-phellandrene synthase, betaphellandrene synthase, (E)-beta-ocimene synthase, Trans-ocimene synthase, gamma-terpinene synthase, alpha-terpineol synthase, alpha-pinene synthase, beta-pin
  • the terpene synthase is selected from the group consisting of: a (+)-limonene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 20, for example the polypeptide of SEQ ID NO: 20; a (-)-limonene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 21, for example the polypeptide of SEQ ID NO: 21; a 1,8-cineole synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 22, for example the polypeptide of SEQ ID NO: 22; a sabinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 26, for example the polypeptide of SEQ ID NO: 26; a camphene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 23, for example the polypeptide of SEQ ID NO: 23; a
  • polypeptide of SEQ ID NO: 28 for example the polypeptide of SEQ ID NO: 28; an alpha-terpineol synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO:
  • polypeptide of SEQ ID NO: 29 29, for example the polypeptide of SEQ ID NO: 29; a tricyclene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 31, for example the polypeptide of SEQ ID NO: 31; an alpha-thujene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 30, for example the polypeptide of SEQ ID NO: 30; an alpha-phellandrene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 33, for example the polypeptide of SEQ ID NO: 33; a beta-phellandrene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO:
  • polypeptide of SEQ ID NO: 35 for example the polypeptide of SEQ ID NO: 35; a gamma-terpinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 34, for example the polypeptide of SEQ ID NO: 34; a trans-ocimene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 37, for example the polypeptide of SEQ ID NO: 37; an alpha-pinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 38, for example the polypeptide of SEQ ID NO: 38; a beta-pinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 39, for example the polypeptide of SEQ ID NO: 39; a 3-carene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 40, for example the polypeptide of SEQ ID NO: 40;
  • the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 9, 10, 11, or 12 carbon atoms.
  • the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: an aromatic prenyltransferase, a C- prenyltransferase, an O-methyltransferase, and a geranyldiphosphate:olivetolate geranyltransferase.
  • the geranyldiphosphate:olivetolate geranyltransferase has at least 80% sequence identity to the polypeptide of SEQ ID NO: 44. In some embodiments, the geranyldiphosphate:olivetolate geranyltransferase has the sequence of SEQ ID NO: 44.
  • the host cell is provided comprising a Linalool synthase, such as a linalool synthase having at least 80% identity to the polypeptide of SEQ ID NO: 87.
  • the host cell comprising a polynucleotide encoding a Linalool synthase, such as a polynucleotide having at least 80% identity to the polypeptide of SEQ ID NO: 88.
  • the host cell is provided comprising a phosphoribosylanthranilate isomerase, such as a phosphoribosylanthranilate isomerase having at least 80% identity to the polypeptide of SEQ ID NO: 89.
  • a phosphoribosylanthranilate isomerase such as a phosphoribosylanthranilate isomerase having at least 80% identity to the polypeptide of SEQ ID NO: 89.
  • the host cell comprising a polynucleotide encoding a phosphoribosylanthranilate isomerase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 90.
  • the host cell comprising an Imidazoleglycerol-phosphate dehydratase, such as an Imidazoleglycerol-phosphate dehydratase having at least 80% identity to the polypeptide of SEQ ID NO: 91.
  • the host cell comprising a polynucleotide encoding an Imidazoleglycerol-phosphate dehydratase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 92.
  • the host cell is provided comprising a phosphomevalonate kinase, such as a phosphomevalonate kinase having at least 80% identity to the polypeptide of SEQ ID NO: 93.
  • the host cell is provided comprising a polynucleotide encoding a phosphomevalonate kinase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 94.
  • the host cell is provided comprising a Mevalonate kinase, such as a Mevalonate kinase having at least 80% identity to the polypeptide of SEQ ID NO: 95.
  • the host cell is provided comprising a polynucleotide encoding a Mevalonate kinase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 96.
  • the host cell is provided comprising a Mevalonate pyrophosphate decarboxylase, such as a Mevalonate pyrophosphate decarboxylase having at least 80% identity to the polypeptide of SEQ ID NO: 97.
  • a Mevalonate pyrophosphate decarboxylase such as a Mevalonate pyrophosphate decarboxylase having at least 80% identity to the polypeptide of SEQ ID NO: 97.
  • the host cell comprising a polynucleotide encoding a Mevalonate pyrophosphate decarboxylase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 98.
  • a host cell comprising one or more of the above polynucleotide and/or polypeptides.
  • FPP Farnesyl diphosphate
  • the branch point compound is farnesyl diphopsphate (FPP).
  • the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is an FPP synthase
  • the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
  • the FPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 46, for example wherein the polypeptide has the sequence of SEQ ID NO: 46.
  • the terpene synthase is selected from the group consisting of: farnesol synthase, farnesene synthase, beta-caryophyllene synthase, alpha-humulene synthase, bisabolol synthase, bisabolene synthase, germacrene synthase, bergamotene synthase, muurolol synthase, santalol synthase, thujopsene synthase, cedrene synthase, santalene synthase, valencene synthase, aristolene synthase, aristolochene synthase, cadinene synthase, sinensal synthase.
  • farnesol synthase farnesene synthase
  • beta-caryophyllene synthase alpha-humulene synthase
  • bisabolol synthase bis
  • the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: an aromatic prenyltransferase, a C- prenyltransferase, and an O-methyltransferase.
  • DMAPP Dimethylallyl diphosphate
  • the branch point compound is dimethylallyl diphopsphate (DMAPP).
  • the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a DMAPP synthase or an isopentelyl diphosphate isomerase (IDI)
  • IDI isopentelyl diphosphate isomerase
  • the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
  • the terpene synthase is an isoprene synthase.
  • the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 4, 5, 6, or 7 carbon atoms.
  • the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: lavandulyl diphosphate synthase, chrysanthemyl diphosphate synthase, maconellyl diphosphate synthase, planococcyl diphosphate synthase, O-methyltransferase, C-methyltranaferase, and aromatic prenyltransferase.
  • the branch point compound is geranylgeranyl diphosphate (GGPP).
  • the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a GGPP synthase
  • the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
  • the GGPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ. ID NO: 47, for example wherein the polypeptide has the sequence of SEQ ID NO: 47.
  • the host cell comprises one or more polynucleotides selected from the group consisting of: a) a polynucleotide which is at least 80% identical to SEQ ID NO: 82; b) a polynucleotide which is at least 80% identical to SEQ ID NO: 80; c) a polynucleotide which is at least 80% identical to SEQ ID NO: 82; d) a polynucleotide which is at least 80% identical to SEQ ID NO: 96; e) a polynucleotide which is at least 80% identical to SEQ ID NO: 94; f) a polynucleotide which is at least 80% identical to SEQ ID NO: 98; g) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5-phosphate decarboxylase (M5PD) of SEQ ID NO: 7; h)
  • M5PD mevalonate
  • the host cell is provided wherein the corresponding: a) acetoacetyl-CoA thiolase has at least 80% identity to the polypeptide of SEQ ID NO: 1; b) HMG-CoA synthase (HMGS) has at least 80% identity to the polypeptide of SEQ ID NO: 2; c) HMG-CoA reductase (HMGR) has at least 80% identity to the polypeptide of SEQ ID NO: 3; d) mevalonate 5-kinase (MV5K) has at least 80% identity to the polypeptide of SEQ ID NO: 4; e) phosphomevalonate kinase (PMVK) has at least 80% identity to the polypeptide of SEQ ID NO: 5; f) mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) has at least 80% identity to the polypeptide of SEQ ID NO: 6; g) mevalonate
  • the host cell is provided wherein the corresponding: a) acetoacetyl-CoA thiolase has at least 80% identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO: 81; b) HMG-CoA synthase (HMGS) has at least 80% identity to the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 79; c) HMG-CoA reductase (HMGR) has at least 80% identity to the polypeptide of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 81; d) mevalonate 5-kinase (MV5K) has at least 80% identity to the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 95; e) phosphomevalonate kinase (PMVK) has at least 80% identity to the polypeptide of SEQ ID NO: 5 or SEQ ID NO: 93; f
  • the host cell comprises one or more polynucleotides selected from the group consisting of: a) a polynucleotide encoding a polypeptide which is at least 80% identical to acetyl-CoA thiolase of SEQ ID NO: 1, or SEQ ID NO: 81; b) a polynucleotide encoding a polypeptide which is at least 80% identical to HMG-CoA synthase (HMGS) of SEQ ID NO: 2 or SEQ ID NO: 79; c) a polynucleotide encoding a polypeptide which is at least 80% identical to HMG-CoA reductase (HMGR) of SEQ ID NO: 3, or SEQ ID NO: 81; d) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5-kinase (MV5K) of SEQ ID NO: 4, or SEQ
  • the operative metabolic pathway is pathway 1 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) mevalonate 5-phosphate decarboxylase (M5PD) converting mevalonate 5-phosphate to isopentenyl phosphate (IP); f) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to is
  • the host cell is provided wherein the corresponding: a) mevalonate 5-phosphate decarboxylase has at least 80% identity to the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 49; and/or b) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
  • a) mevalonate 5-phosphate decarboxylase has at least 80% identity to the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 49
  • IPK isopentenyl phosphate kinase
  • the host cell comprising: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and d) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized GPP synthase (GPPS).
  • GPPS peroxisomally localized GPP synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized neryl diphosphate synthase (NPPS).
  • NPPS peroxisomally localized neryl diphosphate synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized geranylgeranyl diphosphate synthase (GGPPS).
  • GGPPS peroxisomally localized geranylgeranyl diphosphate synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized farnesyl diphopsphate synthase (FPPS). Pathway 2
  • the operative metabolic pathway is pathway 2 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 3-kinase (MV3K) converting mevalonate to mevalonate-3-phosphate; e) mevalonate-3-phosphate-5-kinase (M3P5K) converting mevalonate-3-phosphate to mevalonate-3, 5-bisphosphate; f) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) converting mevalonate
  • the host cell is provided wherein the corresponding: a) mevalonate 3-kinase (MV3K) has at least 80% identity to the polypeptide of SEQ ID NO: 9, or SEQ ID NO: 61; b) mevalonate-3-phosphate-5-kinase (M3P5K) has at least 80% identity to the polypeptide of SEQ ID NO: 10, or SEQ ID NO: 63; c) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) has at least 80% identity to the polypeptide of SEQ ID NO: 11, or SEQ ID NO: 65; and/or d) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
  • MV3K mevalonate 3-kinase
  • M3P5K mevalonate-3-phosphate-5-kinase
  • BMD meval
  • the host cell comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and/or d) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
  • the host cell further comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ERG20(N127W)-SKL (SEQ ID NO: 85), and/or b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to FpTPS-SKL (SEQ ID NO: 87).
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized GPP synthase (GPPS).
  • GPPS peroxisomally localized GPP synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized neryl diphosphate synthase (NPPS).
  • NPPS peroxisomally localized neryl diphosphate synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized geranylgeranyl diphosphate synthase (GGPPS).
  • GGPPS peroxisomally localized geranylgeranyl diphosphate synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized farnesyl diphopsphate synthase (FPPS).
  • FPPS peroxisomally localized farnesyl diphopsphate synthase
  • the operative metabolic pathway is pathway 3 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) converting mevalonate 5-phosphate to trans-anhydromevalonate 5-phosphate; f) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) converting mevalonate 5-
  • the corresponding: a) mevalonate 5-phosphate dehydratase (PMDh) has at least 80% identity to the polypeptide of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; and/or b) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59.
  • PMDh mevalonate 5-phosphate dehydratase
  • AMPD trans-anhydromevalonate 5-phosphate decarboxylase
  • AMPD trans-anhydromevalonate 5-phosphate decarboxylase
  • the host cell comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and/or d) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
  • the host cell further comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ERG20(N127W)-SKL (SEQ ID NO: 85), and/or b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to FpTPS-SKL (SEQ ID NO: 87).
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 3, and a peroxisomally localized GPP synthase (GPPS).
  • GPPS peroxisomally localized GPP synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-i) defined herein for pathway 3, and a peroxisomally localized neryl diphosphate synthase (NPPS).
  • NPPS peroxisomally localized neryl diphosphate synthase
  • a yeast cell comprising all the peroxisomally-localized polypeptides a)-i) defined herein for pathway 3, and a peroxisomally localized geranylgeranyl diphosphate synthase (GGPPS).
  • GGPPS peroxisomally localized geranylgeranyl diphosphate synthase
  • a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-i) defined herein for pathway 3, and a peroxisomally localized farnesyl diphopsphate synthase (FPPS).
  • the peroxisomally-localized enzyme having kinase activity is heterologous to the host cell.
  • the peroxisomally-localized enzyme of iii) having kinase activity is a peroxisomally-localized kinase.
  • IPK Isopentenyl phosphate kinase
  • the kinase is an isopentenyl phosphate kinase (IPK).
  • IPK isopentenyl phosphate kinase
  • the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), Methanothermobacter thermautotrophicus IPK (MtIPK), Thermoplasma acidophilum IPK variant IVG (TalPK(IVG), and TalPK wild-type.
  • the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 17, for example the polypeptide of SEQ UD NO: 17; Thermoplasma acidophilum IPK variant IVG (TalPK(IVG), such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 19, or such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 18, such as the polypeptide of SEQ ID NO: 18, or such as the polypeptide of SEQ UD NO: 19; TalPK wild-type, and Methanothermobacter thermautotrophicus IPK (MtIPK), such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 51, for example the polypeptide of SEQ ID NO: 51.
  • AtIPK Arabidopsis
  • the host cell comprises a polynucleotide encoding an IPK of the present disclosure.
  • the polynucleotide has at least 80% identity to the polynucleotide of SEQ ID NO: 52.
  • the host cell is a yeast cell comprising a peroxisomally-localized isopentenyl phosphate kinase (IPK), optionally wherein the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), Methanothermobacter thermautotrophicus IPK (MtIPK), Thermoplasma acidophilum IPK variant IVG (TalPK(IVG)), and TalPK wild-type.
  • IPK peroxisomally-localized isopentenyl phosphate kinase
  • yeast strain used as the basis to derive the strains described in this application is EGY48, a Saccharomyces cerevisiae (S. cerevisiae) strain disclosed in (Thomas & Rothstein, 1989) and (Ignea et al., 2011) with the genotype specified in Table 3.
  • Yeast codon-optimized genes were purchased from Twist Biosciences, Inc. (USA) in synthetic form, amplified by PCR using the corresponding primers listed in Table 6, placed under the control of an inducible promoter (P AL1, PGAL2, or PGAL10), and inserted into one of the following integration plasmids, pAssl, pAss2, pAss3, pAss2A, pAss2B, or pAss2C.
  • This plasmid integration system is disclosed in (Forman et al., 2022) and the plasmids generated in this application are listed in Table 4. Two integration sites for high expression were used throughout this study: X-4 and XII-2, as previously described (Jensen et al., 2012)
  • the cloning method used for vector construction in this application is the Uracil-Specific- Excision-Reagent (USER) method, described in (Nour-Eldin et al., 2010)
  • the gene products generated by the integrated genes contain a C-terminally fused amino acid sequence (Serine-Lysine-Leucine (SKL)) to target them to the yeast peroxisome, as demonstrated in (Dusseaux et al., 2020).
  • Transformations were conducted using the standard protocol outlined by (Daniel Gietz & Woods, 2002). Transformants were selected by picking eight colonies of S. cerevisiae for each transformation and plating them on selective auxotrophic minimal media. To confirm the presence of the integrative vectors, each of the eight colonies was genotyped using primers specific to the genomic locus where integration was targeted (The primers used for genotyping are listed in Table 6).
  • S. cerevisiae pre-cultures of each studied strain were individually selected and grown overnight at 30°C, in a shaking incubator at 250 revolutions per minute, in a 24-well plate containing 1 mL YPD and 6% glucose. After incubation, 150 pL of cell suspension was transferred to a new 24- well plate where 0.6 mL of sunflower oil and 2.7 mL of rich media containing 0.3 g/L raffinose and 0.8 g/L galactose were added. Cultures were grown at 30°C with shaking at 250 revolutions per minute for 72 hours, and the sunflower overlay phase was isolated by centrifugation and subsequently analyzed using GC-FID.
  • a GC-FID SCION 436-GC was utilized for GC-FID analysis using an HP-5MS Ultra Inert column with a 5 % Phenyl-methylpolysiloxane stationary phase. Injection volume was set to 1 pL and the sample was injected by a CP8400 autosampler. The oven temperature was initially set at 40 °C for 3 min and subsequently increased to 80 °C at a rate of 3 °C/min. When reaching 80 °C the rate was increased to 30 °C/min until reaching 300 °C, followed by a hold of 10 min.
  • Example 2 Construction of a control strain to evaluate peroxisomal pathway performance
  • control strain xEVO838 was constructed through the integration of the following codon-optimized genes within the yeast genome: Enterococcus faecalis EfmvaE and EfmvaS genes (equivalent to the function of ERG10, ERG13, and HMG1 in yeast), ScERG12, SclDI, and two copies of the ERG20(N127W) variant (serving as a geranyl diphosphate synthase). Furthermore, two copies of the gene encoding for the FpTPS linalool synthase were introduced, allowing assessment of the flux through the pathway by quantifying linalool production.
  • Strain xEVO838 contains a terpene synthase (FpTPS) and a prenyltransferase (ERG20(N127W)) together with an incomplete mevalonate pathway (the strain lacks the peroxisomal-targeted versions of ScERG8 and ScERG19). Therefore, strain xEVO838 serves as the basis of comparison with other strains that contain complete designs of alternative forms of the mevalonate pathway in the peroxisome.
  • FpTPS terpene synthase
  • ERP20(N127W) prenyltransferase
  • xEVO820 and xEVO823 also contain peroxisomally-localized variants of the enzymes EfmvaE-SKL, EfmvaS-SKL, SclDI-SKL, ERG20(N127W)-SKL, and FpTPS-SKL to enable linalool production. Additionally, strain xEVO820 contains a peroxisomally-localized variant of IPK from Methanothermobacter thermautotrophicus and strain xEVO823 contains a peroxisomally- localized variant of IPK from Thermoplasma acidophilum.
  • strains xEVO820 and xEVO823 in terpene production were evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 1, linalool production by strains xEVO820 and xEVO823 was similar to the control strain xEVO838.
  • strains xEVO805 and xEVO808 contain the enzymes mevalonate 5-phosphate dehydratase subunit 1 (PMDhl-SKL), mevalonate 5- phosphate dehydratase subunit 2 (PMDh2-SKL), and trans-anhydromevalonate 5-phosphate decarboxylase (AMPD-SKL) from Methanosarcina mazei, equipped with C-terminal peroxisomal localization signals (SKL).
  • PMDhl-SKL mevalonate 5-phosphate dehydratase subunit 1
  • PMDh2-SKL mevalonate 5- phosphate dehydratase subunit 2
  • AMPD-SKL trans-anhydromevalonate 5-phosphate decarboxylase
  • xEVO805 and xEVO808 also contain the peroxisomally- localized variants enzymes EfmvaE-SKL, EfmvaS-SKL, ScERG12-SKL, SclDI-SKL, ERG20(N127W)-SKL, and FpTPS-SKL to enable linalool production.
  • strain xEVO805 contains a peroxisomally- localized variant of IPK from M. thermautotrophicus
  • strain xEVO808 contains a peroxisomally- localized variant of IPK from T. acidophilum.
  • the combination of these enzymes (PMDhl, PMDh2, AMPD, and IPK) converts mevalonate 5-phosphate to IPP in the Aeropyrum-type pathway.
  • strains xEVO805 and xEVO808 in terpene production were evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 2, linalool production by strains xEVO805 and xEVO808 was 82% and 77% higher, respectively, than the control strain xEVO838.
  • Example 5 Reconstruction of a Thermoplasma-type pathway (Pathway 2) in S. cerevisiae peroxisome
  • strains xEVO810 and xEVO813 contain the enzymes mevalonate-3-kinase (TaMV3K-SKL), mevalonate-3-phosphate 5-kinase (TaM3P5K), and Mevalonate 3,5-bisphosphate decarboxylase (TaBMDl-SKL) from T. acidophilum, all equipped with C-terminal peroxisomal localization signals (SKL).
  • xEVO810 and xEVO813 also contain peroxisomally-localized variants of the enzymes EfmvaE-SKL, EfmvaS-SKL, ScERG12-SKL, SclDI-SKL, ERG20(N127W)-SKL, and FpTPS-SKL to enable linalool production.
  • strain xEVO810 contains the IPK from M. thermautotrophicus
  • strain xEVO813 contains the IPK from T. acidophilum.
  • the combination of these enzymes (TaMV3K, TaM3P5K, TaBMDl, and IPK) converts mevalonate to IPP in the Thermoplasma-type pathway.
  • strains xEVO810 and xEVO813 in terpene production were evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 3, linalool production by strains xEVO810 and xEVO813 was 3.45 and 3.47 times higher, respectively, than the control strain xEVO838.
  • Example 6 Reconstruction of an Haloarchea-type pathway (Pathway 1) in the S. cerevisiae peroxisome.
  • Haleoarchaea-type MVA pathway (Pathway 1) within the yeast peroxisome, we constructed a strain that incorporates this alternative pathway in the peroxisomes.
  • This alternative, Haloarchea-type, pathway differs from the classical MVA pathway in the last two steps. Instead of ScERG8 and ScERG19 (in the case of S. cerevisiae), it utilizes a phosphomevalonate decarboxylase (M5PD) and an isopentenyl phosphate kinase (IPK).
  • M5PD phosphomevalonate decarboxylase
  • IPK isopentenyl phosphate kinase
  • strains xEVO792 and xEVO789 in terpene production were evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 4, linalool production by strains xEVO792 and xEVO789 was 4.05 and 4.37 times higher, respectively, than the control strain.
  • Table 4 Yeast plasmids used in this application.
  • Table 5 List of protein sequences used in this application
  • Table 6 -List of primer sequences used in this application.

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Abstract

The present disclosure describes a method for preparing branch point compounds and derivatives using alternative isopentenyl diphosphate-supplying pathways. Further, the present disclosure describes host cells capable of performing said method as well as a fermentation liquid comprising the compounds prepared by the method.

Description

Optimized production of branch point compounds and derivatives using alternative isopentenyl diphosphate-supplying pathways
Field
[0001] The present disclosure describes a method for preparing branch point compounds and derivatives using alternative isopentenyl diphosphate-supplying pathways. Further, the present disclosure describes host cells capable of performing said method as well as a fermentation liquid comprising the compounds prepared by the method.
Background
[0002] Terpenes, terpenoids, derivatives thereof and other prenylated aromatic compounds are widely used e.g. as pharmaceuticals, cosmetics, nutraceuticals, flavors, fragrances and pesticides. Methods for increasing the production of these compounds in natural or engineered cells are abundant in the art.
[0003] Using engineered microorganisms for producing valuable molecules from renewable feedstock is a desirable alternative from conventional means of production. However, achieving economically viable yield, titers and productivity is a major roadblock towards industrialization. Obstacles often encountered arise from the standoff between the engineered pathway and the native metabolism that are pulling in opposite directions. Metabolism has evolved towards meeting the needs for growth and rerouting it can be challenging due to multiple layers of control, such as gene regulation, negative feedback loops at the enzyme level by downstream products, and efficient competing pathways.
[0004] Monoterpenes and other geranyl diphosphate (GPP)-derived compounds, which are widely used as flavors, fragrances, pesticides and could find applications as drop in jet fuel or biopolymers, are a prime example of these issues. On one hand, extraction from plant natural sources can hardly meet the increasing demands and represents an environmental challenge, whereas, on the other hand, production by microbial host leads to low yield and is hindered by native metabolism constraints.
[0005] Monoterpene production by engineered microbes relies on either the MEP pathway (mainly prokaryotes) orthe MVA pathway, both leading to the formation of DMAPP and IPP, which are, in turn, condensed to form GPP. GPP is converted either into a wide array of monoterpenes by monoterpene synthases (MTSs) that rearrange the 10-carbons backbone of GPP into various monoterpenes or precursors thereof, or it is further elongated into FPP or GGPP by successive addition of IPP molecules to form sesquiterpenes and diterpenes respectively. GPP also serves as the precursor for the synthesis of a number of compounds that contain a terpene moiety, such as cannabinoids, iridoids, monoterpene indole alkaloids, prenylated aromatic compounds, and other meroterpenoids.
[0006] Yeast is considered a good host for terpene production because of its ease to be engineered, its native mevalonate pathway, and a good capacity to harbor functional cytochromes P450 in its endoplasmic reticulum (ER) membrane for terpene scaffold decoration. It has shown great capacity at producing sesquiterpenes, such as artemisinin and farnesene, at industrial scale. However, the production of monoterpenes has so far been considerably less successful.
[0007] This can be mainly explained by the rope-pulling game that is played at the GPP branch-point between native sterol biosynthesis and the heterologous pathway leading to monoterpenes, and which is largely in favor of the native metabolism. In wild-type yeast, there are no GPP-based compounds produced and the only purpose of GPP is to serve as an intermediate that is further elongated into FPP for the production of squalene in the sterol pathway. Because of this, no dedicated GPP synthase is present in yeast, and GPP is produced by a bi-functional GPP-FPP synthase, Erg20p, that has been shown to convert very efficiently GPP into FPP as soon as it is formed and channel it into sterol synthesis. Various strategies have been employed to downregulate Erg20p, either by converting it into a strict GPP synthase, or by reducing its activity, but the intrinsic essentiality of sterol synthesis have rendered those attempts only moderate fruitful, while decreasing cells viability by posing a burden on sterol synthesis.
[0008] Compartmentalization is a strategy used by eukaryotic cells to optimize their own metabolism. Organelles, such as mitochondria, peroxisomes, and the endoplasmic reticulum (ER), are designed to protect the rest of the cells from toxic compounds, isolate intermediates from competing pathways, shield enzymes from inhibitors, and, overall, provide a more suitable environment for a reaction to occur away from the main bulk of the metabolism.
[0009] An example of such a strategy has been reported recently, where geraniol production has been improved 11.5-fold by compartmentalizing an extra copy of the entire MVA pathway into the mitochondria together with a geraniol synthase, in comparison with the same modification in the cytosol.
[0010] While this proved to be a successful strategy, hijacking the mitochondria appeared to pose a metabolic burden to the strain with lower cells viability and growth. This can be attributed to the essential nature of the mitochondria as the powerhouse of the cells, which may hinder further engineering to reach the significantly higher titer needed for industrial application. These findings also showed that mitochondria might come with limitations on how far they can be engineered without compromising the integrity of the metabolism. [0011] US 20150010978 discloses methods for producing terpenoids in a vast number of cells by transforming the cells with genes encoding enzymes involved in the biosynthesis of the terpenoids. The genes may be introduced into the genomes of chloroplasts for cells having chloroplasts. The exemplification discloses production of di-terpenes.
[0012] KR20190079575A discloses a recombinant yeast wherein the number of peroxisomes is increased, leading to increased terpenoid production. Also disclosed is insertion of a heterologous geranylgeranyl pyrophosphate synthase.
[0013] US20130302861A1 discloses terpenoid production in yeast by localizing a terpene synthase to the mitochondria. The exemplification focuses on FPP-derived sesquiterpenes.
[0014] Guo-Song Liu et al (J. Agric. Food Chem. 2020, 68, 7, 2132-2138) reported the production of squalene, the FPP-based precursor of ergosterol, in yeast peroxisome demonstrating the functionality of the MVA pathway in this organelle. However, the resulting strain did not outperform its cytosolic counterpart, most probably due to the fact that the original pathway is already well-tuned and designed to efficiently produce squalene in the cytosol.
Summary
[0015] The present inventors have surprisingly demonstrated that alternative MVA pathways can also successfully be incorporated into the peroxisome, efficiently producing terpenes and related compounds.
[0016] Thus, in a first aspect a host cell is provided comprising, i) a peroxisomally-localized enzyme catalyzing the formation of a branch point compound which in a metabolic pathway can be metabolized by a prioritized metabolic pathway and one or more nonprioritized pathways leading to different metabolites, ii) a peroxisomally-localized enzyme catalyzing the first step of a non-prioritized metabolic pathway for the branch point compound, and iii) a peroxisomally-localized enzyme having kinase activity, and/or genes encoding the peroxisomally-localized enzymes of i), ii) and iii).
[0017] In a further aspect, a cell culture is provided, comprising the host cell or yeast cell defined herein, and a growth medium.
[0018] In a further aspect, a method is provided for producing a compound selected from the group consisting of: a monoterpenoid, a sesquiterpenoid, a diterpenoid, a cannabinoid, a monoterpene indole alkaloid, and a prenylated aromatic compound, comprising the steps of: a) providing a host cell or a yeast cell as defined herein; b) fermenting the host cell or yeast cell in a substrate supporting growth of the cell; c) when required, providing the cell with a substrate to be prenylated, for example wherein the substrate to be prenylated is selected from: olivetolic acid, olivetolic acid derivatives, naringenin, genistein, resveratrol or p-coumaric acid, and d) recovering the compound from the fermentation broth.
[0019] In some embodiments, a fermentation liquid is provided comprising the compound as defined herein and the host cell as defined herein.
Description of drawings and figures
[0020] The figures included herein are illustrative and simplified for clarity, and they merely show details which are essential to the understanding of the invention, while other details may have been left out. Throughout the specification, claims and drawings the same reference numerals are used for identical or corresponding parts. In the figures and drawing include herein:
Figure 1 shows an evaluation of the performance of an isoprenoid alcohol-type pathway in the yeast peroxisome for terpenoid production. Linalool production in the form of linalool titer (i.e., mg of linalool produced per L of culture) per unit of Optical Density (OD) of the culture was evaluated by GC-FID using commercially available linalool (Sigma-Aldrich, USA) as a standard. Strain xEVO838 serves as the control. Strains xEVO820 and xEVO823 contain the isoprenoid alcohol-type pathway constructed with two different IPK enzymes. Strain xEVO820 contains a peroxisomally-localized variant of IPK from M. thermautotrophicus and strain xEVO823 contains a peroxisomally-localized variant of IPK from T. acidophilum. Bars show the average of three experiments (n=3) and error bars correspond to the standard deviation (SD). Figure 2 shows an evaluation of the performance of an Aeropyrum-type pathway (Pathway 3) in the yeast peroxisome for terpenoid production. Linalool production in the form of linalool titer (i.e., mg of linalool produced per L of culture) per unit of Optical Density (OD) of the culture was evaluated by GC-FID using commercially available linalool (Sigma-Aldrich, USA) as a standard. Strain xEVO838 serves as the control. Strains xEVO805 and xEVO808 contain the Aeropyrum-type pathway (Pathway 3) constructed with two different IPK enzymes. Strain xEVO805 contains a peroxisomally-localized variant of IPK from M. thermautotrophicus and strain xEVO808 contains a peroxisomally-localized variant of IPK from T. acidophilum. Bars show the average of three experiments (n=3) and error bars correspond to the standard deviation (SD).
Figure 3 shows an evaluation of the performance of a Thermoplasma-type pathway (Pathway 2) in the yeast peroxisome for terpenoid production. Linalool production in the form of linalool titer (i.e., mg of linalool produced per L of culture) per unit of Optical Density (OD) of the culture was evaluated by GC-FID using commercially available linalool (Sigma-Aldrich, USA) as a standard. Strain xEVO838 serves as the control. Strains xEVO810 and xEVO813 contain the Thermoplasma-type pathway (Pathway 2) constructed with two different IPK enzymes. Strain xEVO810 contains a peroxisomally-localized variant of IPK from M. thermautotrophicus and strain xEVO813 contains a peroxisomally-localized variant of IPK from T. acidophilum. Bars show the average of three experiments (n=3) and error bars correspond to the standard deviation (SD).
Figure 4 shows an evaluation of the performance of a Haloarchea-type pathway (Pathway 1) in the yeast peroxisome for terpenoid production. Linalool production in the form of linalool titer (i.e., mg of linalool produced per L of culture) per unit of Optical Density (OD) of the culture was evaluated by GC-FID using commercially available linalool (Sigma-Aldrich, USA) as a standard. Strain xEVO838 serves as the control. Strains xEVO792 and xEVO789 contain the Haloarchea-type pathway (Pathway 1) constructed with two different IPK enzymes. Strain xEVO792 contains a peroxisomally-localized variant of IPK from M. thermautotrophicus and strain xEVO789 contains a peroxisomally-localized variant of IPK from T. acidophilum. Bars show the average of three experiments (n=3) and error bars correspond to the standard deviation (SD).
Incorporation by reference
[0021] All publications, patents, and patent applications referred to herein are 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. In the event of a conflict between a term herein and a term in an incorporated reference, the term herein prevails and controls.
Details
Definitions
[0022] The terms "heterologous" or "recombinant" or "genetically modified" and their grammatical equivalents as used herein interchangeably refers to entities "derived from a different species or cell". For example, a heterologous or recombinant polynucleotide gene is a gene in a host cell not naturally containing that gene, i.e. the gene is from a different species or cell type than the host cell. The term "heterologous" also covers one or more endogenous genes under a non-native or altered promoter, or located in a locus other than the native locus. The terms as used herein about microbial host cells refers to microbial host cells comprising and expressing heterologous or recombinant polynucleotide genes. [0023] The term "operative metabolic pathway" as used herein is intended to mean two or more enzymes acting sequentially in a live cell to convert chemical substrate(s) into chemical product(s). Enzymes are characterized by having catalytic activity, which can change the chemical structure of the substrate(s). An enzyme may have more than one substrate and produce more than one product. The enzyme may also depend on cofactors, which can be inorganic chemical compounds or organic compounds such as proteins for example enzymes (co-enzymes). The term "operative biosynthetic metabolic pathway" refers to a metabolic pathway that occurs in a live recombinant host, as described herein.
[0024] The term "in vivo", as used herein refers to within a living cell or organism, including, for example animal, a plant or a microorganism.
[0025] The term "in vitro", as used herein refers to outside a living cell or organism, including, without limitation, for example, in a microwell plate, a tube, a flask, a beaker, a tank, a reactor and the like. [0026] The term "in planta", as used herein refers to within a plant or plant cell.
[0027] Term "endogenous" or "native" as used herein refers to a gene or a polypepetide in a host cell which originates from the same host cell.
[0028] The terms "substantially" or "approximately" or "about", as used herein refers to a reasonable deviation around a value or parameter such that the value or parameter is not significantly changed. These terms of deviation from a value should be construed as including a deviation of the value where the deviation would not negate the meaning of the value deviated from. For example, in relation to a reference numerical value the terms of degree can include a range of values plus or minus 10% from that value. For example, deviation from a value can include a specified value plus or minus a certain percentage from that value, such as plus or minus 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from the specified value.
[0029] The term "and/or" as used herein is intended to represent an inclusive "or". The wording X and/or Y is meant to mean both X or Y and X and Y. Further the wording X, Y and/or Z is intended to mean X, Y and Z alone or any combination of X, Y, and Z.
[0030] The term "isolated" as used herein about a compound, refers to any compound, which by means of human intervention, has been put in a form or environment that differs from the form or environment in which it is found in nature. Isolated compounds include but is no limited to compounds of the disclosure for which the ratio of the compounds relative to other constituents with which they are associated in nature is increased or decreased. In an important embodiment the amount of compound is increased relative to other constituents with which the compound is associated in nature. In an embodiment the compound of the disclosure may be isolated into a pure or substantially pure form. In this context a substantially pure compound means that the compound is separated from other extraneous or unwanted material present from the onset of producing the compound or generated in the manufacturing process. Such a substantially pure compound preparation contains less than 10%, such as less than 8%, such as less than 6%, such as less than 5%, such as less than 4%, such as less than 3%, such as less than 2%, such as less than 1 %, such as less than 0.5% by weight of other extraneous or unwanted material usually associated with the compound when expressed natively or recombinantly. In an embodiment the isolated compound is at least 90% pure, such as at least 91% pure, such as at least 92% pure, such as at least 93% pure, such as at least 94% pure, such as at least 95% pure, such as at least 96% pure, such as at least 97% pure, such as at least 98% pure, such as at least 99% pure, such as at least 99.5% pure, such as 100 % pure by weight.
[0031] The term "non-naturally occurring" as used herein about a substance, refers to any substance that is not normaly found in nature or natural biological systems. In this context the term "found in nature or in natural biological systems" does not include the finding of a substance in nature resulting from releasing the substance to nature by deliberate or accidental human intervention. Non-naturally occurring substances may include substances completely or partially synthetized by human intervention and/or substances prepared by human modification of a natural substance.
[0032] The term "% identity" is used herein about the relatedness between two amino acid sequences or between two nucleotide sequences.
[0033] The term "% sequence identity" as used herein about amino acid sequences refers to the degree of identity in percent between two amino acid sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or later. The polypeptides or polynucleotides specified herein as having at least 80% sequence identity to a specified sequence, such as having at least 80% sequence identity to any one of SEQ ID NO: 1-98 have in some embodiments at least 85% sequence identity to any one of SEQ ID NO: 1-98, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99%, such as 100%.
[0034] The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical amino acid residues - x 100 Length of alignment — total number of gaps in alignment [0035] The term "% identity" as used herein about nucleotide sequences refers to the degree of identity in percent between two nucleotide sequences obtained when using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: identical deoxyribonucleotides
- - - x 100
Length of alignment — total number of gaps in alignment
[0036] The protein sequences of the present disclosure can further be used as a "query sequence" to perform a search against sequence databases, for example to identify other family members or related sequences. Such searches can be performed using the BLAST programs. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov). BLASTP is used for amino acid sequences and BLASTN for nucleotide sequences. The BLAST program uses as defaults:
Cost to open gap: default= 5 for nucleotides/ 11 for proteins
Cost to extend gap: default = 2 for nucleotides/ 1 for proteins
Penalty for nucleotide mismatch: default = -3
Reward for nucleotide match: default= 1
Expect value: default = 10
Wordsize: default = 11 for nucleotides/ 28 for megablast/ 3 for proteins.
[0037] Furthermore, the degree of local identity between the amino acid sequence query or nucleic acid sequence query and the retrieved homologous sequences is determined by the BLAST program. However only those sequence segments are compared that give a match above a certain threshold. Accordingly, the program calculates the identity only for these matching segments. Therefore, the identity calculated in this way is referred to as local identity.
[0038] The term "mature polypeptide" or "mature enzyme" as used herein refers to a polypeptide in its final active form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc. It is known in the art that a host cell may produce a mixture of two of more different mature polypeptides (i.e., with a different C- terminal and/or N-terminal amino acid) expressed by the same polynucleotide.
[0039] The term "cDNA" refers to a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps, including splicing, before appearing as mature spliced mRNA.
[0040] The term "coding sequence" refers to a nucleotide sequence, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon such as ATG, GTG, or TTG and ends with a stop codon such as TAA, TAG, or TGA. The coding sequence may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.
[0041] The term "control sequence" as used herein refers to a nucleotide sequence necessary for expression of a polynucleotide encoding a polypeptide. A control sequence may be native (i.e., from the same gene) or heterologous or foreign (i.e., from a different gene) to the polynucleotide encoding the polypeptide. Control sequences include, but are not limited to leader sequences, polyadenylation sequence, pro-peptide coding sequence, promoter sequences, signal peptide coding sequence, translation terminator (stop) sequences and transcription terminator (stop) sequences. To be operational control sequences usually must include promoter sequences, transcriptional and translational stop signals. Control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with a coding region of a polynucleotide encoding a polypeptide.
[0042] The term "expression" includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post- translational modification, and secretion.
[0043] The term "expression vector" refers to a DNA molecule, either single- or double stranded, either linear or circular, which comprises a polynucleotide encoding a polypeptide and is operably linked to control sequences that provide for its expression. Expression vectors include expression cassettes for the integration of genes into a host cell as well as plasmids and/or chromosomes comprising such genes.
[0044] The term "host cell" refers to any cell type that is susceptible to transformation, transfection, transduction, or the like with a nucleic acid construct or expression vector comprising a polynucleotide of the present disclosure. Host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication. [0045] The term "polynucleotide construct" refers to a polynucleotide, either single- or double stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises a polynucleotide encoding a polypeptide and one or more control sequences.
[0046] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding polynucleotide such that the control sequence directs expression of the coding polynucleotide.
[0047] The terms "nucleotide sequence and "polynucleotide" are used herein interchangeably.
[0048] The term "comprise" and "include" as used throughout the specification and the accompanying items as well as variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. These words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.
[0049] The articles "a" and "an" are used herein refers to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.
[0050] Terms like "preferably", "commonly", "particularly", and "typically" are not utilized herein to limit the scope of the itemed disclosure or to imply that certain features are critical, essential, or even important to the structure or function of the itemed disclosure. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of the present disclosure.
[0051] The term "cell culture" as used herein refers to a culture medium comprising a plurality of host cells of the disclosure. A cell culture may comprise a single strain of host cells or may comprise two or more distinct host cell strains. The culture medium may be any medium that may comprise a recombinant host, e.g., a liquid medium (i.e., a culture broth) or a semi-solid medium, and may comprise additional components, e.g., a carbon source such as dextrose, sucrose, glycerol, or acetate; a nitrogen source such as ammonium sulfate, urea, or amino acids; a phosphate source; vitamins; trace elements; salts; amino acids; nucleobases; yeast extract; aminoglycoside antibiotics such as G418 and hygromycin B.
Host cells
[0052] In the context of the present disclosure, a host cell is any cell that comprises a peroxisome.
[0053] In some embodiments, a host cell is provided comprising, i) a peroxisomally-localized enzyme catalyzing the formation of a branch point compound which in a metabolic pathway can be metabolized by a prioritized metabolic pathway and one or more non- prioritized pathways leading to different metabolites, ii) a peroxisomally-localized enzyme catalyzing the first step of a non-prioritized metabolic pathway for the branch point compound, and iii) a peroxisomally-localized enzyme having kinase activity, and/or genes encoding the peroxisomally-localized enzymes of i), ii) and iii).
[0054] In some embodiments, the present disclosure provides peroxisomal localization by inserting a peroxisomal localization signal in the genes encoding the peroxisomally-localized enzymes.
[0055] In some embodiments, the peroxisomally-localized enzyme of i), ii), and/or iii) are heterologous to the host cell.
[0056] In some embodiments, the host cell comprises or expresses or is configured to express one or more sequences selected from the group consisting of: SEQ. ID NO: 1 to SEQ ID NO: 98.
Yeast cells
[0057] In some embodiments, the host cell is a yeast cell. In some embodiments, the yeast cell belongs to a genus selected from the group consisting of: Saccharomyces, Pichia, Candida, Ogatea, Yarrowia, Kluyveromyces, Rhodotorula, Rhodosporidium, Cryptococcus, Schizosaccharomyces, Trichosporon and Lipomyces.
[0058] In some embodiments, the yeast cell is of a species selected from the group consisting of: Saccharomyces spp., Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Ogataea polymorpha, Kluyveromyces marxianus, Kluyveromyces lactis, Candida albicans, Candida boidinii, Schizosaccharomyces pombe, Scheffersomyces stipidis, and Dekkera bruxellensis.
Branch point compounds
[0059] In some embodiments, the branch point compound of the present disclosure is selected from the group consisting of: geranyl diphopsphate (GPP), neryl diphosphate (NPP), dimethylallyl diphosphate (DMAPP), geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP).
Geranyl diphopsphate (GPP) and neryl diphosphate (NPP)
[0060] In some embodiments, the branch point compound is geranyl diphopsphate (GPP). In some embodiments, the branch point compound is neryl diphosphate (NPP).
[0061] In some embodiments, i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a GPP synthase or an NPP synthase, ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase. In some embodiments, the NPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 48, for example wherein the polypeptide has the sequence of SEQ ID NO: 48. In some embodiments, the GPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 45, for example wherein the polypeptide has the sequence of SEQ ID NO: 45.
[0062] In some embodiments, the terpene synthase is selected from the group consisting of: (+)- limonene synthase, (-)-limonene synthase, 1,8-cineole synthase, sabinene synthase, camphene synthase, geraniol synthase, linalool synthase, myrcene synthase, bornyl diphosphate synthase, alphaterpineol synthase, tricyclene synthase, alpha-thujene synthase, alpha-phellandrene synthase, betaphellandrene synthase, (E)-beta-ocimene synthase, gamma-terpinene synthase, alpha-terpineol synthase, alpha-pinene synthase, beta-pinene synthase, 3-carene synthase, beta-thujene synthase, terpinolene synthase, nerol synthase, and 2-methylisoborneol synthase.
[0063] In some embodiments, the terpene synthase is selected from the group consisting of: (+)- limonene synthase, (-)-limonene synthase, 1,8-cineole synthase, sabinene synthase, camphene synthase, geraniol synthase, linalool synthase, myrcene synthase, bornyl diphosphate synthase, alphaterpineol synthase, tricyclene synthase, alpha-thujene synthase, alpha-phellandrene synthase, betaphellandrene synthase, (E)-beta-ocimene synthase, Trans-ocimene synthase, gamma-terpinene synthase, alpha-terpineol synthase, alpha-pinene synthase, beta-pinene synthase, 3-carene synthase, beta-thujene synthase, terpinolene synthase, nerol synthase, and 2-methylisoborneol synthase.
[0064] In some embodiments, the terpene synthase is selected from the group consisting of: a (+)-limonene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 20, for example the polypeptide of SEQ ID NO: 20; a (-)-limonene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 21, for example the polypeptide of SEQ ID NO: 21; a 1,8-cineole synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 22, for example the polypeptide of SEQ ID NO: 22; a sabinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 26, for example the polypeptide of SEQ ID NO: 26; a camphene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 23, for example the polypeptide of SEQ ID NO: 23; a geraniol synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 24, for example the polypeptide of SEQ ID NO: 24; a linalool synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 25, for example the polypeptide of SEQ ID NO: 25; a myrcene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 27 , for example the polypeptide of SEQ ID NO: 27; a bornyl diphosphate synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO:
28, for example the polypeptide of SEQ ID NO: 28; an alpha-terpineol synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO:
29, for example the polypeptide of SEQ ID NO: 29; a tricyclene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 31, for example the polypeptide of SEQ ID NO: 31; an alpha-thujene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 30, for example the polypeptide of SEQ ID NO: 30; an alpha-phellandrene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 33, for example the polypeptide of SEQ ID NO: 33; a beta-phellandrene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO:
35, for example the polypeptide of SEQ ID NO: 35; a gamma-terpinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 34, for example the polypeptide of SEQ ID NO: 34; a trans-ocimene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 37, for example the polypeptide of SEQ ID NO: 37; an alpha-pinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 38, for example the polypeptide of SEQ ID NO: 38; a beta-pinene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 39, for example the polypeptide of SEQ ID NO: 39; a 3-carene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 40, for example the polypeptide of SEQ ID NO: 40; a beta-thujene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 41, for example the polypeptide of SEQ ID NO: 41; a terpinolene synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 42, for example the polypeptide of SEQ ID NO: 42; a nerol synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO: 43, for example the polypeptide of SEQ ID NO: 43, and a 2-methylisoborneol synthase having at least 80% sequence identity to the polypeptide of SEQ ID NO:
36, for example the polypeptide of SEQ ID NO: 36.
[0065] In some embodiments, the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 9, 10, 11, or 12 carbon atoms. [0066] In some embodiments, the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: an aromatic prenyltransferase, a C- prenyltransferase, an O-methyltransferase, and a geranyldiphosphate:olivetolate geranyltransferase. In some embodiments, the geranyldiphosphate:olivetolate geranyltransferase has at least 80% sequence identity to the polypeptide of SEQ ID NO: 44. In some embodiments, the geranyldiphosphate:olivetolate geranyltransferase has the sequence of SEQ ID NO: 44.
[0067] In some embodiments, the host cell is provided comprising a Linalool synthase, such as a linalool synthase having at least 80% identity to the polypeptide of SEQ ID NO: 87.
[0068] In some embodiments, the host cell is provided comprising a polynucleotide encoding a Linalool synthase, such as a polynucleotide having at least 80% identity to the polypeptide of SEQ ID NO: 88.
[0069] In some embodiments, the host cell is provided comprising a phosphoribosylanthranilate isomerase, such as a phosphoribosylanthranilate isomerase having at least 80% identity to the polypeptide of SEQ ID NO: 89.
[0070] In some embodiments, the host cell is provided comprising a polynucleotide encoding a phosphoribosylanthranilate isomerase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 90.
[0071] In some embodiments, the host cell is provided comprising an Imidazoleglycerol-phosphate dehydratase, such as an Imidazoleglycerol-phosphate dehydratase having at least 80% identity to the polypeptide of SEQ ID NO: 91.
[0072] In some embodiments, the host cell is provided comprising a polynucleotide encoding an Imidazoleglycerol-phosphate dehydratase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 92.
[0073] In some embodiments, the host cell is provided comprising a phosphomevalonate kinase, such as a phosphomevalonate kinase having at least 80% identity to the polypeptide of SEQ ID NO: 93.
[0074] In some embodiments, the host cell is provided comprising a polynucleotide encoding a phosphomevalonate kinase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 94.
[0075] In some embodiments, the host cell is provided comprising a Mevalonate kinase, such as a Mevalonate kinase having at least 80% identity to the polypeptide of SEQ ID NO: 95.
[0076] In some embodiments, the host cell is provided comprising a polynucleotide encoding a Mevalonate kinase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 96.
[0077] In some embodiments, the host cell is provided comprising a Mevalonate pyrophosphate decarboxylase, such as a Mevalonate pyrophosphate decarboxylase having at least 80% identity to the polypeptide of SEQ ID NO: 97.
[0078] In some embodiments, the host cell is provided comprising a polynucleotide encoding a Mevalonate pyrophosphate decarboxylase, such as a polynucleotide having at least 80% identity to the polynucleotide of SEQ ID NO: 98.
[0079] In some embodiments, a host cell is provided comprising one or more of the above polynucleotide and/or polypeptides.
Farnesyl diphosphate (FPP)
[0080] In some embodiments, the branch point compound is farnesyl diphopsphate (FPP).
[0081] In some embodiments, i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is an FPP synthase, ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase. In some embodiments, the FPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 46, for example wherein the polypeptide has the sequence of SEQ ID NO: 46.
[0082] In some embodiments, the terpene synthase is selected from the group consisting of: farnesol synthase, farnesene synthase, beta-caryophyllene synthase, alpha-humulene synthase, bisabolol synthase, bisabolene synthase, germacrene synthase, bergamotene synthase, muurolol synthase, santalol synthase, thujopsene synthase, cedrene synthase, santalene synthase, valencene synthase, aristolene synthase, aristolochene synthase, cadinene synthase, sinensal synthase.
[0083] In some embodiments, the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 14, 15, 16, or 17 carbon atoms.
[0084] In some embodiments, the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: an aromatic prenyltransferase, a C- prenyltransferase, and an O-methyltransferase.
Dimethylallyl diphosphate (DMAPP)
[0085] In some embodiments, the branch point compound is dimethylallyl diphopsphate (DMAPP).
[0086] In some embodiments, i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a DMAPP synthase or an isopentelyl diphosphate isomerase (IDI), ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase. In some embodiments, the terpene synthase is an isoprene synthase. In some embodiments, the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 4, 5, 6, or 7 carbon atoms.
[0087] In some embodiments, the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: lavandulyl diphosphate synthase, chrysanthemyl diphosphate synthase, maconellyl diphosphate synthase, planococcyl diphosphate synthase, O-methyltransferase, C-methyltranaferase, and aromatic prenyltransferase.
Geranylgeranyl diphosphate (GGPP)
[0088] In some embodiments, the branch point compound is geranylgeranyl diphosphate (GGPP).
[0089] In some embodiments, i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a GGPP synthase, ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase. In some embodiments, the GGPP synthase is a polypeptide having at least 80% sequence identity to the polypeptide of SEQ. ID NO: 47, for example wherein the polypeptide has the sequence of SEQ ID NO: 47. In some embodiments, the terpene synthase is selected from the group consisting of: taxadiene synthase, ent-copalyl diphosphate synthase, casbene synthase, miltiradiene synthase (bifunctional), levopimaradiene synthase, abietadiene synthase. In some embodiments, the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 19, 20, 21, or 22 carbon atoms.
[0090] In some embodiments, the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: phytoene synthase, O-methyltransferase, C-methyltransferase, and aromatic prenyltransferase.
Metabolic operative pathway
[0091] In some embodiments, the host cell comprises an operative metabolic pathway comprising one or more peroxisomally-localized enzymes selected from the group consisting of: a kinase, a decarboxylase, and a dehydratase.
[0092] In some embodiments, the enzymes, such as the one or more peroxisomally-localized enzymes specifically listed herein may be capable of catalyzing a plurality of steps of the operative metabolic pathway. In some embodiments, the host cell comprises a bifunctional acetoacetyl-CoA thiolase/HMG-CoA reductase enzyme such that step a) and c) of the operative metabolic pathway are catalyzed by the same bifunctional enzyme.
[0093] In some embodiments, the host cell comprises an operative metabolic pathway comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) Mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) phosphomevalonate kinase (PMVK) converting mevalonate-5-phosphate to mevalonate 5- diphosphate; f) mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) converting mevalonate-5- diphosphate (or pyrophosphate) to isopentenyl diphosphate (or pyrophosphate) (IPP); g) mevalonate 5-phosphate decarboxylase (M5PD) converting mevalonate 5-phosphate to isopentenyl phosphate (IP); h) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); i) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP); j) mevalonate 3-kinase (MV3K) converting mevalonate to mevalonate-3-phosphate; k) mevalonate-3-phosphate-5-kinase (M3P5K) converting mevalonate-3-phosphate to mevalonate-3, 5-bisphosphate; l) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) converting mevalonate-3, 5- bisphosphate to isopentenyl phosphate (IP); m) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) converting mevalonate 5-phosphate to trans-anhydromevalonate 5-phosphate; n) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) converting mevalonate 5-phosphate to trans-anhydromevalonate 5-phosphate; and o) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) converting trans- anhydromevalonate 5-phosphate to isopentenyl phosphate (IP).
[0094] In some embodiments, the host cell comprises one or more polynucleotides selected from the group consisting of: a) a polynucleotide which is at least 80% identical to SEQ ID NO: 82; b) a polynucleotide which is at least 80% identical to SEQ ID NO: 80; c) a polynucleotide which is at least 80% identical to SEQ ID NO: 82; d) a polynucleotide which is at least 80% identical to SEQ ID NO: 96; e) a polynucleotide which is at least 80% identical to SEQ ID NO: 94; f) a polynucleotide which is at least 80% identical to SEQ ID NO: 98; g) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5-phosphate decarboxylase (M5PD) of SEQ ID NO: 7; h) a polynucleotide which is at least 80% identical to SEQ ID NO: 52, or SEQ ID NO: 54; i) a polynucleotide which is at least 80% identical to SEQ ID NO: 62; j) a polynucleotide which is at least 80% identical to SEQ ID NO: 64; k) a polynucleotide which is at least 80% identical to SEQ ID NO: 66; l) a polynucleotide which is at least 80% identical to SEQ ID NO: 56, or SEQ ID NO: 58; m) a polynucleotide which is at least 80% identical to SEQ ID NO: 60; and n) a polynucleotide which is at least 80% identical to SEQ ID NO: 84.
[0095] In some embodiments, the host cell is provided wherein the corresponding: a) acetoacetyl-CoA thiolase has at least 80% identity to the polypeptide of SEQ ID NO: 1; b) HMG-CoA synthase (HMGS) has at least 80% identity to the polypeptide of SEQ ID NO: 2; c) HMG-CoA reductase (HMGR) has at least 80% identity to the polypeptide of SEQ ID NO: 3; d) mevalonate 5-kinase (MV5K) has at least 80% identity to the polypeptide of SEQ ID NO: 4; e) phosphomevalonate kinase (PMVK) has at least 80% identity to the polypeptide of SEQ ID NO: 5; f) mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) has at least 80% identity to the polypeptide of SEQ ID NO: 6; g) mevalonate 5-phosphate decarboxylase (M5PD) has at least 80% identity to the polypeptide of SEQ ID NO: 7; h) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8; i) mevalonate 3-kinase (MV3K) has at least 80% identity to the polypeptide of SEQ ID NO: 9; j) mevalonate-3-phosphate-5-kinase (M3P5K) has at least 80% identity to the polypeptide of SEQ ID NO: 10; k) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) has at least 80% identity to the polypeptide of SEQ ID NO: 11; l) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) has at least 80% identity to the polypeptide of SEQ ID NO: 12; m) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) has at least 80% identity to the polypeptide of SEQ ID NO: 15; n) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13; o) isopentenyl diphosphate isomerase ( I DI ) has at least 80% identity to the polypeptide of SEQ ID NO: 14; and/or p) bifunctional acetoacetyl-CoA thiolase/HMG-CoA reductase enzyme has at least 80% identity to the polypeptide of SEQ ID NO: 16.
[0096] In some embodiments, the host cell is provided wherein the corresponding: a) acetoacetyl-CoA thiolase has at least 80% identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO: 81; b) HMG-CoA synthase (HMGS) has at least 80% identity to the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 79; c) HMG-CoA reductase (HMGR) has at least 80% identity to the polypeptide of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 81; d) mevalonate 5-kinase (MV5K) has at least 80% identity to the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 95; e) phosphomevalonate kinase (PMVK) has at least 80% identity to the polypeptide of SEQ ID NO: 5 or SEQ ID NO: 93; f) mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) has at least 80% identity to the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 97; g) mevalonate 5-phosphate decarboxylase (M5PD) has at least 80% identity to the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 49; h) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53; i) mevalonate 3-kinase (MV3K) has at least 80% identity to the polypeptide of SEQ ID NO: 9, or SEQ ID NO: 61; j) mevalonate-3-phosphate-5-kinase (M3P5K) has at least 80% identity to the polypeptide of SEQ ID NO: 10, or SEQ ID NO: 63; k) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) has at least 80% identity to the polypeptide of SEQ ID NO: 11, or SEQ ID NO: 65; l) mevalonate 5-phosphate dehydratase (PMDh) has at least 80% identity to the polypeptide of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; m) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59; and/or n) isopentenyl diphosphate isomerase ( I DI ) has at least 80% identity to the polypeptide of SEQ ID NO:
14 or SEQ ID NO: 83.
[0097] In some embodiments, the host cell comprises one or more polynucleotides selected from the group consisting of: a) a polynucleotide encoding a polypeptide which is at least 80% identical to acetyl-CoA thiolase of SEQ ID NO: 1, or SEQ ID NO: 81; b) a polynucleotide encoding a polypeptide which is at least 80% identical to HMG-CoA synthase (HMGS) of SEQ ID NO: 2 or SEQ ID NO: 79; c) a polynucleotide encoding a polypeptide which is at least 80% identical to HMG-CoA reductase (HMGR) of SEQ ID NO: 3, or SEQ ID NO: 81; d) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5-kinase (MV5K) of SEQ ID NO: 4, or SEQ ID NO: 95; e) a polynucleotide encoding a polypeptide which is at least 80% identical to phosphomevalonate kinase (PMVK) of SEQ ID NO: 5 or SEQ ID NO: 93; f) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) of SEQ ID NO: 6 or SEQ ID NO: 97; g) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5-phosphate decarboxylase (M5PD) of SEQ ID NO: 7; h) a polynucleotide encoding a polypeptide which is at least 80% identical to isopentenyl phosphate kinase (IPK) of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53; i) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 3-kinase (MV3K) of SEQ ID NO: 9, or SEQ ID NO: 61; j) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate-3-phosphate- 5-kinase (M3P5K) of SEQ ID NO: 10, or SEQ ID NO: 63; k) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate-3-phosphate- 5-phosphate decarboxylase (BMD) of SEQ ID NO: 11, or SEQ ID NO: 65; l) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5-phosphate dehydratase (PMDh) of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; m) a polynucleotide encoding a polypeptide which is at least 80% identical to transanhydromevalonate 5-phosphate decarboxylase (AMPD) of SEQ ID NO: 13 or SEQ ID NO: 59; and n) a polynucleotide encoding a polypeptide which is at least 80% identical to isopentenyl diphosphate isomerase ( ID I) of SEQ ID NO: 14 or SEQ ID NO: 83.
Pathway 1
[0098] In some embodiments, the operative metabolic pathway is pathway 1 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) mevalonate 5-phosphate decarboxylase (M5PD) converting mevalonate 5-phosphate to isopentenyl phosphate (IP); f) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); and g) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP).
[0099] In some embodiments, the host cell is provided wherein the corresponding: a) mevalonate 5-phosphate decarboxylase has at least 80% identity to the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 49; and/or b) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
[0100] In some embodiments, the host cell is provided comprising: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and d) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
[0101] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized GPP synthase (GPPS).
[0102] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized neryl diphosphate synthase (NPPS).
[0103] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized geranylgeranyl diphosphate synthase (GGPPS).
[0104] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-g) defined herein for pathway 1, and a peroxisomally localized farnesyl diphopsphate synthase (FPPS). Pathway 2
[0105] In some embodiments, the operative metabolic pathway is pathway 2 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 3-kinase (MV3K) converting mevalonate to mevalonate-3-phosphate; e) mevalonate-3-phosphate-5-kinase (M3P5K) converting mevalonate-3-phosphate to mevalonate-3, 5-bisphosphate; f) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) converting mevalonate-3, 5- bisphosphate to isopentenyl phosphate (IP); g) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); and h) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP).
[0106] In some embodiments, the host cell is provided wherein the corresponding: a) mevalonate 3-kinase (MV3K) has at least 80% identity to the polypeptide of SEQ ID NO: 9, or SEQ ID NO: 61; b) mevalonate-3-phosphate-5-kinase (M3P5K) has at least 80% identity to the polypeptide of SEQ ID NO: 10, or SEQ ID NO: 63; c) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) has at least 80% identity to the polypeptide of SEQ ID NO: 11, or SEQ ID NO: 65; and/or d) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
[0107] In some embodiments, the host cell comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and/or d) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
[0108] In some embodiments, the host cell further comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ERG20(N127W)-SKL (SEQ ID NO: 85), and/or b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to FpTPS-SKL (SEQ ID NO: 87).
[0109] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized GPP synthase (GPPS).
[0110] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized neryl diphosphate synthase (NPPS).
[0111] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized geranylgeranyl diphosphate synthase (GGPPS).
[0112] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 2, and a peroxisomally localized farnesyl diphopsphate synthase (FPPS).
Pathway 3
[0113] In some embodiments, the operative metabolic pathway is pathway 3 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) converting mevalonate 5-phosphate to trans-anhydromevalonate 5-phosphate; f) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) converting mevalonate 5-phosphate to trans-anhydromevalonate 5-phosphate; g) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) converting trans- anhydromevalonate 5-phosphate to isopentenyl phosphate (IP); h) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); and i) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP).
[0114] In some embodiments, the corresponding: a) mevalonate 5-phosphate dehydratase (PMDh) has at least 80% identity to the polypeptide of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; and/or b) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59.
[0115] In some embodiments, the corresponding: a) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) has at least 80% identity to the polypeptide of SEQ ID NO: 12 or SEQ ID NO: 55; b) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) has at least 80% identity to the polypeptide of SEQ ID NO: 15 or SEQ ID NO: 57; c) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59 and/or d) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
[0116] In some embodiments, the host cell comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and/or d) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
[0117] In some embodiments, the host cell further comprises: a) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ERG20(N127W)-SKL (SEQ ID NO: 85), and/or b) a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to FpTPS-SKL (SEQ ID NO: 87).
[0118] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-h) defined herein for pathway 3, and a peroxisomally localized GPP synthase (GPPS).
[0119] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-i) defined herein for pathway 3, and a peroxisomally localized neryl diphosphate synthase (NPPS).
[0120] In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-i) defined herein for pathway 3, and a peroxisomally localized geranylgeranyl diphosphate synthase (GGPPS). In some embodiments, a yeast cell is provided comprising all the peroxisomally-localized polypeptides a)-i) defined herein for pathway 3, and a peroxisomally localized farnesyl diphopsphate synthase (FPPS).
Kinase
[0121] In some embodiments, the peroxisomally-localized enzyme having kinase activity is heterologous to the host cell.
[0122] In some embodiments, the peroxisomally-localized enzyme of iii) having kinase activity is a peroxisomally-localized kinase.
Isopentenyl phosphate kinase (IPK)
[0123] In some embodiments, the kinase is an isopentenyl phosphate kinase (IPK). In some embodiments, the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), Methanothermobacter thermautotrophicus IPK (MtIPK), Thermoplasma acidophilum IPK variant IVG (TalPK(IVG), and TalPK wild-type. In some embodiments, the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 17, for example the polypeptide of SEQ UD NO: 17; Thermoplasma acidophilum IPK variant IVG (TalPK(IVG), such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 19, or such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 18, such as the polypeptide of SEQ ID NO: 18, or such as the polypeptide of SEQ UD NO: 19; TalPK wild-type, and Methanothermobacter thermautotrophicus IPK (MtIPK), such as a polypeptide having at least 80% sequence identity to the polypeptide of SEQ ID NO: 51, for example the polypeptide of SEQ ID NO: 51. In some embodiments, the host cell comprises a polynucleotide encoding an IPK of the present disclosure. In some embodiments, the polynucleotide has at least 80% identity to the polynucleotide of SEQ ID NO: 52.
[0124] In some embodiments, the host cell is a yeast cell comprising a peroxisomally-localized isopentenyl phosphate kinase (IPK), optionally wherein the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), Methanothermobacter thermautotrophicus IPK (MtIPK), Thermoplasma acidophilum IPK variant IVG (TalPK(IVG)), and TalPK wild-type.
Sequence listing
[0125] The present application contains a Sequence Listing prepared in WIPO Sequence included below in Table A but also submitted electronically in ST26 format which is hereby incorporated by reference in its entirety.
Table A
Examples
Example 1: Materials and methods
[0126] Chemicals used in the examples herein, e.g. for buffers and substrates, are commercial products of at least reagent grade.
• The names and origins of the genes used in the following examples are listed in Table 1.
• The sequences of the genes used are given in Table 2.
• The genotypes of the yeast strains constructed in this application are listed in Table 3.
• The yeast plasmids used in this application are listed in Table 4. • The sequences of the proteins encoded by the genes listed in Table 2 are provided in Table 5.
• The primers used for PCR amplification are listed in Table 6.
Yeast strains
[0127] The yeast strain used as the basis to derive the strains described in this application is EGY48, a Saccharomyces cerevisiae (S. cerevisiae) strain disclosed in (Thomas & Rothstein, 1989) and (Ignea et al., 2011) with the genotype specified in Table 3.
Construction of plasmids [0128] Yeast codon-optimized genes were purchased from Twist Biosciences, Inc. (USA) in synthetic form, amplified by PCR using the corresponding primers listed in Table 6, placed under the control of an inducible promoter (P AL1, PGAL2, or PGAL10), and inserted into one of the following integration plasmids, pAssl, pAss2, pAss3, pAss2A, pAss2B, or pAss2C. This plasmid integration system is disclosed in (Forman et al., 2022) and the plasmids generated in this application are listed in Table 4. Two integration sites for high expression were used throughout this study: X-4 and XII-2, as previously described (Jensen et al., 2012)
[0129] The cloning method used for vector construction in this application is the Uracil-Specific- Excision-Reagent (USER) method, described in (Nour-Eldin et al., 2010) The gene products generated by the integrated genes contain a C-terminally fused amino acid sequence (Serine-Lysine-Leucine (SKL)) to target them to the yeast peroxisome, as demonstrated in (Dusseaux et al., 2020).
Genotyping of generated strains
[0130] Transformations were conducted using the standard protocol outlined by (Daniel Gietz & Woods, 2002). Transformants were selected by picking eight colonies of S. cerevisiae for each transformation and plating them on selective auxotrophic minimal media. To confirm the presence of the integrative vectors, each of the eight colonies was genotyped using primers specific to the genomic locus where integration was targeted (The primers used for genotyping are listed in Table 6).
Culture conditions
[0131] S. cerevisiae pre-cultures of each studied strain were individually selected and grown overnight at 30°C, in a shaking incubator at 250 revolutions per minute, in a 24-well plate containing 1 mL YPD and 6% glucose. After incubation, 150 pL of cell suspension was transferred to a new 24- well plate where 0.6 mL of sunflower oil and 2.7 mL of rich media containing 0.3 g/L raffinose and 0.8 g/L galactose were added. Cultures were grown at 30°C with shaking at 250 revolutions per minute for 72 hours, and the sunflower overlay phase was isolated by centrifugation and subsequently analyzed using GC-FID.
GC-FID method
[0132] A GC-FID SCION 436-GC was utilized for GC-FID analysis using an HP-5MS Ultra Inert column with a 5 % Phenyl-methylpolysiloxane stationary phase. Injection volume was set to 1 pL and the sample was injected by a CP8400 autosampler. The oven temperature was initially set at 40 °C for 3 min and subsequently increased to 80 °C at a rate of 3 °C/min. When reaching 80 °C the rate was increased to 30 °C/min until reaching 300 °C, followed by a hold of 10 min. Example 2: Construction of a control strain to evaluate peroxisomal pathway performance
[0133] Initially, the control strain xEVO838 was constructed through the integration of the following codon-optimized genes within the yeast genome: Enterococcus faecalis EfmvaE and EfmvaS genes (equivalent to the function of ERG10, ERG13, and HMG1 in yeast), ScERG12, SclDI, and two copies of the ERG20(N127W) variant (serving as a geranyl diphosphate synthase). Furthermore, two copies of the gene encoding for the FpTPS linalool synthase were introduced, allowing assessment of the flux through the pathway by quantifying linalool production. All the aforementioned genes encode for gene products that contain a C-terminal peroxisomal localized signal (-SKL), as described in Example 1. This construction provides the basal strain that will serve as the control to which all alternative MVA pathway designs will be compared. As shown previously, the localization of a terpene synthase together with a prenyltransferase that supplies the terpene synthase substrate in the yeast peroxisome results in efficient terpene production. Further assembly of a complete classical MVA pathway in the yeast peroxisome enhances terpene production (Dusseaux et al., 2020). Strain xEVO838 contains a terpene synthase (FpTPS) and a prenyltransferase (ERG20(N127W)) together with an incomplete mevalonate pathway (the strain lacks the peroxisomal-targeted versions of ScERG8 and ScERG19). Therefore, strain xEVO838 serves as the basis of comparison with other strains that contain complete designs of alternative forms of the mevalonate pathway in the peroxisome. Thus, if a strain that contains an alternative mevalonate pathway design shows increased terpene production compared to xEVO838, this will be an indication that the corresponding alternative mevalonate pathway design is active in the peroxisome and supplies additional amounts of precursors to the prenyltransferase and the terpene synthase.
Example 3: Reconstruction of an isoprenoid alcohol pathway in the S. cerevisiae peroxisome
[0134] To assess the efficiency of an isoprenoid alcohol pathway in improving terpene production in the yeast peroxisome, we constructed strains xEVO820 and xEVO823. Both these strains contain the enzymes Myxococcus xanthus enoyl-CoA hydratase (McLiuC-SKL), M. xanthus malonate decarboxylase alpha subunit (McAibA-SKL), M. xanthus CoA-transferase (McAibB-SKL), Clostridium beijerinckii acyl- CoA reductase (MccbjALD-SKL), Escherichia coli hydroxyethylthiazole kinase (EcThiM-SKL), and E. coli NADPH-dependent aldehyde reductase (EcYahK-SKL), all equipped with C-terminal peroxisomal localization signals (SKL). Furthermore, xEVO820 and xEVO823 also contain peroxisomally-localized variants of the enzymes EfmvaE-SKL, EfmvaS-SKL, SclDI-SKL, ERG20(N127W)-SKL, and FpTPS-SKL to enable linalool production. Additionally, strain xEVO820 contains a peroxisomally-localized variant of IPK from Methanothermobacter thermautotrophicus and strain xEVO823 contains a peroxisomally- localized variant of IPK from Thermoplasma acidophilum. The combination of these enzymes (McLiuC, McAibA, McAibB, MccbjALD, EcThiM, EcYahK, and IPK) converts 3-hydroxy-3-methylglutaryl- coenzyme A (HMG-CoA) to IPP in the isoprenoid alcohol-type pathway.
Results:
[0135] The performance of strains xEVO820 and xEVO823 in terpene production was evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 1, linalool production by strains xEVO820 and xEVO823 was similar to the control strain xEVO838.
Conclusion:
[0136] These results suggest that the assembled alternative isoprenoid-type pathway was not able to efficiently convert HMG-CoA into IPP and enhance terpene production in the yeast peroxisome.
Example 4: Reconstruction of an Aeropyrum-type pathway (Pathway 3) in the S. cerevisiae peroxisome
[0137] To assess the efficiency of an Aeropyrum-type pathway (Pathway 3) in improving terpene production in the yeast peroxisome, we constructed strains xEVO805 and xEVO808. Both these strains contain the enzymes mevalonate 5-phosphate dehydratase subunit 1 (PMDhl-SKL), mevalonate 5- phosphate dehydratase subunit 2 (PMDh2-SKL), and trans-anhydromevalonate 5-phosphate decarboxylase (AMPD-SKL) from Methanosarcina mazei, equipped with C-terminal peroxisomal localization signals (SKL). Furthermore, xEVO805 and xEVO808 also contain the peroxisomally- localized variants enzymes EfmvaE-SKL, EfmvaS-SKL, ScERG12-SKL, SclDI-SKL, ERG20(N127W)-SKL, and FpTPS-SKL to enable linalool production. Additionally, strain xEVO805 contains a peroxisomally- localized variant of IPK from M. thermautotrophicus, and strain xEVO808 contains a peroxisomally- localized variant of IPK from T. acidophilum. The combination of these enzymes (PMDhl, PMDh2, AMPD, and IPK) converts mevalonate 5-phosphate to IPP in the Aeropyrum-type pathway.
Results:
[0138] The performance of strains xEVO805 and xEVO808 in terpene production was evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 2, linalool production by strains xEVO805 and xEVO808 was 82% and 77% higher, respectively, than the control strain xEVO838. Conclusion:
[0139] These results suggest that Pathway 3 is able to improve IPP supply to the downstream steps in the peroxisome.
Example 5: Reconstruction of a Thermoplasma-type pathway (Pathway 2) in S. cerevisiae peroxisome
[0140] To assess the efficiency of a Thermoplasma-type pathway (Pathway 2) in improving terpene production in the yeast peroxisome, we constructed strains xEVO810 and xEVO813. Both these strains contain the enzymes mevalonate-3-kinase (TaMV3K-SKL), mevalonate-3-phosphate 5-kinase (TaM3P5K), and Mevalonate 3,5-bisphosphate decarboxylase (TaBMDl-SKL) from T. acidophilum, all equipped with C-terminal peroxisomal localization signals (SKL). Furthermore, xEVO810 and xEVO813 also contain peroxisomally-localized variants of the enzymes EfmvaE-SKL, EfmvaS-SKL, ScERG12-SKL, SclDI-SKL, ERG20(N127W)-SKL, and FpTPS-SKL to enable linalool production. Additionally, strain xEVO810 contains the IPK from M. thermautotrophicus, and strain xEVO813 contains the IPK from T. acidophilum. The combination of these enzymes (TaMV3K, TaM3P5K, TaBMDl, and IPK) converts mevalonate to IPP in the Thermoplasma-type pathway.
Results:
[0141] The performance of strains xEVO810 and xEVO813 in terpene production was evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 3, linalool production by strains xEVO810 and xEVO813 was 3.45 and 3.47 times higher, respectively, than the control strain xEVO838.
Conclusion:
[0142] These results suggest that the Pathway 2 is able to convert mevalonate into IPP and can have a strong impact on enhancing terpene production in the yeast peroxisome.
Example 6: Reconstruction of an Haloarchea-type pathway (Pathway 1) in the S. cerevisiae peroxisome.
[0143] To assess the functional activity of the Haleoarchaea-type MVA pathway (Pathway 1) within the yeast peroxisome, we constructed a strain that incorporates this alternative pathway in the peroxisomes. This alternative, Haloarchea-type, pathway differs from the classical MVA pathway in the last two steps. Instead of ScERG8 and ScERG19 (in the case of S. cerevisiae), it utilizes a phosphomevalonate decarboxylase (M5PD) and an isopentenyl phosphate kinase (IPK). To establish this Haloarchaea-type MVA pathway, two distinct IPKs were identified from Methanothermobacter thermautotrophicus and Thermoplasma acidophilum, respectively, and introduced into yeast in combination with the M5PD from Haloferax volcanii (HvM5PD). To assemble this pathway, all other common genes between the two types of mevalonate pathway were also introduced (i.e. EfmvaE-SKL, EfmvaS-SKL, ScERG12-SKL, and SclDI-SKL). All the gene products contained peroxisomal localization signals (SKL). This led to the construction of strains xEVO789 and xEVO792. Strain xEVO789 contains the Haloarchea-type pathway (Pathway 1) assembled with the IPK from M. thermautotrophicus, and strain xEVO792 contains Pathway 1 assembled with the IPK from T. acidophilum.
Results:
[0144] The performance of strains xEVO792 and xEVO789 in terpene production was evaluated by GC-FID analysis, as described in Example 1, and compared to the performance of the control strain xEVO838. As shown in Figure 4, linalool production by strains xEVO792 and xEVO789 was 4.05 and 4.37 times higher, respectively, than the control strain.
Conclusion:
[0145] This result proves that the combination of H. volcanii M5PD with M.thermautotrophicus IPK or T. acidophilum IPK can convert mevalonate-5-phosphate into IPP and that the alternative Haloarchaea-type pathway is active in the yeast peroxisome. These results underscore the strong potential of harnessing Pathway 1 in the yeast peroxisome to increase the flux towards IPP and obtain improved production of terpenes.
Tables
Table 1 - Genes used in this application.
Table 2: Sequences of genes used in this application
Table 3 - Strains used in this application
Table 4 - Yeast plasmids used in this application. Table 5 - List of protein sequences used in this application Table 6 -List of primer sequences used in this application.
References
Gietz, R.D., Woods, R.A. (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol. 350:87-96. doi: 10.1016/s0076- 6879(02)50957-5.
Dusseaux, S., Wajn, W. T., Liu, Y., Ignea, C., & Kampranis, S. C. (2020). Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proceedings of the National Academy of Sciences of the United States of America, 117(50), 31789-31799. https://doi.Org/10.1073/PNAS.2013968117/-/DCSUPPLEMENTAL
Forman, V., Luo, D., Geu-Flores, F., Lemcke, R., Nelson, D. R., Kampranis, S. C., Staerk, D., Lindberg Mpller, B., & Pateraki, I. (2022). A gene cluster in Ginkgo biloba encodes unigue multifunctional cytochrome P450s that initiate ginkgolide biosynthesis, https://doi.org/10.1038/s41467-022- 32879-9
Ignea, C., Cvetkovic, L, Loupassaki, S., Kefalas, P., Johnson, C. B., Kampranis, S. C., & Makris, A. M. (2011). Improving yeast strains using recyclable integration cassettes, for the production of plant terpenoids. Microbial Cell Factories, 10(1), 1-18. https://doi.org/10.1186/1475-2859-10- 4
Jensen, N. B., Strucko, T., Kildegaard, K. R., David, F., Maury, J., Mortensen, U. H., Forster, J., Nielsen, J., & Borodina, I. (2014). EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. https://doi.org/10.llll/1567-1364.12118 Nour-Eldin, H. H., Geu-Flores, F., & Halkier, B. A. (2010). Chapter 13 USER Cloning and USER Fusion: The Ideal Cloning Technigues for Small and Big Laboratories, https://doi.org/10.1007/978-l- 60761-723-5_13
Thomas, B. J., & Rothstein, R. (1989). Elevated Recombination Rates in Transcriptionally Active DNA. Cell, 56, 619-630.

Claims

Claims
1. A host cell comprising, i) a peroxisomally-localized enzyme catalyzing the formation of a branch point compound which in a metabolic pathway can be metabolized by a prioritized metabolic pathway and one or more non-prioritized pathways leading to different metabolites, ii) a peroxisomally-localized enzyme catalyzing the first step of a non-prioritized metabolic pathway for the branch point compound, and iii) a peroxisomally-localized enzyme having kinase activity, and/or genes encoding the peroxisomally-localized enzymes of i), ii) and iii).
2. The host cell according to claim 1, wherein the host cell is a yeast cell.
3. The host cell according to claim 2, wherein the yeast cell belongs to a genus selected from the group consisting of: Saccharomyces, Pichia, Candida, Ogatea, Yarrowia, Kluyveromyces, Rhodotorula, Rhodosporidium, Cryptococcus, Schizosaccharomyces, Trichosporon and Lipomyces.
4. The host cell according to claim 3, wherein the yeast cell is of a species selected from the group consisting of: Saccharomyces spp., Saccharomyces cerevisiae, Yarrowia lipolytica, Pichia pastoris, Ogataea polymorpha, Kluyveromyces marxianus, Kluyveromyces lactis, Candida albicans, Candida boidinii, Schizosaccharomyces pombe, Scheffersomyces stipidis, and Dekkera bruxellensis.
5. The host cell according to any one of the preceding claims, wherein peroxisomal localization is provided by inserting a peroxisomal localization signal in the genes encoding the peroxisomally-localized enzymes.
6. The host cell according to any one of the preceding claims, wherein the branch point compound is selected from the group consisting of: geranyl diphopsphate (GPP), neryl diphosphate (NPP), dimethylallyl diphosphate (DMAPP), and farnesyl diphosphate (FPP).
7. The host cell according to any one of the preceding claims, wherein i) the peroxisomally- localized enzyme catalyzing the formation of the branch point compound is a GPP synthase or an NPP synthase, ii) the peroxisomally-localized enzyme catalyzing the first step of the non- prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
8. The host cell according to claim 7, wherein the terpene synthase is selected from the group consisting of: (+)-limonene synthase, (-)-limonene synthase, 1,8-cineole synthase, sabinene synthase, camphene synthase, geraniol synthase, linalool synthase, myrcene synthase, bornyl diphosphate synthase, alpha-terpineol synthase, tricyclene synthase, alpha-thujene synthase, alpha-phellandrene synthase, beta-phellandrene synthase, (E)-beta-ocimene synthase, gamma-terpinene synthase, alpha-terpineol synthase, alpha-pinene synthase, beta-pinene synthase, 3-carene synthase, beta-thujene synthase, terpinolene synthase, nerol synthase, and 2-methylisoborneol synthase, .
9. The host cell according to any one of claims 7-8, wherein the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 9, 10, 11, or 12 carbon atoms.
10. The host cell according to claim 7, wherein the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: an aromatic prenyltransferase, a C-prenyltransferase, an O-methyltransferase, and a geranyldiphosphate:olivetolate geranyltransferase.
11. The host cell according to claim 6, wherein the branch point compound is farnesyl diphosphate (FPP).
12. The host cell according to any one of claims 1-5 and 11, wherein i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is an FPP synthase, ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
13. The host cell according to claim 12, wherein the terpene synthase is selected from the group consisting of: farnesol synthase, farnesene synthase, beta-caryophyllene synthase, alpha- humulene synthase, bisabolol synthase, bisabolene synthase, germacrene synthase, bergamotene synthase, muurolol synthase, santalol synthase, thujopsene synthase, cedrene synthase, santalene synthase, valencene synthase, aristolene synthase, aristolochene synthase, cadinene synthase, sinensal synthase.
14. The host cell according to any one of claims 12-13, wherein the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 14, 15, 16, or 17 carbon atoms.
15. The host cell according to claim 12, wherein the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: an aromatic prenyltransferase, a C-prenyltransferase, and an O-methyltransferase.
16. The host cell according to claim 6, wherein the branch point compound is dimethylallyl diphopsphate (DMAPP).
17. The host cell according to claim 16, wherein i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is an isopentenyl diphosphate isomerase (IDI), ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
18. The host cell according to claim 17, wherein the terpene synthase is an isoprene synthase.
19. The host cell according to any one of claims 17-18, wherein the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 4, 5, 6, or 7 carbon atoms.
20. The host cell according to claim 17, wherein the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: lavandulyl diphosphate synthase, chrysanthemyl diphosphate synthase, maconellyl diphosphate synthase, planococcyl diphosphate synthase, O-methyltransferase, C-methyltranaferase, and aromatic prenyltransferase.
21. The host cell according to claim 6, wherein the branch point compound is geranylgeranyl diphosphate (GGPP).
22. The host cell according to any one of claims 1-5 and 21, wherein i) the peroxisomally-localized enzyme catalyzing the formation of the branch point compound is a GGPP synthase, ii) the peroxisomally-localized enzyme catalyzing the first step of the non-prioritized metabolic pathway is selected from the group consisting of: a terpene synthase, a prenyltransferase, and another isoprenoid or non-isoprenoid prenyltransferase.
23. The host cell according to claim 22, wherein the terpene synthase is selected from the group consisting of: taxadiene synthase, ent-copalyl diphosphate synthase, casbene synthase, miltiradiene synthase (bifunctional), levopimaradiene synthase, abietadiene synthase.
24. The host cell according to any one of claims 22-23, wherein the terpene synthase is capable of accepting non-canonical isoprenoid substrates with 19, 20, 21, or 22 carbon atoms.
25. The host cell according to claim 22, wherein the prenyltransferase, or another isoprenoid or non-isoprenoid prenyl-transferase is selected from the group consisting of: phytoene synthase, O-methyltransferase, C-methyltranaferase, and aromatic prenyltransferase.
26. The host cell according to any one of the preceding claims, wherein the host cell comprises an operative metabolic pathway comprising one or more peroxisomally-localized enzymes selected from the group consisting of: a kinase, a decarboxylase, and a dehydratase.
27. The host cell according to any one of the preceding claims, wherein the host cell comprises an operative metabolic pathway comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) Mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) phosphomevalonate kinase (PMVK) converting mevalonate-5-phosphate to mevalonate 5- diphosphate; f) mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) converting mevalonate-5- diphosphate (or pyrophosphate) to isopentenyl diphosphate (or pyrophosphate) (IPP); g) mevalonate 5-phosphate decarboxylase (M5PD) converting mevalonate 5-phosphate to isopentenyl phosphate (IP); h) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); i) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP); j) mevalonate 3-kinase (MV3K) converting mevalonate to mevalonate-3-phosphate; k) mevalonate-3-phosphate-5-kinase (M3P5K) converting mevalonate-3-phosphate to mevalonate-3, 5-bisphosphate; l) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) converting mevalonate-3, 5- bisphosphate to isopentenyl phosphate (IP); m) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) converting mevalonate 5- phosphate to trans-anhydromevalonate 5-phosphate; n) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) converting mevalonate 5- phosphate to trans-anhydromevalonate 5-phosphate; and o) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) converting trans- anhydromevalonate 5-phosphate to isopentenyl phosphate (IP).
28. The host cell according to claim 27, wherein the corresponding: a) acetoacetyl-CoA thiolase has at least 80% identity to the polypeptide of SEQ ID NO: 1, SEQ ID NO: 16, or SEQ ID NO: 81; b) HMG-CoA synthase (HMGS) has at least 80% identity to the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 79; c) HMG-CoA reductase (HMGR) has at least 80% identity to the polypeptide of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 81; d) mevalonate 5-kinase (MV5K) has at least 80% identity to the polypeptide of SEQ ID NO: 4 or SEQ ID NO: 95; e) phosphomevalonate kinase (PMVK) has at least 80% identity to the polypeptide of SEQ ID NO: 5 or SEQ ID NO: 93; f) mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) has at least 80% identity to the polypeptide of SEQ ID NO: 6 or SEQ ID NO: 97; g) mevalonate 5-phosphate decarboxylase (M5PD) has at least 80% identity to the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 49; h) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53; i) mevalonate 3-kinase (MV3K) has at least 80% identity to the polypeptide of SEQ ID NO: 9, or SEQ ID NO: 61; j) mevalonate-3-phosphate-5-kinase (M3P5K) has at least 80% identity to the polypeptide of SEQ ID NO: 10, or SEQ ID NO: 63; k) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) has at least 80% identity to the polypeptide of SEQ ID NO: 11, or SEQ ID NO: 65; l) mevalonate 5-phosphate dehydratase (PMDh) has at least 80% identity to the polypeptide of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; m) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59; and/or n) isopentenyl diphosphate isomerase (I DI) has at least 80% identity to the polypeptide of SEQ ID NO: 14 or SEQ ID NO: 83.
29. The host cell according to any one of the preceding claims, wherein the host cell comprises one or more polynucleotides selected from the group consisting of: a) a polynucleotide encoding a polypeptide which is at least 80% identical to acetyl-CoA thiolase of SEQ ID NO: 1, or SEQ ID NO: 81; b) a polynucleotide encoding a polypeptide which is at least 80% identical to HMG-CoA synthase (HMGS) of SEQ ID NO: 2 or SEQ ID NO: 79; c) a polynucleotide encoding a polypeptide which is at least 80% identical to HMG-CoA reductase (HMGR) of SEQ ID NO: 3, or SEQ ID NO: 81; d) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5- kinase (MV5K) of SEQ ID NO: 4, or SEQ ID NO: 95; e) a polynucleotide encoding a polypeptide which is at least 80% identical to phosphomevalonate kinase (PMVK) of SEQ ID NO: 5 or SEQ ID NO: 93; f) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate pyrophosphate (diphosphate) decarboxylase (MPD) of SEQ ID NO: 6 or SEQ ID NO: 97; g) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5- phosphate decarboxylase (M5PD) of SEQ ID NO: 7; h) a polynucleotide encoding a polypeptide which is at least 80% identical to isopentenyl phosphate kinase (IPK) of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53; i) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 3- kinase (MV3K) of SEQ ID NO: 9, or SEQ ID NO: 61; j) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate-3- phosphate-5-kinase (M3P5K) of SEQ ID NO: 10, or SEQ ID NO: 63; k) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate-3- phosphate-5-phosphate decarboxylase (BMD) of SEQ ID NO: 11, or SEQ ID NO: 65; l) a polynucleotide encoding a polypeptide which is at least 80% identical to mevalonate 5- phosphate dehydratase (PMDh) of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; m) a polynucleotide encoding a polypeptide which is at least 80% identical to transanhydromevalonate 5-phosphate decarboxylase (AMPD) of SEQ ID NO: 13 or SEQ ID NO: 59; and n) a polynucleotide encoding a polypeptide which is at least 80% identical to isopentenyl diphosphate isomerase (IDI) of SEQ ID NO: 14 or SEQ ID NO: 83.
30. The host cell according to any one of claims 26-29, wherein the operative metabolic pathway is pathway 1 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) mevalonate 5-phosphate decarboxylase (M5PD) converting mevalonate 5-phosphate to isopentenyl phosphate (IP); f) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); and g) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP).
31. The host cell according to claim 30, wherein the corresponding: a) mevalonate 5-phosphate decarboxylase has at least 80% identity to the polypeptide of SEQ ID NO: 7 or SEQ ID NO: 49; and/or b) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
32. The host cell according to claims 30-31, wherein host cell comprises: a. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and d. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
33. The host cell according to any one of claims 26-29, wherein the operative metabolic pathway is pathway 2 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 3-kinase (MV3K) converting mevalonate to mevalonate-3-phosphate; e) mevalonate-3-phosphate-5-kinase (M3P5K) converting mevalonate-3-phosphate to mevalonate-3, 5-bisphosphate; f) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) converting mevalonate-3, 5- bisphosphate to isopentenyl phosphate (IP); g) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); and h) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP).
34. The host cell according to claim 33, wherein the corresponding: a) mevalonate 3-kinase (MV3K) has at least 80% identity to the polypeptide of SEQ ID NO: 9, or SEQ ID NO: 61; b) mevalonate-3-phosphate-5-kinase (M3P5K) has at least 80% identity to the polypeptide of SEQ ID NO: 10, or SEQ ID NO: 63; c) mevalonate-3-phosphate-5-phosphate decarboxylase (BMD) has at least 80% identity to the polypeptide of SEQ ID NO: 11, or SEQ ID NO: 65; and/or d) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
35. The host cell according to claims 33-34, wherein host cell comprises: a. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and/or d. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
36. The host cell according to claims 35, wherein host cell further comprises: a. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ERG20(N127W)-SKL (SEQ ID NO: 85), and/or b. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to FpTPS-SKL (SEQ ID NO: 87).
37. The host cell according to any one of claims 26-29, wherein the operative metabolic pathway is pathway 3 comprising one or more peroxisomally-localized polypeptides selected from the group consisting of: a) acetoacetyl-CoA thiolase converting acetyl-CoA to acetoacetyl-CoA; b) HMG-CoA synthase (HMGS) converting acetoacetyl-Coa to HMG-CoA; c) HMG-CoA reductase (HMGR) converting HMG-CoA to mevalonate; d) mevalonate 5-kinase (MV5K) converting mevalonate to mevalonate-5-phosphate; e) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) converting mevalonate 5- phosphate to trans-anhydromevalonate 5-phosphate; f) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) converting mevalonate 5- phosphate to trans-anhydromevalonate 5-phosphate; g) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) converting trans- anhydromevalonate 5-phosphate to isopentenyl phosphate (IP); h) isopentenyl phosphate kinase (IPK) converting isopentenyl phosphate (IP) to isopentenyl diphosphate (IPP); and i) isopentenyl diphosphate isomerase (IDI) converting isopentenyl diphosphate (IPP) into dimethylallyl diphosphate (DMAPP).
38. The host cell according to claim 37, wherein the corresponding: a) mevalonate 5-phosphate dehydratase (PMDh) has at least 80% identity to the polypeptide of SEQ ID NO: 12, SEQ ID NO: 15, SEQ ID NO: 55, or SEQ ID NO: 57; and/or b) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59.
39. The host cell according to claim 37, wherein the corresponding: a) mevalonate 5-phosphate dehydratase subunit 1 (PMDhl) has at least 80% identity to the polypeptide of SEQ ID NO: 12 or SEQ ID NO: 55; b) mevalonate 5-phosphate dehydratase subunit 2 (PMDh2) has at least 80% identity to the polypeptide of SEQ ID NO: 15 or SEQ ID NO: 57; c) trans-anhydromevalonate 5-phosphate decarboxylase (AMPD) has at least 80% identity to the polypeptide of SEQ ID NO: 13 or SEQ ID NO: 59 and/or d) isopentenyl phosphate kinase (IPK) has at least 80% identity to the polypeptide of SEQ ID NO: 8, SEQ ID NO: 51, or SEQ ID NO: 53.
40. The host cell according to claims 37-39, wherein host cell comprises: a. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaE-SKL (SEQ ID NO: 81), b. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to EfmvaS-SKL (SEQ ID NO: 79), c. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ScERG12-SKL (SEQ ID NO: 95), and/or d. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to SclDI-SKL (SEQ ID NO: 83).
41. The host cell according to claims 35, wherein host cell further comprises: a. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to ERG20(N127W)-SKL (SEQ ID NO: 85), and/or b. a peroxisomally-localized polypeptide or a polynucleotide encoding said polypeptide which is at least 80% identical to FpTPS-SKL (SEQ ID NO: 87).
42. The host cell according to any one of the preceind claims, wherein the host cell comprises or expresses or is configured to express one or more sequences selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO: 98.
43. The host cell according to any one of the preceding claims, wherein the peroxisomally- localized enzyme of i), ii), and/or iii) are heterologous to the host cell.
44. The host cell according to claim 43, wherein the peroxisomally-localized enzyme having kinase activity is heterologous to the host cell.
45. The host cell according to any one of the preceding claims, wherein the peroxisomally- localized enzyme of iii) having kinase activity is a peroxisomally-localized kinase.
46. The host cell according to claim 45, wherein the kinase is an isopentenyl phosphate kinase (IPK).
47. The host cell according to claim 46, wherein the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), Methanothermobacter thermautotrophicus PK (MtIPK), Thermoplasma acidophilum IPK variant IVG (TalPK(IVG), and TalPK wild-type.
48. The host cell according to any one of the preceding claims, wherein the host cell is a yeast cell comprising a peroxisomally-localized isopentenyl phosphate kinase (IPK), optionally wherein the IPK is selected from the group consisting of: IPK from Arabidopsis thaliana (AtIPK), Methanothermobacter thermautotrophicus IPK (MtIPK), Thermoplasma acidophilum IPK variant 204G (TalPK(204G)), and TalPK wild-type.
49. A yeast cell comprising comprising all the peroxisomally-localized polypeptides a)-g) defined in claim 30 (pathway 1), and a peroxisomally localized GPP synthase (GPPS).
50. A yeast cell comprising comprising all the peroxisomally-localized polypeptides a)-h) defined in claim 33 (pathway 2), and a peroxisomally localized GPP synthase (GPPS).
51. A yeast cell comprising comprising all the peroxisomally-localized polypeptides a)-i) defined in claim 37 (pathway 3), and a peroxisomally localized GPP synthase (GPPS).
52. The yeast cell according to any one of claims 49, 50, or 51, further comprising a terpene synthase, a prenyltransferase, and/or another isoprenoid or non-isoprenoid prenyltransferase.
53. A cell culture, comprising the host cell of any one of claims 1-48, or the yeast cell of any one of claims 49-52, and a growth medium.
54. A method for producing a compound selected from the group consisting of: a monoterpenoid, a cannabinoid, a monoterpene indole alkaloid, and a prenylated aromatic compound, comprising the steps of: a) providing a host cell as defined in any of the claims 1-48 or a yeast cell as defined in any one of claims 49-52; b) fermenting the host cell or yeast cell in a substrate supporting growth of the cell; c) when required, providing the cell with a substrate to be prenylated, for example wherein the substrate to be prenylated is selected from: olivetolic acid, olivetolic acid derivatives, naringenin, genistein, resveratrol or p-coumaric acid, and d) recovering the compound from the fermentation broth.
55. The method according to claim 54, wherein the compound is selected from the group consisting of: sabinene, alpha-pinene, beta-pinene, camphene, (+)-limonene, (-)-limonene, geraniol, linalool, myrcene, 1,8-cineole, borneol, bornyl diphosphate, alpha-terpineol, isoborneol, tricyclene, alpha-thujene, alpha-fenchene, delta-2-carene, alpha-phellandrene, 3- carene, 1,4-cineole, alpha-terpinene, beta-phellandrene, (Z)-beta-ocimene, (E)-beta-ocimene, gamma-terpinene, terpinen-4-ol, terpinolene, allo-ocimene, cis-beta-terpineol, cis-terpine-1- ol, delta-terpineol, alpha-terpineol, nerol, 2-methylisoborneol, 2-methylenebornene, 2- methyl-2-bornene, beta-phellandrene, 2-methyllimonene, 2-methylmyrcene, 2- methylgeraniol, 2-methylinalool, cannabigerolic acid, cannabiberolic acid analogs, prenyl trypto-phan, artepillin C, drupanin, osthrutin, geranyl-resveratrol, geranylated querce-tin, geranyl-naringenin, geranyl-isoliqiritigenin, isobavachalcone, isoprene, la-vandulol, chrysanthemol dimethylallyltryptophan, 4'-dimethylallyl-apigenin, 6-prenyl-apigenin, 4'- dimethylallyl-naringenin, 4'-dimethylallyl-kaempferol, 4'-dimethylallyl-daidzein, 7- dimethylallyl-daidzein, 7,4'-di-(dimethylallyl)-daidzein, 4'-dimethylallyl-genistein, 7- dimethylallyl-genistein, 7,4'-di-(dimethylallyl)-genistein, 4-dimethylallyl-isoliquiritigenin, 4'- dimethylallyl-equol, 7-dimethylallyl-equol, 6-dimethylallyl-equol, 4'-dimethylallyl-daidzin, 7- dimethylallyl-umbelliferone, 8-dimethylallyl-curcumin, 8'-dimethylallyl-demethoxycurcumin, 8-dimethylallyl-demethoxycurcumin, 4'-dimethylallyl-resveratrol, or 5-dimethylallyl- diethylstilbestrol.
56. A fermentation liquid comprising the compound as defined in any one of claims 54-55 and the host cell as defined in any one of claims 1-48.
57. The fermentation liquid according to claim 56, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of the host cells are disrupted.
58. The fermentation liquid according to any one of claims 56-57, wherein at least 50%, such as at least 75%, such as at least 95%, such as at least 99% of solid cellular material has separated from the liquid.
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