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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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