Production of geranyl diphosphate-derived compounds

Abstract

Yeast cells are disclosed that have a peroxisome-localized GPP synthase and a peroxisome-localized enzyme or precursor that converts GPP to monoterpenoids, cannabinoids, monoterpene indole alkaloids, and prenylated aromatic compounds, such that the yeast cells can produce improved amounts of monoterpenoids, cannabinoids, monoterpene indole alkaloids, and prenylated aromatic compounds compared to the same yeast cells in which the GPP synthase and the enzyme that converts GPP are located in the cytoplasm. Uses of the yeast cells for producing monoterpenoids, cannabinoids, monoterpene indole alkaloids, and prenylated aromatic compounds are further disclosed.

Description

[Technical Field]

[0001] Sequence Listing Reference This application contains a Sequence Listing in computer readable format, which is incorporated by reference.

[0002] The present invention relates to the production of monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and prenylated aromatic compounds in eukaryotic cells, such as yeast cells. The present invention further relates to engineered yeast cells particularly adapted for such production. [Background technology]

[0003] Terpenes, terpenoids, their derivatives, and other prenylated aromatic compounds are widely used, for example, as pharmaceuticals, cosmetics, dietary supplements, flavors, fragrances, and insecticides. Methods for increasing the production of these compounds in natural or engineered cells are abundant in the art.

[0004] The use of engineered microorganisms to produce valuable molecules from renewable feedstocks is a desirable alternative to traditional production methods. However, achieving economically viable yields, titers, and productivity is a major obstacle to industrialization. A frequently encountered obstacle arises from tradeoffs between engineered pathways and native metabolism, which pull in opposite directions. Metabolism has evolved to meet the needs of growth and rerouting, which can be challenging due to multiple layers of control, such as gene regulation, negative feedback loops at the enzyme level with downstream products, and efficient competing pathways.

[0005] Monoterpenes and other geranyl diphosphate (GPP)-derived compounds, widely used as flavors, fragrances, and insecticides, and which may find application as droplets in jet fuel or biopolymers, are prime examples of these problems: on the one hand, extraction from natural plant sources is hardly able to meet the increasing demand and represents an environmental challenge, while on the other hand, production by microbial hosts results in low yields and is hampered by natural metabolic constraints.

[0006] Monoterpene production by engineered microorganisms relies on either the MEP pathway (primarily in prokaryotes), the MVA pathway, or the alternative MVA pathway, all three of which result in the formation of DMAPP and IPP, which then condense to form GPP. GPP is converted to a wide range of monoterpenes by monoterpene synthases (MTSs), which rearrange the 10-carbon skeleton of GPP into various monoterpenes or their precursors, or is further elongated to FPP or GGPP by the successive addition of IPP molecules to form sesquiterpenes and diterpenes, respectively. GPP also serves as a precursor for the synthesis of several compounds containing a terpene moiety, such as cannabinoids, iridoids, monoterpene indole alkaloids, prenylated aromatic compounds, and other meroterpenoids.

[0007] Yeast is considered a good host for terpene production due to its ease of manipulation, its native mevalonate pathway, and its ability to harbor functional cytochrome P450 in its endoplasmic reticulum (ER) membrane for terpene scaffold decoration. It has shown great potential for producing sesquiterpenes such as artemisinin and farnesene on an industrial scale. However, the production of monoterpenes has been much less successful to date.

[0008] This can be explained primarily by a game of tug-of-the-rope that takes place at the GPP branch point between native sterol biosynthesis and the heterologous pathway leading to monoterpenes, favoring native metabolism. In wild-type yeast, no GPP-based compounds are produced; GPP's sole purpose is to serve as an intermediate for further elongation to FPP to produce squalene in the sterol pathway. Therefore, yeast lacks a dedicated GPP synthase; GPP is produced by a bifunctional GPP-FPP synthase, Erg20p, which has been shown to highly efficiently convert GPP to FPP upon formation, directing it toward sterol synthesis. Various strategies have been employed to downregulate Erg20p by converting it into a strict GPP synthase or by reducing its activity; however, the essential nature of sterol synthesis has limited these attempts, often reducing cell viability by overburdening sterol synthesis. Compartmentalization is a strategy used by eukaryotic cells to solve similar problems within their own metabolism. Organelles such as mitochondria, peroxisomes, and the endoplasmic reticulum (ER) are designed to protect the rest of the cell from toxic compounds, isolate intermediates from competing pathways, shield enzymes from inhibitors, and overall provide a more suitable environment for reactions to occur away from the main body of metabolism.

[0009] An example of such a strategy was recently reported, where compartmentalization of an extra copy of the entire MVA pathway within mitochondria together with geraniol synthase improved geraniol production by 11.5-fold compared with the same modification in the cytosol.

[0010] While this proved to be a successful strategy, hijacking mitochondria appeared to result in metabolic burdens on strains, resulting in lower cell viability and growth. This may be due to the essential nature of mitochondria as the powerhouse of the cell, which may hinder further engineering to reach the significantly higher titers required for industrial applications. These findings also indicated that there may be limitations to the extent to which mitochondria can be engineered without compromising metabolic integrity.

[0011] U.S. Patent Application Publication No. 20150010978 discloses a method for producing terpenoids in large numbers of cells by transforming the cells with genes encoding enzymes involved in terpenoid biosynthesis. These genes can be introduced into the chloroplast genome of cells that have chloroplasts. An example is the production of diterpenes.

[0012] Korean Patent Publication No. 20190079575 discloses a recombinant yeast that increases the number of peroxisomes, resulting in increased production of terpenoids. The insertion of a heterologous geranylgeranyl pyrophosphate synthase is also disclosed.

[0013] US Patent Application Publication No. 20130302861 discloses terpenoid production in yeast by localizing terpene synthase to mitochondria. The examples focus on sesquiterpenes derived from FPP.

[0014] Guo-Song Liu et al. (J. Agric. Food Chem. 2020, 68, 7, 2132-2138) reported the production of squalene, an FPP-based precursor of ergosterol, in yeast peroxisomes, demonstrating the functionality of the MVA pathway in this organelle. However, the resulting strain did not outperform its cytosolic counterpart. This is most likely due to the fact that the original pathway was already well-tuned and engineered to efficiently produce squalene in the cytosol. Summary of the Invention

[0015] In a first aspect, the present invention relates to a yeast cell comprising a peroxisomal enzyme that catalyzes the formation of a branch point compound, the peroxisomal enzyme being capable of converting the branch point compound via a preferred pathway and a non-preferential pathway, and a peroxisomal enzyme that catalyzes the first step of the non-preferential pathway. In a preferred embodiment, the present invention relates to a yeast cell comprising a peroxisomal GPP synthase and a peroxisomal monoterpene synthase.

[0016] In a second aspect, the invention relates to methods for producing monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and prenylated aromatic compounds using the yeast cells of the invention. [Brief explanation of the drawings]

[0017] [Figure 1] 1 shows a chart disclosing the production of limonene in yeast, where limonene synthase (MTS) and / or GPP synthase were localized in either the cytoplasm or peroxisomes. For further details, see Example 1. [Figure 2]

[0023] Figure 1 shows a chart disclosing the effect of localizing genes of the MVA pathway along with limonene synthase (MTS) and GPP synthase to peroxisomes. For further details, see Example 2. [Figure 3] 1 shows a chart disclosing the improved production of six monoterpenoids, camphene, sabinene, (S)-(-)-limonene, alpha-pinene, (R)-(+)-limonene, and (R)-(+)-linalool, by peroxisomal localization of the respective synthases. For further details, see Example 3. [Figure 4A] 4A and 4B show graphs of titer development in fermentations of yeast according to the present invention: Figure 4A shows the production of (R)-(+)-limonene; [Figure 4B]Figure 4A shows a graph of titer development in fermentation of yeast according to the present invention. Figure 4B shows the production of geraniol. For further details, see Example 4. [Figure 5] 1 shows a chart disclosing the effect of localizing a bifunctional GPP synthase / terpene synthase enzyme (GPP synthase-terpene synthase fusion) to peroxisomes on terpene production. For further details, see Example 5. [Figure 6A] Figure 6A shows graphs disclosing the production of trans-isopiperitenol and 8-hydroxy-geraniol. Figure 6B shows the production of (-)-limonene and trans-isopiperitenol in strains CYTLim06, PERLim29, and PERLim30. [Figure 6B] Figure 6A shows a graph disclosing the production of trans-isopiperitenol and 8-hydroxy-geraniol. Figure 6B shows the production of geraniol and 8-hydroxy-geraniol in strains PERMGe03 and PERGer04. [Figure 7A] 7A and 7B show graphs disclosing the production of cannabinoid precursors. Figure 7A shows CBGA production in strains PERMva01 and PERCan01 in cultures supplemented with 0.5 mM OA. [Figure 7B] 7A and 7B show graphs disclosing the production of cannabinoid precursors. Figure 7B shows the production of CBGA at different concentrations of OA added to the culture. [Figure 7C] Figure 7C shows graphs disclosing the production of cannabinoid precursors. Figure 7C shows the improvement of peroxisomal CBGA production by targeting CsPT4 to peroxisomes using an N-terminal targeting signal in the PER-Can02 strain.

[0018] Sequence Listing Overview SEQ ID NO: 1 is derived from the Saccharomyces cerevisiae Erg20p protein, Erg20p N127W 1 is the amino acid sequence of the engineered geranyl diphosphate synthase shown as

[0019] SEQ ID NO:2 is derived from the Saccharomyces cerevisiae Erg20p protein, Erg20p N127W , which is the amino acid sequence of geranyl diphosphate synthase to which the SKL peroxisomal localization signal is attached.

[0020] SEQ ID NO:3 is the amino acid sequence of (+)-limonene synthase from Citrus limon, encoded by the C / LimS gene.

[0021] SEQ ID NO: 4 is the amino acid sequence of (+)-limonene synthase from Citrus limon, encoded by the C / LimS gene and provided with the SKL peroxisomal localization signal.

[0022] SEQ ID NO: 5 is the amino acid sequence of camphene synthase derived from Solanum elaeagnifolium and encoded by the SeCamS gene.

[0023] SEQ ID NO: 6 is the amino acid sequence of camphene synthase from Solarium area agnifolium, encoded by the SeCamS gene, and provided with the SKL peroxisomal localization signal.

[0024] SEQ ID NO: 7 is the amino acid sequence of α-pinene synthase from Pinus taeda encoded by the PtPinS gene.

[0025] SEQ ID NO: 8 is the amino acid sequence of α-pinene synthase from Pinus staeda encoded by the PtPinS gene and provided with the SKL peroxisomal localization signal.

[0026] SEQ ID NO: 9 is the amino acid sequence of sabinene synthase encoded by the SpSabS gene from Salvia pomifera.

[0027] SEQ ID NO: 10 is the amino acid sequence of sabinene synthase from Salvia pomifera, encoded by the SpSabS gene and provided with the SKL peroxisomal localization signal.

[0028] SEQ ID NO: 11 is the amino acid sequence of geraniol synthase derived from Ocimum basilicum and encoded by the tObGES gene.

[0029] SEQ ID NO: 12 is the amino acid sequence of geraniol synthase from Ocimum basilicum, encoded by the tObGES gene, and provided with the SKL peroxisomal localization signal.

[0030] SEQ ID NO: 13 is the amino acid sequence of geranyl diphosphate:olivetolate geranyltransferase derived from Cannabis sativa and encoded by the CsPT4 gene.

[0031] SEQ ID NO: 14 is the amino acid sequence of geranyl diphosphate:olivetolate geranyltransferase from Cannabis sativa, encoded by the CsPT4 gene, and provided with the SKL peroxisomal localization signal.

[0032] SEQ ID NO: 15 is a GPP synthase domain (Erg20p) linked by a 5xGS polypeptide and targeted to peroxisomes by a C-terminal PTS1. N127W ) and the terpene synthase domain (C / LimS).

[0033] SEQ ID NO: 16 is a sequence of a terpene synthase domain (C / LimS) and a GPP synthase domain (Erg20p) linked by a 5xGS polypeptide and targeted to peroxisomes by a C-terminal PTS1. N127W ) and the amino acid sequence of the fusion protein having

[0034] SEQ ID NO: 17 is the amino acid sequence of geraniol 8-hydroxylase from Catharanthus roseus and encoded by the CrG80H gene.

[0035] SEQ ID NO: 18 is the amino acid sequence of the cytochrome P450 reductase from Catharanthus roseus and encoded by the Ci OPR gene.

[0036] SEQ ID NO: 19 is the amino acid sequence of the aromatic prenyltransferase AtaPT from Aspergillus terreus.

[0037] SEQ ID NO: 20 is the amino acid sequence of 7-dimethylallyltryptophan synthase (7-DMATS) from Neosartorya fumigatus.

[0038] SEQ ID NO: 21 is the amino acid sequence of the phenylpropane-specific prenyltransferase AcPT1 from Artemisia capillaris.

[0039] SEQ ID NO: 22 is the amino acid sequence of (R)-(+)-linalool synthase from Mentha citrate and encoded by the McLiS gene.

[0040] SEQ ID NO: 23 is the amino acid sequence of (R)-(+)-linalool synthase from Mentha citrate, encoded by the McLiS gene and provided with the SKL peroxisomal localization signal.

[0041] SEQ ID NO: 24 is the amino acid sequence of (S)-(-)-limonene synthase from spearmint encoded by the MsLimS gene.

[0042] SEQ ID NO: 25 is the amino acid sequence of (S)-(-)-limonene synthase from spearmint, encoded by the MsLimS gene and provided with the SKL peroxisomal localization signal.

[0043] SEQ ID NO: 26 is the amino acid sequence of beta-myrcene synthase from Ocimum basilicum, encoded by the ObMyrS gene, and provided with the SKL peroxisomal localization signal.

[0044] SEQ ID NO: 27 is the amino acid sequence of limonene-3-hydroxylase from spearmint, encoded by the MsLim3H gene.

[0045] SEQ ID NO: 28 is the amino acid sequence of the cytochrome P450 reductase from Taxus cuspidata and encoded by the tcCPR gene.

[0046] SEQ ID NO: 29 is the amino acid sequence of geranyl diphosphate:olivetolate geranyltransferase from Cannabis sativa, encoded by the CsPT4 gene, and carrying an N-terminal peroxisomal localization signal. Definitions and Abbreviations

[0047] Branch point molecule: According to the present invention, a branch point molecule is intended to mean a molecule of a biochemical pathway that can be converted into two or more different other molecules or pathways. An example is GPP, which can be converted into FPP, thereby leading to the synthesis of sesquiterpenes and higher terpenes, or it can be converted into monoterpenes by monoterpene synthases, into cannabinoids by prenyltransferase enzymes, or into prenylated aromatic compounds by corresponding prenyltransferases. Branch point molecules typically have a preferred or preferred pathway, which in native yeast cells is preferred, for example, due to biosynthetic needs, and one or more other non-preferential pathways.

[0048] DMAPP and IPP: Dimethylallyl pyrophosphate (or dimethylallyl diphosphate; DMAPP) and isopentenyl pyrophosphate (or isopentenyl diphosphate; IPP) are five-carbon precursors used to make isoprenoids.

[0049] GPP: geranyl diphosphate (or geranyl pyrophosphate; GPP). GPP is formed by the condensation of DMAPP and an IPP molecule. GPP is a branch point molecule in isoprenoid synthesis, and by adding an IPP molecule, it is converted to FPP, which can lead to the biosynthesis of sesquiterpenes, diterpenes, or triterpenes, or to sterol synthesis, or, through the action of monoterpene synthase, to the synthesis of monoterpenoids, iridoids, and monoterpene indole alkaloids. Other prenyltransferases can also direct GPP to the production of cannabinoids, prenylated aromatic compounds, or meroterpenoids in general.

[0050] FPP: Farnesyl pyrophosphate (or farnesyl diphosphate; FPP) is formed by condensing GPP with an IPP molecule. FPP is a precursor for the synthesis of sesquiterpenes, diterpenes, triterpenes, and sterols.

[0051] GGPP: Geranylgeranyl pyrophosphate (or geranylgeranyl diphosphate; GGPP).

[0052] GGPP is formed by condensing FPP with an IPP molecule. GGPP is a precursor for the synthesis of diterpenes.

[0053] Higher terpene: As used herein, this refers to a molecule containing an isoprenoid structure of more than 10 carbon atoms. Examples include sesquiterpenes, diterpenes, and triterpenes. In addition to the terpene structure, higher terpenes may also contain moieties that do not have an isoprenoid structure.

[0054] Monoterpene: A monoterpene (or monoterpenoid) is a molecule containing a 10-carbon isoprenoid structure. In addition to the 10-carbon isoprenoid structure, monoterpenoids may contain moieties that do not have an isoprenoid structure. In many cases, the biosynthesis of monoterpenoids involves several additional steps after the initial conversion of GPP to the basic monoterpene skeleton. These additional steps can be oxidation (e.g., catalyzed by cytochrome P450 enzymes), reduction, isomerization, acetylation, methylation, etc.

[0055] Iridoid: a group of compounds found in plants and some animals that are biosynthetically derived from 8-oxogeraniol.

[0056] Monoterpene indole alkaloids are a large and diverse group of phytochemical compounds derived from tryptamine units and 10- or 9-carbon units of terpenoid origin, as well as from 8-oxo-geraniol.

[0057] Cannabinoids: A group of compounds whose members were first isolated from the plant Cannabis sativa. Many cannabinoids are biosynthesized by the addition of GPP to olivetolic acid.

[0058] MEP pathway: The methylerythritol 4-phosphate (MEP) pathway that forms IPP and DMAPP. The pathway is found, for example, in most bacteria, algae, and plastids of higher plants.

[0059] MVA pathway: The mevalonate pathway (MVA pathway) is an essential metabolic pathway present in eukaryotes and some bacteria that starts from acetyl-CoA to form IPP and DMAPP.

[0060] Alternative MVA Pathway: An alternative MVA pathway is found in archaea, providing IPP and DMAPP that starts from acetyl-CoA but utilizes isopentenyl phosphate as an intermediate.

[0061] Monoterpene synthase. This term includes any enzyme that can catalyze the rearrangement of GPP to monoterpenoids. Monoterpene synthases typically synthesize multiple products, although the diversity of products varies among terpene synthases. Some terpene synthases have high product specificity and catalyze the synthesis of a limited number of products, while others have low product specificity and catalyze the synthesis of a wide variety of different terpenes. Examples of products of monoterpene synthases include, but are not limited to, the following compounds: tricyclene, alpha-thuene, alpha-pinene, alpha-phencene, camphene, sabinene, beta-pinene, myrcene, delta-2-carene, alpha-phellandrene, 3-carene, 1,4-cineole, alpha-ter-pinene, beta-phellandrene, 1,8-cineole, limonene, (Z)-beta-ocimene, (E)-beta-ocimene, gamma-terpinene, terpinolene, linalool, perylene, allo-ocimene, cis-beta-terpineol, cis-terpin-1-ol, isoborneol, delta-terpineol, borneol, chrysanthemol, lavandulol, alpha-terpineol, neol, and geraniol. In addition to GPP, certain terpene synthases (or terpene synthase variants developed by protein engineering) have been reported to convert non-standard prenyl diphosphate substrates, such as the 11-carbon substrate 2-methyl-GPP, to terpenes with non-standard prenyl scaffolds (Ignea et al., 2018). In the context of this disclosure, enzymes capable of converting non-standard prenyl diphosphates with lengths different from 10 to non-standard terpenoids with 8, 9, 11, or 12 carbons are also included in the definition of monoterpene synthase.

[0062] Prenyltransferase: An enzyme that adds a prenyl moiety to an isoprenoid or non-isoprenoid backbone. Many prenyltransferases that add prenyl moieties to other isoprenoid chains are involved in the synthesis of prenyl diphosphate precursors, such as GPP (GPP synthase), FPP (FPP synthase), GGPP (GGPP synthase), or geranyl-farnesyl diphosphate synthase (GFPP synthase). These enzymes typically add an IPP unit to extend DMAPP to a larger prenyl diphosphate in the trans configuration. For this reason, they are also called trans-polyprenyl synthases or trans-polyprenyltransferases. Several prenyltransferase enzymes exist that catalyze the cis-condensation and elongation of DMAPP with IPP. These enzymes are called cis-prenyltransferases, or cis-polyprenyl diphosphate synthases, or cis-polyprenyltransferases, and are responsible for the synthesis of neryl diphosphate, cis,cis-farnesyl diphosphate, and neryl-neryl diphosphate.

[0063] Furthermore, certain isoprenoid prenyltransferases have been reported to condense two DMAPP molecules into lavandulyl diphosphate or chrysanthemyl diphosphate.

[0064] Prenyltransferases, which add prenyl moieties to non-isoprenoid skeletons, add DMAPP, GPP, FPP, or GGPP to non-isoprenoid compounds, including flavonoids, amino acid residues and peptides, aromatic compounds, and generally other chemical compounds. Such prenyltransferase enzymes are involved in the biosynthesis of many different natural products, including, but not limited to, cannabinoids, prenylated flavonoids, or other meroterpenoids. In the case of cannabinoid synthesis, this enzyme is geranyl diphosphate:olivetrate geranyltransferase.

[0065] Prenyltransferase can be part of a separate polypeptide or fused to one polypeptide chain. Prenyltransferase can also be fused to GPP synthase, terpene synthase, or other non-terpene synthesis protein. Prenyltransferase can also be fused to the enzyme that naturally localizes in the peroxisomal matrix or its membrane in yeast or other organisms, or can be fused to a polypeptide chain that itself is fused to a peroxisomal targeting signal.

[0066] Aromatic prenyltransferase can be selected from any enzyme with prenyltransferase activity identified from any organism or engineered organism, which can transfer isoprenoid moiety to another isoprenoid or non-isoprenoid compound.Prenyltransferase can be part of a separate polypeptide, or can be fused to one polypeptide chain.Prenyltransferase can also be fused to GPP synthase, terpene synthase, or other non-terpene synthesis protein.Prenyltransferase can also be fused to an enzyme that is naturally localized in the peroxisomal matrix or its membrane in yeast or other organisms, or can be fused to a polypeptide chain that is itself fused to a peroxisomal targeting signal. DETAILED DESCRIPTION OF THE INVENTION

[0067] The present invention is based on the recognition that in biochemical pathways, there are branch points where the branch point molecule can be diverted to a different biochemical pathway, where the preferred pathway leads to compounds essential for the growth of a particular organism, while other pathways are not prioritized (designated non-prioritized pathways). As a result, special measures are required to induce the cell to prioritize the non-preferred pathway.

[0068] According to the present invention, production of non-preferential pathway compounds is increased by peroxisomal localization of an enzyme that catalyzes the formation of a branch point compound and an enzyme that catalyzes the first step of the non-preferential pathway.

[0069] The enzyme that catalyzes the formation of the branch point compound and the enzyme that catalyzes the first step of the non-preferential pathway may exist as separate molecules; they may exist as a single molecule containing a domain that catalyzes the formation of the branch point compound and another domain that catalyzes the first step of the non-preferential pathway; or they may exist in the form of a multidomain molecule that contains one or two of the two enzymatic activities plus one or more additional domains with different functions.

[0070] GPP is such a branch point molecule in terpene synthesis and can be converted into compounds containing a monoterpenoid or a 10-carbon monoterpenoid structure linked to a non-prenyl moiety, a sesquiterpenoid, a disesquiterpenoid, or a triterpenoid, or a 15-carbon sesquiterpenoid, a 20-carbon disesquiterpenoid, or a 30-carbon triterpenoid structure, or into sterols.

[0071] In yeast, conversion of GPP to FPP and ultimately to sterols is prioritized because sterols are essential for cell growth and viability.

[0072] Thus, in a first aspect, the present invention relates to a yeast cell in which an enzyme that catalyzes the formation of GPP and an enzyme that catalyzes the first step of a pathway starting from GPP and forming a monoterpenoid, cannabinoid, iridoid, monoterpene indole alkaloid, or prenylated aromatic compound are localized in the peroxisome.

[0073] Enzymes that catalyze the formation of GPP, also known as GPP synthases, are known to those skilled in the art. The present invention is not limited to any particular GPP synthase; in principle, any GPP synthase localized in peroxisomes can be used in accordance with the present invention. The GPP synthase may be a homologous GPP synthase, i.e., an enzyme derived from the same species as the host cell; a heterologous enzyme, i.e., an enzyme derived from a different species than the host cell; or a synthase, i.e., an enzyme not found in nature but artificially created using techniques known in the field of genetic engineering. GPP synthases may be single subunits or multisubunit enzymes composed of identical or non-identical subunits (several such examples exist in nature and are known to those skilled in the art, i.e., the combination of large and small subunits of the snapdragon GGPP synthase (Orlova I. et al., 2009)). The subunits of a GPP synthase may be part of separate polypeptides or fused together into a single polypeptide chain. The subunits may also be fused to a terpene synthase, prenyltransferase, or another non-terpene synthesis protein. In particular, it may be fused to an enzyme that is naturally localized to peroxisomes in yeast or another organism, or may be fused to a polypeptide chain that is in turn fused to a peroxisomal targeting signal.

[0074] An example of a preferred GPP synthase according to the present invention is the engineered GPP synthase Erg20p, which is a native S. cerevisiae GPP synthase containing the N127W substitution. N127W (SEQ ID NO: 1). The N127W substitution blocks the catalytic site of the enzyme, preventing further conversion of GPP to FPP by the addition of an IPP molecule.

[0075] Other preferred GPP synthases include polypeptides having GPP synthase activity, alone or in combination with other polypeptides, from organisms belonging to any of the kingdoms of life: bacteria, archaea, protozoa, chromistas, plants, fungi, or animals. Other preferred synthases include enzymes that have been engineered using protein engineering to have GPP synthase activity, alone or in combination with other polypeptides.

[0076] Those skilled in the art are also familiar with enzymes that catalyze the first step of the pathway that starts from GPP and forms monoterpenoids, iridoids, cannabinoids, monoterpene indole alkaloids, prenylated aromatic compounds, or other meroterpenoids. Non-limiting examples include monoterpene synthases, such as (+)-limonene synthase, (-)-limonene synthase, α-pinene synthase, 1,8-cineole synthase, sabinene synthase, camphene synthase, linalool synthase, myrcene synthase, or geraniol synthase, and prenyltransferases, such as geranyl diphosphate:olivetolate geranyltransferase or broad specificity aromatic prenyltransferase.

[0077] Examples of preferred monoterpene synthases according to the present invention include (+)-limonene synthase from Citrus limon and having the amino acid sequence of SEQ ID NO: 3, camphene synthase from Solarium area agnifolium and having the amino acid sequence of SEQ ID NO: 5, (-)-limonene synthase from spearmint (Mentha spicata) and having the amino acid sequence of SEQ ID NO: 25, (+)-linalool synthase from Mentha citrata and having the amino acid sequence of SEQ ID NO: 23, myrcene synthase from Ocimum basilicum and having the amino acid sequence of SEQ ID NO: 26, α-pinene synthase from Loblolly pine and having the amino acid sequence of SEQ ID NO: 7, sabinene synthase from Salvia pomifera and having the amino acid sequence of SEQ ID NO: 9, and geraniol synthase from Ocimum basilicum and having the amino acid sequence of SEQ ID NO: 11.

[0078] Other preferred monoterpene synthases include beta-pinene synthase, (-)-limonene synthase, linalool synthase, myrcene synthase, bornyl diphosphate synthase, alpha-terpineol synthase, isoborneol synthase, tricyclene synthase, alpha-thudiene synthase, alpha-fenchene synthase, delta-2-carene synthase, alpha-phellandrene synthase, 3-carene synthase, 1,4-cineole synthase, alpha-terpinene synthase, beta-phellandrene synthase, 1,8-cineole synthase, (Z)-beta -Includes polypeptides having activity as ocimene synthase, (E)-beta-ocimene synthase, gamma-terpinene synthase, terpinolene synthase, allo-ocimene synthase, cis-beta-terpineol synthase, cis-terpin-1-ol synthase, delta-terpineol synthase, borneol synthase, voltanol synthase, alpha-terpineol synthase, nerol synthase, 2-methyl-isoborneol synthase, 2-methylenebornene synthase, 2-methyl-2-bornene synthase, or beta-phellandrene synthase.

[0079] Prenyltransferases capable of attaching geranyl moieties to non-isoprenoid backbones include the geranyl diphosphate:olivetol geranyltransferase CsPT4 from Cannabis sativa, which has the amino acid sequence of SEQ ID NO: 13, or the aromatic prenyltransferase AtaPT from Aspergillus terreus, which has the amino acid sequence of SEQ ID NO: 19.

[0080] Other preferred prenyltransferases capable of adding a prenyl group to an isoprenoid backbone include neryl diphosphate synthase, chrysanthemyl diphosphate synthase, or lavandulyl diphosphate synthase, and preferred prenyltransferases capable of adding a prenyl group to a non-isoprenoid backbone include 7-dimethylallyltryptophan synthase (7-DMATS) from Aspergillus fumigatus (SEQ ID NO: 20) and the phenylpropane species-specific prenyltransferase AcPT1 from Artemisia capillaris (SEQ ID NO: 21).

[0081] In a preferred embodiment, Saccharomyces cerevisiae cells are produced in which GPP synthase and limonene synthase are localized in peroxisomes. We found that this alone was sufficient to induce a 32-fold improvement in monoterpene (limonene) production compared to the production obtained when these two enzymes were expressed in the cytosol, or when only one of the two enzymes was present in the peroxisome and the other in the cytosol. Furthermore, further peroxisomal compartmentalization of the complete MVA pathway, consisting of EfmvaS, EfmvaE, Erg12p, Erg9p, and Idi1p, improved monoterpene production by 14-fold, 17-fold, 17-fold, 20.5-fold, 22-fold, and 125-fold for camphene, pinene, (-)-limonene, (+)-linalool, sabinene, and (+)-limonene, respectively, compared to identical yeast cells in which the enzymes were localized in the cytosol.

[0082] In another preferred embodiment, S. cerevisiae cells are provided in which GPP synthase and geraniol synthase are localized in the peroxisome, and the yeast cells exhibit improved levels of geraniol, a precursor of iridoid and monoterpene indole alkaloids, compared to the same cells in which the enzymes are localized in the cytosol.

[0083] In a further preferred embodiment, S. cerevisiae cells are provided in which GPP synthase and olivetolic acid prenyltransferase are localized in the peroxisome. The yeast cells are efficient at producing cannabigerolic acid, a precursor of several cannabinoid compounds.

[0084] In a further embodiment, S. cerevisiae cells are produced in which Aspergillus terreus GPP synthase and aromatic prenyltransferase AtaPT are localized in the peroxisome. When umbelliferone, quercetin, isoquercetin, resveratrol, or naringenin were produced, the yeast cells efficiently synthesized osrutin, geranylated questin, geranylated isoquercetin, geranylresveratrol, and geranyl-naringenin, respectively. In a further embodiment, S. cerevisiae cells are produced in which isopentenyl diphosphate isomerase (IDI), a DMAPP synthase, and terpene synthase (isoprene synthase; ISPS), which catalyzes the synthesis of isoprene, are localized in the peroxisome. The resulting yeast cells efficiently synthesized isoprene. In another embodiment, S. cerevisiae cells are provided in which isopentenyl diphosphate isomerase (IDI) and lavandulyl diphosphate synthase from Lavandula x intermedia are localized in peroxisomes.The yeast cells are provided with efficient synthesis of lavandulol.

[0085] In a further embodiment, S. cerevisiae cells are provided in which isopentenyl diphosphate isomerase (IDI) and chrysanthemyl diphosphate synthase from Tanacetum cinerariifolium are localized in the peroxisome. The yeast cells provided efficient synthesis of chrysanthemol.

[0086] In a further embodiment, S. cerevisiae cells are provided in which isopentenyl diphosphate isomerase (IDI) and 7-dimethylallyltryptophan synthase (7-DMATS) from Aspergillus fumigatus are localized in the peroxisome. The yeast cells provided efficient synthesis of prenyl-tryptophan.

[0087] In a further embodiment, S. cerevisiae cells are provided in which isopentenyl diphosphate isomerase (IDI) and the phenylpropane-specific prenyltransferase AcPT1 from Artemisia capillaris are localized in peroxisomes. When p-coumaric acid was provided, the yeast cells efficiently synthesized dorupanin and artepillin C.

[0088] In a further embodiment, S. cerevisiae cells are provided in which isopentenyl diphosphate isomerase (IDI) and the O-prenyltransferase AcaPT from Antrodia camphorata are localized in peroxisomes. When apigenin, kaempferol, daidzein, naringenin, genistein, isoliquiritigenin, equol, umbelliferone, curcumin, resveratrol, or diethylstilbestrol is provided, the yeast cells are capable of expressing 4'-dimethylallyl-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)-daidzein, 4'-dimethylallyl-genistein, 7,4'-di-(dimethylallyl) ... This effectively resulted in the synthesis of 4-(dimethylallyl)-genistein, 4-dimethylallyl-isoliquitigenin, 4'-dimethylallyl-equol, 7-dimethylallyl-equol, 6-dimethylallyl-equol, 4'-dimethylallyl-daidzin, 7-dimethylallyl-umbelliferone, 8-dimethylallyl-curcumin, 8'-di-methylallyl-demethoxycurcumin, 8-dimethylallyl-demethoxycurcumin, 7-dimethylallyl-L-tryptophan, 4'-dimethylallyl-resveratrol, and 5-dimethylallyl-diethylstilbestrol.

[0089] Peroxisome localization According to the present invention, the expression peroxisomal localization or grammatically equivalent terms in relation to biosynthetic enzymes of the terpene pathway is intended to mean that the enzymes in question are translocated to peroxisomes or peroxisomal membranes after synthesis, and that the enzymes then exert their catalytic function in the peroxisomes.

[0090] Peroxisome localization can be achieved by providing a gene encoding a peroxisome-localized enzyme with a peroxisome localization signal. Peroxisome localization and peroxisome localization signals are known in the art, for example, WO 9424289 and KR 101308971 (incorporated herein by reference), and such signals and methods known in the art can also be used in accordance with the present invention.

[0091] A preferred peroxisomal localization signal is SKL (SerLysLeu) or any C-terminal tripeptide with the standard sequence (S / A / C)-(K / R / H)-(L / M) added to the C-terminus of a polypeptide to localize it to peroxisomes.

[0092] In yeast, another preferred localization signal consists of the conserved peptide (R / K)-(L / V / I)-X5-(H / Q)-(L / A / F) added to the N-terminus of the polypeptide to localize it to peroxisomes.

[0093] A further method for achieving peroxisomal localization of a protein is to fuse the protein with another protein that is naturally found in the peroxisomes of yeast or other organisms, or to construct a protein fusion between the protein and another protein (or protein domain) that is not normally present in peroxisomes but that has been engineered to localize to peroxisomes by the addition of a localization signal as described above.

[0094] The peroxisome-localized enzyme may be homogeneous, meaning that the peroxisome-localized enzyme is identical to the enzyme naturally found in the cytoplasm of the host cell, or it may be heterologous, meaning that it is different from the enzyme naturally found in the cytoplasm of the host cell.

[0095] According to the present invention, peroxisomal localization of an enzyme means that a gene encoding the enzyme in question is introduced into a host cell, which provides a coded peroxisomal localization signal.If the enzyme in question is an enzyme naturally found in the host cell, the peroxisomal localized enzyme will result in improved synthesis of monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and other prenylated compounds according to the present invention, while the natural enzyme localized in the cytoplasm of the host cell will result in normal biosynthesis of biomolecules necessary for the survival and growth of the host cell.For example, if the enzyme in question is GPP synthase, the peroxisomal GPP synthase will result in improved synthesis of monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and other prenylated compounds compatible with the present invention, and the natural GPP synthase localized in the cytoplasm will ensure that GPP is supplied for the biosynthesis of necessary molecules, such as sterols, which are necessary to ensure the survival and normal growth of the host cell.

[0096] If the selected host cell is a polyploid cell, such as a diploid or tetraploid cell, it may even be possible to effect peroxisomal localization by gene editing techniques that provide a peroxisomal localization signal in one or more alleles of the gene encoding the enzyme of interest, leaving at least one allele unchanged, thereby ensuring that the edited allele comprises a peroxisomal-localized enzyme and the unedited allele comprises the native enzyme localized to the cytoplasm.

[0097] host cell According to the present invention, the host cell is a yeast cell, i.e., a eukaryotic single-cell organism, as reviewed, for example, in The yeasts. 5th edition. A taxonomic study. Editors: Kurtz-man, Fell, Boekhout. Elsevier, 2011.

[0098] Preferred host cells include cells belonging to the genera Saccharomyces, Pichia, Candida, Yarrowia, and Ogatea. More preferably, the host cell is selected from the species Saccharomyces cerevisiae, Pichia pastoris, Yarrowia lipolytica, Ogataea polymorpha, Candida albicans, and Candida boidinii.

[0099] The present invention is not limited to any particular method for providing peroxisomal localization for the enzymes according to the present invention. Any method known in the art for providing suitable genes, optimizing codon usage, providing suitable regulatory elements such as promoters, terminators, adenylation sites, introns, exons, enhancer elements, ribosome binding sites, Kozak sequences, transformed yeast, etc. can be used in accordance with the present invention.

[0100] production The invention also relates to the production of monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and other prenylated compounds using the yeast cells of the invention.

[0101] According to the present invention, monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and prenylated compounds are a. providing a yeast cell according to the present invention; b. growing yeast cells on a substrate that supports yeast cell growth; and c. optionally providing a co-substrate to be prenylated; d. Recovering the compounds from the fermentation broth or converting the compounds to more complex products within the yeast cells by the action of additional native or heterologously expressed enzymes. The method can be generated using a method including:

[0102] The compounds resulting from this method can be further converted to more complex products within the yeast cell by the action of additional native or heterologously expressed enzymes.

[0103] Growth of yeast can in principle be carried out by any known method for growing yeast, although for ease of recovery it is preferred to grow the yeast cells in liquid medium in vessels such as shake flasks or fermentors.

[0104] It is particularly preferred to grow the yeast cells in a fermentor, and the fermentation process can be carried out as a batch, fed-batch, or continuous fermentation, as is known in the art.

[0105] The substrate that supports the growth of yeast cells can be any suitable medium containing a carbon source, a nitrogen source, minerals, and nutrients required by the particular yeast cells.

[0106] The matrix may be a complex matrix containing components that are not completely purified, or it may be a defined medium containing only defined components.

[0107] Examples of complex media components include molasses, dextrin, starch and / or protein hydrolysates.

[0108] Examples of components of a defined medium can include glucose, sucrose, ammonia, salts, minerals and vitamins.

[0109] The fermentation process produces a fermentation broth containing cells, water, product, remaining nutrients and minerals, and waste products produced by the cells. Recovery of monoterpenoids, cannabinoids, iridoids, monoterpene indole alkaloids, and other prenylated compounds from the fermentation broth is carried out using methods known in the art for recovering such compounds.

[0110] material and method Genes used: [Table 1]

[0111] yeast strains The yeast strain used in this study was based on the EGY 48 Saccharomyces cerevisiae strain described in (Ignea et al. (2011), Thomas BJ and R. Rothstein (1989), and (Ellerstrom M et al. (1992)) and modified according to Table 2. [Table 2]

[0112] Plasmid construction: Plasmids were generated using standard methods used in genetic engineering and known in the art. Detailed protocols for plasmid construction can be found in general handbooks containing methods for molecular cloning.

[0113] The plasmid designed to confer peroxisomal localization of the enzyme, called pPER, contained a peroxisomal localization signal (-SKL) or an N-terminal peroxisomal localization signal fused C-terminally to the amino acid sequence of the enzyme, whereas the plasmid designed to confer cytoplasmic localization of the enzyme (pCYT) did not contain this signal.

[0114] The gene was amplified by PCR and encoded by the dual inducible promoter P GAL1 and P GAL10 The coding gene sequences were then ligated into the backbone of pESC-URA, pESC-LEU, pESC-TRP, and pESC-HIS vectors (Agilent Technologies) using USER cloning (Nour-Eldin et al., 2010) to construct the plasmids listed in Table 3. [Table 3] TIFF0007811016000004.tif57170

[0115] Name of strain carrying the plasmid used in this application The plasmids (Table 3) were then used to transform yeast cells (Table 2) using the lithium acetate / PEG method. Transformants were selected by their respective auxotrophy on the corresponding minimal medium. [Table 4]

[0116] Culture conditions Yeast cells were first grown overnight at 30°C in selective minimal medium containing glucose. Complete minimal medium consisted of 0.13% w / v dropout powder, 0.67% w / v yeast nitrogen base (YNB+AS) without amino acids containing ammonium sulfate, and 2% w / v glucose. Dropout powder was purchased to lack leucine, histidine, uracil, and tryptophan. These four nutrients were added at 0.01–0.02% w / v as needed. Cells were then harvested by centrifugation, the medium removed, and the medium was removed until the initial OD reached approximately 0.5. 600nm The cells were resuspended in selective minimal production medium containing 0.13% w / v of dropout powder, 0.64% w / v of YNB+AS, 2% galactose, and 1% w / v of raffinose. When appropriate, the same four nutrients as above were added at 0.01–0.02% w / v.

[0117] Isopropyl myristate (IPM) was added as an overlay equivalent to 10% of the culture volume. Cultures were grown at 30°C and 150 rpm for the indicated times, after which the cells were harvested by centrifugation and the IPM phase was collected and analyzed using GC-FID and / or GC-MS. [Example]

[0118] Example 1: Peroxisomal co-localization of GPP synthase and terpene synthase improves terpene production

[0119] Yeast strain construction The Saccharomyces cerevisiae strain used was derived from the EGY48 strain (Mat α, ura3, trp1, his3, 6xLexA operators::LEU2). The engineered GPP synthase, ERG20, from S. cerevisiae N127W , and limonene synthase, a monoterpene synthase (MT) characterized as C / LimS from Citrus limon. GAL1 -P GAL10 It was expressed under the control of a promoter.

[0120] First, we expressed C / LimS in the cytosol of strain EGY48 (strain CYTLim01). As shown in Figure 1, this strain produced only 0.31 mg / L of limonene. Very similar results were observed when C / LimS was targeted to peroxisomes by the addition of the C-terminal PTS1 SKL (strain PERLim01). Indeed, although GPP can translocate from the cytosol to peroxisomes, the naturally occurring, very low cytosolic GPP pool most likely results in very limited transport of this molecule to other compartments.

[0121] Enterococcus faecalis EfmvaE and EfmvaS (as GPP synthase) genes (equivalent to Erg10p, Erg13p, and HmgRp in yeast), Erg8p, Erg12p, Erg9p, Idi1p, and Erg20p N127W Using to overexpress the entire MVA pathway in the cytosol, the fold of limonene production increased to 1.12 mg / L, a 3.6-fold increase (strain CYTLim02).

[0122] However, the GPP synthase ERG20p N127WBy simply targeting C / LimS to peroxisomes (strain PERLim02), limonene production was dramatically improved by 32-fold compared to CYTLim02, reaching 35 mg / L, indicating that the precursors IPP and / or DMAPP can be transported to peroxisomes and converted to GPP in this organelle.

[0123] The significant increase in limonene production also indicates that peroxisomes can effectively act as a barrier, protecting this newly formed GPP from cytoplasmic ERG20p and thus allowing uptake by C / LimS.

[0124] The results are also shown in FIG.

[0125] Example 2: Localization of the mevalonate pathway to peroxisomes enhances terpenoid production

[0126] To assess the possibility of harvesting peroxisomal acetyl-CoA to produce GPP in this organelle, eight MVA pathway enzymes were targeted to peroxisomes by the addition of a type 1 C-terminal peroxisomal targeting signal (PTS1) consisting of the tripeptide SKL (see sequence section). N127W The presence of C / LimS and C / LimS was sufficient to observe a significant jump in limonene production, whereas stepwise localization of additional enzymes of the MVA pathway to peroxisomes only marginally improved limonene production when the pathway was not completed in this organelle. However, when all eight enzymes were targeted to peroxisomes, a further 4-fold increase in limonene production to 141 mg / L was observed (Figure 2).

[0127] Transferring the entire acetyl-CoA to limonene pathway from the cytosol (strain CYTLim02) to the peroxisome (strain PERLim05) resulted in an overall 125-fold improvement in production.

[0128] Example 3: Extending the invention to other monoterpenoids. Construction of yeast strains for improved peroxisome-based production of camphene, pinene, (S)-(-)-limonene, (R)-(+)-limonene, (R)-(+)-linalool, and sabinene.

[0129] To assess whether the peroxisome-related improvements reported in Examples 1 and 2 are specific to limonene production or applicable to monoterpenes in general, we investigated the effects of Erg20p with overexpression of the rest of the MVA pathway. N127W Five additional MTPs were targeted to either the cytosol or peroxisomes. Camphene synthase (SeCamS), (S)-(-)-limonene synthase (MsLimS), (R)-(+)-limonene synthase (C / LimS), (R)-(+)-linalool synthase (McLimS), alpha-pinene synthase (PtPinS), and sabinene synthase (SpSabS) were selected and evaluated by determining the production titers of their major products. GPP synthase (Erg20p) N127W The same positive effect of peroxisomal targeting of these five monoterpene synthases (MTSs) was observed with 14-fold (PERCam02 vs. CYTCam02), 17-fold (PERPin02 vs. CYTPin02), 22-fold (PERSab02 vs. CYTSab02), 17-fold (PERLim27 vs. CYTLim04), 125-fold (PERLim05 vs. CYTLim02), and 20.5-fold (PERLin01 vs. CYTLin01) improvements for camphene, pinene, sabinene, (S)-(-)-limonene, (R)-(+)-linonene, and (R)-(+)-linalool, respectively, compared to the corresponding cytosolic expression of the same enzymes (Figure 3).

[0130] Example 4: Improved monoterpene production using optimized buffered synthetic minimal medium.

[0131] To evaluate production in an industrially relevant medium, a synthetic minimal defined medium was used, which contained the following: 5 g / L (NH4)2SO4, 3 g / L KH2PO4, 1 g / L MgSO4*7H2O, 0.0064 g / L D-biotin, 0.03 g / L nicotinic acid, 0.1 g / L thiamine HCl, 0.04 g / L D-pantothenic acid, 0.08 g / L myo-inositol, 0.02 g / L pyridoxine, 0.067 g / L tritium. Plex III consisted of 0.067 g / L (NH)Fe(SO)6H0, 0.0055 g / L CuSO, 0.02 g / L ZnSO, 0.02 g / L MnSO, ​​0.00125 g / L NiSO, 0.00125 g / L CoCl, 0.00125 g / L boric acid, 0.00125 g / L Kl, and 0.00115 g / L NaMoO, buffered with MES to a starting pH of 6.3.

[0132] The monoterpene production levels of this medium were determined using strains PERLim05, PERGer02, PERPin02, and PERLin01. Additionally, a beta-myrcene-producing strain, designated PERMyr01, was constructed by introducing the beta-myrcene synthase ObMyrS together with the GPP synthase ERG20N127 and the remaining MVA pathway, targeted to peroxisomes via fusion with the C-terminal tripeptide SKL (SEQ ID NO: 26). All strains were grown in shake flasks with a 10% isopropyl myristate overlay at 30°C for 72 hours. The synthetic minimal defined medium described above was used, supplemented with 4% galactose for growth and gene induction.

[0133] Strain PERLim05 produces 770 mg limonene / L of culture, strain PERGer02 produces 1681 mg geraniol / L of culture, strain PERPin02 produces 250 mg alpha-pinene / L of culture, strain PERLin01 produces 547 mg linalool / L of culture, and strain PER-Myr01 produces 251 mg myrcene / L of culture, representing a 5.4-fold, 5.2-fold, 5.1-fold, and 2.9-fold improvement for strains PERLim05, PERGer02, PERPin02, and PERLin01, respectively, compared to the same strains cultured in the unbuffered complete medium used in Example 3.

[0134] Example 5: Production of high levels of (+)-limonene and geraniol by a combined strategy of genomic integration and plasmid-based expression of genes of the MVA pathway involving GPPS and LimS or GES.

[0135] A single copy of each gene in the MVA pathway that is targeted to peroxisomes was expressed as ERG20p N127W and C / LimS / tObGES into the genome of strain EGY48 to obtain strains PERLim06 and PERGer01. Furthermore, the genes of the MVA pathway, Erg20p, N127W and additional copies of C / LimS or tObGES were introduced into the plasmids of strains PERLim06 and PERGer01, giving rise to strains PERLim07 and PERGer02, respectively.

[0136] To determine the maximum limonene and geraniol titers achievable by coupling the pathway to peroxisomes, a semi-continuous fed-batch experiment was performed using strains PERLim07 and PERGer02. Cultures were fed with 40 g / L galactose and 20 g / L raffinose every 48 hours, and the pH was adjusted to 4.5. The IPM layer was harvested every 48 hours to measure monoterpene production.

[0137] Fed-batch flask cultures using strains PERLim07 and PERGer02 resulted in a continuous accumulation of limonene and geraniol that was highly proportional to the amount of biomass formed. After 700 h, titers of 2575 mg limonene / L culture (Figure 4A) and 5516 mg geraniol / L culture (Figure 4B) were determined.

[0138] Example 6: Peroxisomal localization of a fusion protein containing a GPP synthase domain and a terpene synthase domain increases terpene production.

[0139] In this example of the present invention, we investigate the possibility of using a single polypeptide that has both GPP synthase and terpene synthase activity. Such a bifunctional enzyme can be found in nature or can be synthetically produced. For illustrative purposes, we have created such a polypeptide by fusing the GPP synthase domain and the terpene synthase domain together and targeted it to peroxisomes.

[0140] To do so, the GPP synthase Erg20p N127Wwas fused to the terpene synthase C / LimS via a linker polypeptide composed of five glycine-serine repeats (5xGS). The GPP synthase domain can be at the N-terminus of the protein, and the terpene synthase domain can be at the C-terminus of the protein. Alternatively, the terpene synthase domain can be at the N-terminus of the protein, and the GPP synthase domain can be at the C-terminus of the protein. In this example, both of these configurations were tested. Construction of these two synthases resulted in two novel polypeptides, set forth in SEQ ID NO:15 and SEQ ID NO:16. Both sequences were then introduced into yeast expression vectors to yield plasmids pPER15 and pPER16, and the PERLim06 strain was transformed with either one of the two plasmids to yield PERLim10 and PERLim11. After culturing PERLim10 and PERIim11 on the IPM overlay at 30°C for 72 hours, limonene production was measured and compared to that of strains PERLim08 and PERIim09. As can be seen in Figure 5, the production of limonene obtained by peroxisomal targeting of one or the other bifunctional GPP-terpene synthase fusion (PERLim10 and PERLim11) is similar to or better than that observed when GPP synthase and terpene synthase are peroxisomal targeting as separate enzymes (PER-Lim09). Furthermore, targeting the bifunctional GPP-terpene synthase to peroxisomes results in a 16-fold and 11-fold improvement in limonene production compared to targeting only GPP synthase activity to peroxisomes (PER-Lim08).

[0141] Example 7A: Efficient production of trans-isopiperitenol, a precursor to menthol.

[0142] We further evaluated the contribution of our method to the production of trans-isopiperitenol, a precursor of the high-value compound menthol. We introduced limonene-3-hydroxylase (MsLim3H; Q6 IV13.1) from spearmint together with cytochrome P450 reductase (tcCPR / POR) from Taxus cuspidata into strain PERLim27 to obtain strain PERLim30 or an empty vector (pESC-Leu), resulting in strain PER-Lim29. For comparison, we introduced limonene-3-hydroxylase (MsLim3H; Q6 IV13.1) from spearmint together with cytochrome P450 reductase (tcCPR / POR) from Taxus cuspidata into strain CYTLim04 to obtain strain CYTLim06. After 72 hours of growth in complete minimal medium, trans-isopiperitenol production was assessed by GC-FID of culture extracts. As shown in Figure 6A, both (-)-limonene and trans-isopiperitenol could be extracted from the PERLim30 strain, whereas only limonene was detected in the PERLim29 strain, which lacks the limonene-3-hydroxylase MsLim3H and cytochrome P450 reductase tcCPR (Figure 6A). The reduction in (-)-limonene recovered from the PERLim30 culture compared to the PERLim29 strain is in line with the 37% conversion to trans-isopiperitenol (19.24 mg / L) (Figure 6A). However, when limonene was produced in the cytosol of the CYTLim06 strain, only 0.28 mg / L of trans-isopiperitenol was obtained, corresponding to a limonene conversion rate of only 14%. These results demonstrate that increased production of limonene in peroxisomes directs a significant amount of it through the endoplasmic reticulum (ER), where it can be hydroxylated by MsLim3H.

[0143] Example 7B: Efficient production of 8-hydroxygeraniol, a precursor to iridoid and monoterpene indole alkaloids.

[0144] We further evaluated the contribution of our invention to the production of 8-hydroxygeraniol, a precursor to a large group of high-value compounds, including iridoids and monoterpene indole alkaloids. We introduced geraniol 8-hydroxylase from Catharanthus roseus (CrG80H; CYP76B6) into strain PERGer02 along with cytochrome P450 reductase (CrCPR / POR) from the same species to obtain strain PERGer04 or an empty vector (pESC-Leu), resulting in strain PERGer03. After 72 h of growth in complete minimal medium, 8-hydroxygeraniol production was assessed by GC-FID of culture extracts. As shown in Figure 6B, both geraniol and 8-hydroxy-geraniol could be extracted from the PERGer04 strain, whereas only geraniol was detected in the PERGer03 strain, which lacks the geraniol 8-hydroxylase CrG80H and cytochrome P450 reductase CrCPR (Figure 6B). The decrease in geraniol recovered from PERGer04 cultures compared to PERGer03 is in accordance with its partial conversion to 8-hydroxy-geraniol (Figure 6B). These results demonstrate that increased production of geraniol in peroxisomes directs a significant amount of it through the endoplasmic reticulum (ER), where it can be hydroxylated by CrG80H.

[0145] Example 8: Efficient production of cannabinoids by targeting GPP synthase and geranyl diphosphate:olivetolate geranyltransferase to peroxisomes.

[0146] The applicability of the present invention to the production of cannabinoids, another group of high-value compounds derived from GPP beyond monoterpenoids and monoterpene indole alkaloids, was evaluated. In the cannabinoid biosynthetic pathway, olivetolic acid (OA) is prenylated by GPP to form cannabigerolic acid (CBGA) through the action of a dedicated geranyltransferase. CBGA represents a critical step in the pathway because it is the final common precursor of various cannabinoids, such as tetrahydrocannabinolic acid (THCA) and cannabidiolic acid (CBDA). In yeast, this prenylation step poses a major bottleneck in the process of producing high-titer cannabinoids due to the limited pool of GPP in the cytosol.

[0147] In this example, the GPP synthase Erg20p from C. sativa N127W and geranyl diphosphate:olivetolate geranyltransferase CsPT4 were targeted to yeast peroxisomes using the C-terminal targeting signal SKL for the production of CBGA. Both genes were driven by the inducible promoter P GAL1 and P GAL10 The PERCan01 strain was obtained by introducing the CBGA into the PERMva01 strain under the control of β-glucanase (β-glucanase). After 72 hours of growth in complete minimal medium under galactose-inducing conditions and supplementation with various concentrations of olivetolic acid (0.05 mM, 0.1 mM, 0.25 mM, or 0.5 mM), CBGA production was analyzed by LC-MS. Cells were disrupted and CBGA was extracted from the cell pellet using a 1:1 mixture of ethyl acetate and formic acid (0.05% v / v) and glass bead beating. The organic layer was separated by centrifugation and evaporated using a SpinVac. The remaining dry fraction was dissolved in methanol and filtered through a 0.22 μm pore size PVDF filter. Samples were diluted 10-fold before LC-MS analysis.

[0148] As shown in Figure 7A, both OA and CBGA could be extracted from the PERCan01 strain, but the GPP synthase ERG20p N127WIn the PERMva01 strain, which lacks the geranyl diphosphate:olivetrate geranyltransferase CsPT4, only OA was detected. The reduction in OA recovered inside cells of PERCan01 compared to PERMva01 is due to partial conversion to CBGA. These results demonstrate that 1) OA can be transported and / or diffused into peroxisomes, 2) CsPT4 is active in peroxisomes, and 3) the pool of GPP is sufficient to allow efficient OA prenylation in peroxisomes.

[0149] Additionally, alternative N-terminal peroxisomal targeting signals were investigated for CsPT4 (SEQ ID NO: 29). N127W The PERMva01 strain was then introduced with CsPT4 and Erg20p, resulting in peroxisomal localization of both enzymes. This new strain, designated PERCan02, was then evaluated for OA consumption and CBGA production, as previously described. For comparison, CsPT4 and Erg20p were also evaluated. N127W was introduced into the CYTMva01 strain to obtain the CYTCan01 strain for the production of cytoplasmic CBGA. As shown in Figure 7B, the PERCan02 strain produced 82.3 mg / L of CBGA, 19.5-fold more than the CYTCan01 strain, which only had 4.2 mg / L of CBGA. References:

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Claims

1. a peroxisomal enzyme that catalyzes the formation of a branch point compound and is capable of converting said branch point compound through a preferred pathway and a non-preferential pathway, and a peroxisomal enzyme that catalyzes the first step of said non-preferential pathway; the enzyme that catalyzes the formation of the branch point compound is GPP synthase; The yeast cell, wherein the enzyme catalyzing the first step of the non-preferential pathway is selected from among a terpene synthase, a prenyltransferase, or other isoprenoid or non-isoprenoid prenyltransferase.

2. 2. The yeast cell of claim 1, which belongs to one of the genera Saccharomyces, Pichia, Candida, Ogatea, or Yarrowia.

3. 3. The yeast cell of claim 2, wherein the yeast is selected from the species Saccharomyces cerevisiae, Pichia pastoris, Candida albicans, Candida boidinii, Ogataea polymorpha, or Yarrowia lipolytica.

4. The yeast cell according to any one of claims 1 to 3, wherein peroxisomal localization is achieved by inserting a peroxisomal localization signal into the gene encoding the respective enzyme.

5. The peroxisomal localization signal is a. SKL (SerLysLeu) or any C-terminal tripeptide with the standard sequence (S / A / C)-(K / R / H)-(L / M) added to the C-terminus of the polypeptide to localize it to peroxisomes; and b. A conserved peptide (R / K)-(L / V / I)-X added to the N-terminus of the polypeptide to localize it to peroxisomes ５ -(H / Q)-(L / A / F); The yeast cell of claim 4 selected from the group consisting of:

6. The terpene synthase may be (+)-limonene synthase, (-)-limonene synthase, alpha-pinene synthase, 1,8-cineole synthase, sabinene synthase, camphene synthase or geraniol synthase, beta-pinene synthase, linalool synthase, myrcene synthase, bornyl diphosphate synthase, alpha-terpineol synthase, isoborneol synthase, tricyclene synthase, alpha-thudiene synthase, alpha-fenchene synthase, delta-2-carene synthase, alpha-phellandrene synthase, 3-carene synthase, 1,4-cineole synthase, alpha-terpineol synthase, 2. The yeast cell of claim 1, wherein the enzyme is selected from the group consisting of cis-beta-ocimene synthase, beta-phellandrene synthase, (Z)-beta-ocimene synthase, (E)-beta-ocimene synthase, gamma-terpinene synthase, terpinolene synthase, allo-ocimene synthase, cis-beta-terpineol synthase, cis-terpin-1-ol synthase, delta-terpineol synthase, borneol synthase, voltanol synthase, alpha-terpineol synthase, nerol synthase, 2-methyl-isoborneol synthase, 2-methylenebornene synthase, 2-methyl-2-bornene synthase, and beta-phellandrene synthase.

7. The terpene synthase (+)-Limonene synthase having the amino acid sequence of SEQ ID NO: 3; camphene synthase having the amino acid sequence of SEQ ID NO:5; (-)-Limonene synthase from spearmint (Mentha spicata) having the amino acid sequence of SEQ ID NO: 25; (+)-Linalool synthase from Mentha citrate having the amino acid sequence of SEQ ID NO: 23; Myrcene synthase having the amino acid sequence of SEQ ID NO: 26; α-pinene synthase having the amino acid sequence of SEQ ID NO: 7; Sabinene synthase having the amino acid sequence of SEQ ID NO:9; and Geraniol synthase having the amino acid sequence of SEQ ID NO: 11 2. The yeast cell of claim 1, selected from the group consisting of:

8. 2. The yeast cell of claim 1, wherein the terpene synthase can accept non-standard isoprenoid substrates having 9, 11, or 12 carbon atoms.

9. 2. The yeast cell of claim 1, wherein the prenyltransferase, or other isoprenoid or non-isoprenoid prenyltransferase, is selected from among an aromatic prenyltransferase and a geranyl diphosphate:olivetolate geranyltransferase.

10. the enzyme that catalyzes the formation of the branch point compound and the enzyme that catalyzes the first step of the non-preferential pathway a. an enzyme capable of synthesizing DMAPP and an enzyme having isoprene synthase activity, or b. an enzyme capable of synthesizing DMAPP and an enzyme having prenyltransferase activity; c. an enzyme capable of synthesizing DMAPP and an enzyme having C-prenyltransferase activity; d. Enzymes capable of synthesizing DMAPP and enzymes with O-prenyltransferase activity The yeast cell according to any one of claims 1 to 5, wherein the yeast cell is selected from the group consisting of:

11. a. Saccharomyces cerevisiae cells in which GPP synthase and limonene synthase are localized in peroxisomes; b. A Saccharomyces cerevisiae cell in which GPP synthase and geraniol synthase are localized in peroxisomes; and c. Saccharomyces cerevisiae cells in which GPP synthase and olivetolic acid prenyltransferase are localized in peroxisomes; 2. The yeast cell of claim 1, selected from the group consisting of:

12. a. Saccharomyces cerevisiae cells in which GPP synthase from Aspergillus terreus and aromatic prenyltransferase AtaPT are localized in peroxisomes; b. Saccharomyces cerevisiae cells in which isopentenyl diphosphate isomerase (IDI) and terpene synthase, which catalyzes the synthesis of isoprene, are localized in peroxisomes; c. Saccharomyces cerevisiae cells in which isopentenyl diphosphate isomerase (IDI) and lavandulyl diphosphate synthase from Lavandula x intermedia are localized in peroxisomes; d. Saccharomyces cerevisiae cells in which isopentenyl diphosphate isomerase (IDI) and chrysanthemyl diphosphate synthase from Tanacetum cinerea folium are localized in peroxisomes; e. Saccharomyces cerevisiae cells in which isopentenyl diphosphate isomerase (IDI) and 7-dimethylallyltryptophan synthase (7-DMATS) from Aspergillus fumigatus are localized in peroxisomes; f. Saccharomyces cerevisiae cells in which isopentenyl diphosphate isomerase (IDI) and the phenylpropane-specific prenyltransferase AcPT1 from Artemisia capillaris are localized in peroxisomes; and g. Saccharomyces cerevisiae cells in which isopentenyl diphosphate isomerase (IDI) and the O-prenyltransferase AcaPT from Antrodia camphorata are localized in peroxisomes; 2. The yeast cell of claim 1, selected from the group consisting of:

13. A yeast cell comprising a peroxisome-localized GPP synthase and a peroxisome-localized monoterpene synthase.

14. 1. A method for producing a compound selected from a monoterpenoid, a cannabinoid, a monoterpene indole alkaloid, and a prenylated aromatic compound, comprising: a. Providing a yeast cell according to any one of claims 1 to 13 b. fermenting the yeast cells with a substrate that supports the growth of the yeast cells; c. optionally, providing the yeast with a substrate to be prenylated; and d. Recovering said compound from said fermentation broth. A method comprising:

15. e. Converting the compound of step d to a more complex product within the yeast cells by the action of additional native or heterologously expressed enzymes.

15. The method of claim 14, further comprising:

16. The compound may be sabinene, alpha-pinene, beta-pinene, camphene, (+)-limonene, (-)-limonene, geraniol, linalool, myrcene, 1,8-cineole, borneol, bornyl diphosphate, alpha-terpineol, isoborneol, tricyclene, alpha-thuene, alpha-phencene, delta-2-carene, alpha-phellandrene, 3-carene, 1,4-cineole, alpha-terpinene, beta-phellandrene, (Z)-beta-ocimene, (E)-beta-ocimene, gamma-terpinene, ... Pinen-4-ol, terpinolene, alloocimene, cis-beta-terpineol, cis-terpin-1-ol, delta-terpineol, alpha-terpineol, nerol, 2-methylisoborneol, 2-methylenebornene, 2-methyl-2-bornene, beta-phellandrene, 2-methyllimonene, 2-methylmyrcene, 2-methylgeraniol, 2-methyllinalool, cannabigerolic acid, cannabiberolic acid analogs, prenyltryptophan, artepillin C, dorupanin, osrutin, geranyl-resveratrol, geranyl-resveratrol, Ranyl quercetin, geranyl-naringenin, geranyl-isoliquitigenin, isobavachalcone, isoprene, lavandulol, chrysanthemol, dimethylallyltryptophan, 4'-dimethylallyl-apigenin, 6-prenyl-apigenin, 4'-dimethylallyl-naringin, 4'-dimethylallyl-kaempferol, 4'-dimethylallyl-daidzein, 7-dimethylallyl-daidzein, 7,4'-di-(dimethylallyl)-daidzein, 4'-dimethylallyl-genistein, 7-dimethylallyl-genistein, 7,4'-di- 16. The method of claim 14 or 15, wherein the hydroxybenzoate is selected from (dimethylallyl)-genistein, 4-dimethylallyl-isoliquitigenin, 4'-dimethylallyl-equol, 7-dimethylallyl-equol, 6-dimethylallyl-equol, 4'-dimethylallyl-daidzin, 7-dimethylallyl-umbellillone, 8-dimethylallyl-curcumin, 8'-di-methylallyl-demethoxycurcumin, 8-dimethylallyl-demethoxycurcumin, 4'-dimethylallyl-resveratrol, or 5-dimethylallyl-diethylstilbestrol.

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