Sustainable production of cannabinoids from simple precursors using Saccharomyces cerevisiae

Abstract

A recombinant cell of Saccharomyces cerevisiae, wherein the genome of the cell comprises a nucleic acid encoding a cannabinoid biosynthetic pathway gene. The cannabinoid is produced by the recombinant cell in the presence of a cannabinoid precursor substrate, and at least one of the cannabinoid biosynthetic pathway genes is from an organism other than cannabis. The present invention also discloses a method for producing cannabinoids using the recombinant cell and the cannabinoid precursor substrate.

Description

Background Art

[0001] Phytocannabinoids, compounds originally isolated from the cannabis plant (Cannabis sativa), have been greatly hampered in their research and therapeutic applications due to legal and social issues. Some cannabinoids, such as 9-tetrahydrocannabinol (THC), are produced by mixing with other cannabinoids in the plant and have been found to have psychoactive properties. However, at least 113 cannabinoids have been isolated from cannabis (Aizpurua-Olaizola et al., J. Nat. Prod., 2016, 79(2): 324-331), most of which are non-psychoactive and have unique pharmacological properties.

[0002] The widespread expression of human cannabinoid receptors means that these compounds have a wide range of effects on the human body. There are two main types of human cannabinoid receptors - CB1 receptors are mainly expressed in the central nervous system, while CB2 receptors are mainly found in the peripheral immune system. In addition, studies have also found the expression of these receptors in various human tissues, such as the heart, adrenal glands, lungs, spleen and tonsils (Galiègue et al., Eur. J. Biochem., 1995, 232 (1): 54-61). The binding of cannabinoids to these receptors activates signaling pathways that may affect a wide range of biological systems in the human body.

[0003] Phytocannabinoids have been approved by the U.S. Food and Drug Administration for clinical use as antiemetics for patients undergoing intensive cancer chemotherapy (THC and Dronabinol; Pertwee, Forsch. Komplementarmed., 1999, Suppl 3:12-15), and in the UK as highly effective treatments for spasticity in patients with multiple sclerosis who are unresponsive to other drugs (Flachenecker et al., Eur. Neurol., 2014, 71(5-6):271-279). Various studies have also shown the therapeutic potential of phytocannabinoids as anti-tumor drugs (Velasco et al., Nat. Rev. Cancer, 2012, 12(6):436-444; Maria Pyszniak et al., Onco. Targets Therapy, 2016, 9:4323–4336), and as effective antibacterial agents against several methicillin-resistant Staphylococcus aureus (MRSA) strains (Appendino et al., J. Nat. Prod., 2008, 71(8):1427-1430).

[0004] Given these recent advances in cannabinoid research, the legal cannabis market is currently estimated to be worth $7.7 billion and is expected to grow to around $31.4 billion by 2021 (Zhang, Forbes, 2017).

[0005] Currently, most cannabinoids used for research or therapeutic applications are extracted from cannabis extracts. The legal and social implications associated with the illegal use of plant extracts are the biggest challenge facing traditional cultivation due to the high concentrations of psychoactive ingredients such as THC produced in cannabis as a mixture. There is limited selective control over the cannabinoids produced during the cultivation of cannabis. In addition, there is a large market gap in studying the therapeutic potential of cannabis strains that can produce small amounts of cannabinoids such as cannabidiol (CBDV) and cannabichromene (CBL).

[0006] The cultivation of these plants is not only costly but also environmentally unsustainable due to the high energy consumption processes required to control environmental factors. In 2012, research conducted by the Energy and Resources Group at the Lawrence Berkeley National Laboratory estimated that the energy consumption costs of indoor cannabis cultivation in the United States alone are approximately $6 billion per year. In addition, the greenhouse gas emissions of this method to produce one kilogram of the final product are equivalent to the emissions of 3 million cars (Mills, Energy Policy, 2012, 46:58-67).

[0007] There is a need to find alternative methods of cannabinoid production that avoid the complications mentioned above. Summary of the invention

[0008] In order to meet the needs described above, a recombinant cell of Saccharomyces cerevisiae is provided, wherein the genome of the cell contains a plurality of nucleic acids, each of which encodes a cannabinoid biosynthetic pathway gene, so that the recombinant cell produces cannabinoids in the presence of a cannabinoid precursor substrate. At least one cannabinoid biosynthetic pathway gene is not derived from cannabis.

[0009] Also disclosed is a method of producing a cannabinoid by contacting a recombinant cell with a cannabinoid precursor substrate and culturing the recombinant cell.

[0010] The details of one or more embodiments are set forth in the following description and examples.Other features, objects, and advantages are apparent from the detailed description, drawings, and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The following description of the present invention refers to the accompanying drawings, in which:

[0012] Figure 1 The biosynthetic pathways for cannabinoids are shown. The polyketide pathway is in black and the isoprene pathway is in light grey. Modifications to the pathways are shown in dark grey.

[0013] Figure 2The results of geranyl pyrophosphate: dihydroxypentylbenzoate geranyltransferase obtained from the transmembrane helix prediction web server (TMHMM) are shown, showing 6 predicted transmembrane regions in its amino acid sequence.

[0014] Figure 3A Schematic diagram of the yeast Fab pathway assembly system, showing the cloning procedure of pathways (level 2) assembled from biological components (level 0). Image taken from Guo et al., Nucleic Acids Res., 2015, 43(13): e88.

[0015] Figure 3B Sequences of the prefix and suffix of the primary transcription unit (POT) assembled into the secondary pathway are shown.

[0016] Figure 4 Shown are extracted ion counts (left) and mass spectra (right) of LC-MS analysis of cannabinoids produced in Saccharomyces cerevisiae: (first row) negative control construct 4ME expressing 4CL and MCS; (second row) construct 4MOO expressing 4CL, MCS, OLS and OAC; and (last row) OLA standard.

[0017] Figure 5 Shown are extracted ion counts and mass spectra of LC-MS analysis of in vitro enzymatic assays: negative control with no enzyme using GPP and OLA (first row), with GPP and OLA and enzyme GOT (second row), with GPP and OLA and enzyme NphB (third row), and CBGA standard (last row).

[0018] Figure 6 A simplified methylvalerate pathway for overexpression of monoterpene precursors IPP, DMAPP and GPP and geraniol biosynthesis in Saccharomyces cerevisiae is shown (Zhao et al., Appl. Microbiol. Biotechnol., 2016, 100(10):4561-4571). Adapted genes for producing GPP in the system of the present invention are underlined.

[0019] Figure 7 Shown are extracted ion counts and mass spectra for LC-MS analysis of cannabinoids produced in Saccharomyces cerevisiae: negative control is the 4MOON pMKU+empty pCKL construct expressing only cannabinoid genes (first row), the construct expressing cannabinoid genes on the pMKU vector and the mevalonate gene on pCKL (second row), and the 4MOON pMKU+IHUE pCKL construct with the CBGA standard (last row).

[0020] Figure 8The difference in carbon chain length between OLA and DVA is shown (Groom et al., Acta Crystallogr.B Structure. Sci. Cryst. Eng. Mater, 2016, 72(Pt 2): 171-179).

[0021] Fig. 9 The production of OLA analogs using different starting units and S. cerevisiae constructs is shown.

[0022] Fig.10 Column 3 shows the structures of novel dihydroxypentylbenzoic acid analogs produced in E. coli identified by extracted ion counting LC-MS analysis. Column 1 shows the biosynthetic pathway expressed by E. coli and cultured with the substrates shown in column 2. 4MOO = 4-coumaroyl-CoA ligase, malonyl CoA synthetase (MCS), olive alcohol synthase (OLS), and dihydroxypentylbenzoate cyclase (OAC); BMOO = benzoate CoA ligase, MCS, OLS, and OAC; CMOO = cinnamoyl CoA ligase, MCS, OLS, and OAC; PMOO = phenylacetate CoA ligase, MCS, OLS, and OAC.

[0023] Fig.11 The proposed OLS reaction mechanism is shown.

[0024] Fig.12 Shown are the structure of the 3,5-dioxodecanoyl-CoA intermediate (blue) docked to the OLS structure (left) and the protein structure of the 3,5-dioxodecanoyl-CoA intermediate (blue) docked to superimposed malonyl-CoA units (yellow). DETAILED DESCRIPTION

[0025] As described above, the provided recombinant cells contain a plurality of nucleic acids in their genome, each of which encodes a cannabinoid biosynthetic pathway gene. In an exemplary recombinant cell, the cannabinoid biosynthetic pathway gene is from an organism other than cannabis.

[0026] Certain recombinant cells encompassed by the present invention contain nucleic acids encoding coenzyme A (CoA) ligases. The CoA ligases can be, but are not limited to, Nicotiana tabacum 4-coumaroyl-CoA ligase (SEQ ID NO: 1), Rhodopseudomonas palustris benzoate-CoA ligase (SEQ ID NO: 2), and Streptomyces coelicolor phenylacetate-CoA ligase (SEQ ID NO: 3). The CoA ligases can have 70% or higher identity (e.g., 70%, 75%, 80%, 85%, 90%, 95%, and 99% or higher) to the above sequences while maintaining enzymatic activity.

[0027] In addition, the recombinant cell may include a nucleic acid encoding any one of malonyl CoA synthetase (MCS), olive alcohol synthase (OLS) and dihydroxypentylbenzoic acid (OAC). In a specific recombinant cell, the MCS is replaced by acyl activating enzyme (AAE).

[0028] MCS may have the amino acid sequence of SEQ ID NO:5, OLS may have the amino acid sequence of SEQ ID NO:6, OAC may have the amino acid sequence of SEQ ID NO:7, and AAE may have the amino acid sequence of SEQ ID NO:4.

[0029] Alternatively, the MCS, OLS, OAC and AAE may have an amino acid sequence that is 70% or more identical to the amino acid sequence of the corresponding sequence number.

[0030] The above recombinant cells will produce cannabinoids in the presence of cannabinoid precursor substrates. The cannabinoid precursor substrates can be, for example, butyric acid, valeric acid, hexanoic acid, heptanoic acid and octanoic acid. Other cannabinoid precursor substrates are shown in Table 1 below.

[0031] Table 1. Cannabinoid precursor substrates

[0032]

[0033]

[0034]

[0035]

[0036] As described in the Summary of the Invention, a method for producing cannabinoids using the above recombinant cells is disclosed. The method is implemented by contacting the recombinant cells with a cannabinoid precursor substrate and culturing the recombinant cells.

[0037] In an exemplary method, the recombinant cell expresses CoA ligase, malonyl CoA synthetase, olivetol synthase and olivetol acid cyclase, and the cannabinoid precursor substrate is butyrate, valerate, caproate, heptanoate or octanoate. Other cannabinoid precursor substrates shown in Table 1 above can also be used in this method.

[0038] Recombinant synthesis of the cannabinoid biosynthetic pathway in microorganisms such as Saccharomyces cerevisiae can be used for the bioproduction of major and minor cannabinoids. This bioproduction method facilitates the production of specific cannabinoids compared to the cannabinoid mixture produced by cultivated cannabis. In addition, minor cannabinoids can be produced in high yields. Furthermore, enzymes in the cannabinoid biosynthetic pathway can be manipulated and expressed in microorganisms to produce novel unique cannabinoids with therapeutic potential.

[0039] Without further elaboration, it is believed that those skilled in the art can make the best use of the present invention based on the disclosure herein. Therefore, the following specific examples are only interpreted as illustrative and do not limit the remainder of the present disclosure in any way. All publications cited herein are fully incorporated by reference.

[0040] Example

[0041] Example 1: Modification of the cannabinoid biosynthetic pathway

[0042] In order to reconstruct the cannabinoid biosynthetic pathway in microorganisms, it is necessary to first understand the enzymes present in cannabis that catalyze each step in the pathway. Figure 1 .

[0043] The upstream biosynthetic pathway can be divided into two functional parts, the polyketide pathway producing dihydroxypentylbenzoic acid (OLA) as the final product and the isoprenoid pathway producing geranyl pyrophosphate (GPP).

[0044] Polyketone production OLA

[0045] Polyketide pathway (see Figure 1, red) starts with the substrates hexanoate and malonyl CoA. An acyl activating enzyme named CsAAE1 (SEQ ID NO: 4) is responsible for adding the CoA moiety to the hexanoate in the trichome of the cannabis leaf (Stout et al., Plant J., 2012, 71(3): 353-365). Then, a type III polyketide synthase named olive alcohol synthase (OLS; SEQ ID NO: 6) catalyzes the formation of tetraketide thioesters (3,5,7-trioxadodecyl CoA) by utilizing one unit of hexyl CoA and three units of malonyl CoA (Taura et al., FEBS Lett, 2009, 583(12): 2061-2066). Finally, dihydroxypentylbenzoate cyclase (OAC; SEQ ID NO: 7) catalyzes the C2-C7 aldehyde condensation cyclization step to produce OLA (Gagne et al., Proc. Natl. Acad. Sci. USA, 2012, 109(31): 12811-12816).

[0046] Isoprenoid production GPP

[0047] In cannabis, isoprenoids such as dimethylallyl pyrophosphate (DMAPP) and isopentenyl pyrophosphate (IPP) are produced via the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (see Figure 1 , blue) (Van Bakel et al., Genome Biol., 2011, 12(10): R102). A putative GPP synthase combines one unit of DMAPP with one unit of IPP to produce GPP. Isoprenoids can also be produced in nature through the mevalonate pathway, which is commonly found in microorganisms such as yeast and some bacteria (Buhaescu et al., Clin. Biochem., 2007, 40(9-10): 575-584).

[0048] Endpoint Cannabinoids

[0049] Once OLA and GPP are produced, the aryl isopentenyltransferase geranyl pyrophosphate: dihydroxypentylbenzoate geranyltransferase (GOT) catalyzes the production of the first cannabinoid, cannabigerolic acid (CBGA), by transferring the C10 isopentenyl group in GPP to the C3 of dihydroxypentylbenzoate (Fellemier et al., FEBS Lett., 1998, 427(2):283-285; see Figure 1 The biosynthesis of different cannabinoids, such as tetrahydrocannabinolic acid (THCA) and cannabidiol (CBDA), can be carried out by their respective synthases (i.e., THCA synthase and CBDA synthase) by converting CBGA previously transferred from GPP (see Figure 1 , black) undergoes differential cyclization of the C10 carbon chain.

[0050] Modification of the cannabinoid biosynthetic pathway

[0051] Modification of the cannabinoid biosynthetic pathway Figure 1 Green in the figure. Previously, a molecular toolkit was established to produce acyl-CoA thioesters with different functional groups using four different acyl-CoA ligases (Go et al., Biochemistry, 2012, 51(22): 4568-4579). 4-Coumaryl-CoA ligase (4CL) was isolated from Nicotiana tabacum plants (SEQ ID NO: 1), benzoate-CoA ligase (BZL) was isolated from the Gram-negative bacterium Rhodopseudomonas palustris (SEQ ID NO: 2), and phenylacetate CoA ligase (PCL) from the Gram-positive bacterium Streptomyces coelicolor (SEQ ID NO: 3) were all determined to have substrate universality with 70 carboxylic acids (see Table 1 above), including the natural substrate hexanoic acid of OLS. In addition, malonyl-CoA synthetase (MCS) from the soil bacterium Rhizobium trifoliatum produces a number of malonates with CoA moieties as elongation units for type III polyketide synthases such as OLS. Using these well-characterized and substrate-universal CoA ligases to replace CsAAE1 in cannabis could facilitate the downstream production of a variety of cannabinoids.

[0052] Each CoA ligase is paired with a malonyl-CoA synthetase (MCS), which is responsible for adding the CoA moiety to the malonate.

[0053] The expression of the prenyltransferase (i.e., GOT) from cannabis in a heterologous system is challenging. In fact, as shown by the bioinformatics prediction tool TMHMM (Carvalho et al., FEMS Yeast Res., 2017, 17(4)), this enzyme is predicted to be a membrane-associated protein with an intrinsic transmembrane region. Figure 2 .

[0054] A soluble isopentenyl transferase from Streptomyces, NphB, has been previously shown to accept OLA as an isopentenyl acceptor in bacterial systems such as Escherichia coli (Kuzuyama et al., Nature, 2005, 435(7044):983-987; Yang et al., Biochemistry, 2012, 51(12):2606-2618). This enzyme can be used as an alternative to GOT to heterologously express the former acyl transferase in the cannabinoid biosynthetic pathway.

[0055] Example 2: Construction and assembly in Saccharomyces cerevisiae

[0056] The molecular cloning strategy for constructing constructs in Saccharomyces cerevisiae is a set of plasmids based on Golden-Gate assembly, called the YeastFab system (Guo et al.). The assembly system allows modular assembly of transcription units such as promoters and terminators into open reading frames (ORFs), followed by assembly of expression cassettes of up to six different transcription units.

[0057] The YeastFab plasmid suite has been expanded to allow assembly of up to eight transcription units. The modular nature of the assembly facilitates downstream optimization, as it is relatively easy to change transcriptional regulatory elements such as promoters and terminators, enabling differential regulation of expression of each gene in the biosynthetic pathway. In this pathway assembly approach, type II restriction enzymes (e.g., BsaI and BsmBI) cut near the enzyme recognition site, allowing restriction enzymes and DNA ligases to function in a one-step digestion-ligation reaction. See Figure 3A .

[0058] Standard biological parts such as promoters, open reading frames and terminators are cloned separately into individual plasmids (level 0). After a first round of one-step digestion-ligation reactions, they are assembled into individual transcription units, called POTs (level 1). Each POT is designed to assemble with another POT in the following reaction, assembling up to eight transcription units into one pathway (level 2) via prefix and suffix sequences, such as Figure 3B shown.

[0059] Yeast constitutive promoter libraries have been characterized before based on their relative strengths. A set of promoters and terminators with different strengths were selected to express each enzyme in the biosynthetic pathway, reflecting their relative stoichiometry and toxicity to the expression host. Different promoters and terminators were assigned to each ORF (see Table 2 below) to prevent homologous recombination, which often occurs in yeast, from occurring between homologous sequences that are close to each other (Orr Weaver et al., Proc. Natl. Acad. Sci. USA, 1981, 78 (10): 6354-6358).

[0060] Table 2. Selected promoters and terminators for each ORF. (A) Pathway assembled to OLA. (B) Pathway assembled to CBGA. (C) Pathway assembled to the terminus of cannabinoids.

[0061]

[0062]

[0063] After the transcription units and the specified promoter and terminator are assembled, they are assembled into a pathway containing multiple genes in a single plasmid. In yeast, farnesyl pyrophosphate (FPP) is the C15 form of GPP, which is endogenously produced by the enzyme Erg20p by adding additional IPP to GPP. Previous studies have shown that the double mutant expressing this enzyme, Erg20pWW (F96W-N127W), increases endogenous GPP levels (Ignea et al., ACS Synth. Biol., 2014, 3 (5): 298-306). The double mutant is used together with geranyl diphosphate synthase (Ag-GPPase) and chimeric GPP synthase (Chi-GPPase) from Abies grandis (Burke et al., Arch. Biochem. Biophys., 2002, 405 (1): 130-136) to produce GPP in the construct to advance the reaction.

[0064] Example 3: Cannabinoid Bioproduction

[0065] After the expression gene is assembled into the expected plasmid, it is transferred into a single organism (Saccharomyces cerevisiae) for bioproduction of the expected product. Yeast strain BY4741 is an auxotrophic strain of the amino acids methionine, leucine, histidine and uracil (Brachmann et al., Yeast, 1998, 14 (2): 115-132) for bioproduction of cannabinoids. The step-by-step approach can be used as a precaution to ensure that each plasmid-transformed yeast subsequently expresses a functional enzyme. Then, liquid chromatography-mass spectrometry (LC-MS) is used to detect the products of each step (e.g., OLA, CBGA, and CBDA) before entering the next step of the biosynthetic pathway.

[0066] The Saccharomyces cerevisiae culture was grown to stationary phase to express the enzyme. Substrates (i.e., caproic acid and malonic acid) were added to the culture and then grown overnight at 25°C. The culture was quickly centrifuged (spindown) and the pellet was separated from the supernatant culture medium. The cell pellet and supernatant culture medium were checked by LC-MS for the presence of the expected product.

[0067] To prepare samples for LC-MS analysis, the supernatant culture medium was acidified to pH <2.0 and then extracted three times with ethyl acetate. The extracted ethyl acetate was dried using a rotary vacuum concentrator, resuspended in methanol, and analyzed by LC-MS. The cells were first lysed by physical shearing with glass beads or chemical lysis, and the cell pellet was checked for the presence of the expected product. Therefore, the lysate underwent similar acidification, organic extraction, and LC-MS analysis steps.

[0068] Example 4: Biosynthesis of novel cannabinoids

[0069] Computational analysis revealed that diversification of the cannabinoid library could be achieved through three different approaches to generate new and unnatural cannabinoids.

[0070] First, traditional precursor-directed combinatorial biosynthetic approaches, including the use of different substrates, will help explore the substrate universality profile of enzymes in the pathway. Unique and novel products formed at each step then serve as new substrates for the next step in the pathway, thereby increasing the diversity of products produced. Each enzyme has an innate specificity for the substrates it can accept due to spatial constraints and interactions in its binding pocket. Computational methods, such as in silico docking, allow large libraries of small molecule compounds to be screened in a high-throughput manner to identify substrates with good binding affinity, which can then be tested experimentally.

[0071] Second, computer simulation docking of substrates with enzyme structures will clearly elucidate the spatial constraints and interactions of substrates in the active site microenvironment. This information is then used to guide protein engineering. It is crucial to understand the key interactions of substrates with the enzyme binding pocket in order to rationally design specific mutations to increase the diversity of substrates that each enzyme may accept.

[0072] Finally, the degree of protein engineering that can be applied to each enzyme is limited, because the mutation introduced thereby may inadvertently change the architecture of the entire protein structure, and cause protein instability and inactivity. The orthologs of the enzymes that perform each biosynthetic step in different organisms can be identified and integrated into the pathway. The enzyme similarity tool (Enzyme Similarity tool from the Enzyme-Function Initiative, EFI-EST) initiated from enzyme function is a bioinformatics web server that helps to identify homologs of protein query sequences (Gerlt et al., Biochim.Biophys.Acta, 2015, 1854 (8): 1019-1037). The web tool uses the UniProt database to identify amino acid sequences related to the query sequence in nature in terms of function and catalytic reactions. It identifies proteins from different families by generating a sequence similarity network, and these families may have very different substrate specificity maps, thereby facilitating the visualization of the relationship between them. The orthologs thus identified have a completely different set of substrates and may serve as suitable alternatives for specific steps in the cannabinoid biosynthetic pathway, thereby generating unique and novel cannabinoid products.

[0073] Example 5: Bioproduction of cannabinoids in Saccharomyces cerevisiae

[0074] As described in Example 2 above, the transcription units were assembled into POTs. The plasmids were inserted into the second-level assembly plasmids pCKU and pMKU, which contain yeast low-copy or high-copy replication sources CEN and 2μ, respectively, and the URA3 selection marker. The assembled plasmids were transformed into a single strain of Saccharomyces cerevisiae BY4741 using the lithium acetate method described previously (Gietz et al., Nat. Protoc., 2007, 2(1): 35-37). The strains were cultured overnight before the substrates hexanoic acid and malonate were added to the cultures. After overnight incubation with the substrates at 25°C, the cultures were collected and extracted from the growth medium of each culture with ethyl acetate. The results are shown in Figure 2. Figure 4 shown.

[0075] The supernatant of yeast culture expressing four enzymes in the cannabinoid pathway (i.e., CoA ligase, MCS, OLS, and OAC) showed an elution peak at approximately 4 minutes with an m / z value of 223.100 (ppm error 11.7), which corresponds to the m / z value of OLA. Figure 4 This peak was not observed in the supernatant of the negative control yeast lacking polyketide synthase and polyketide cyclase, OLS and OAC. Figure 4 , first line.

[0076] The next step of this pathway produces CBGA. To this end, the first four genes for producing OLA described above were assembled into the second-level assembly plasmids pCKU and pMKU together with a GPP synthetase (Erg20pWW, AgGPPase or ChiGPPase) and an isopentenyl transferase (i.e., GOT or NphB). After assembling the transcription units and transforming them into the host yeast strain, the culture supernatant was analyzed by LC-MS as described above. No CBGA product was observed in all structures assembled inside or outside the cell. Without being bound by theory, the lack of CBGA in some cultures may be due to poor enzyme expression of the isopentenyl transferase (GOT or NphB) or a lack of substrate (OLA or GPP) available for the reaction.

[0077] To detect the isopentenyl transferase activity, the level 1 POT plasmids expressing the GOT and NphB isopentenyl transferase transcription units were transformed into BY4741 cells, respectively. The cultures were grown and lysed in stationary phase as described above. Equal amounts of the lysates were then added to set up in vitro enzymatic assays with OLA and GPP as substrates, respectively. Negative controls were established by replacing the lysates with water. After incubation at 30°C, acidification and organic extraction were performed before LC-MS detection. The results are shown in Figure 2. Figure 5 shown.

[0078] A small peak corresponding to CBGA was observed in the negative control (no enzyme added) and in the lysate containing GOT. Figure 5 , first and second rows. These data suggest that the prenylation of the geranyl moiety on OLA is a spontaneous process without any enzyme catalysis and that a small amount of prenylation was observed when OLA and GPP were present in a mixture. GOT expressed in S. cerevisiae BY4741 was determined to be inactive because the peak area corresponding to CBGA was similar to that of the negative control.

[0079] On the other hand, the lysate containing NphB showed a peak area of ​​CBGA greater than that of the negative control, indicating that NphB is active. Figure 5 , third row. After elution for 0.1 minutes, a secondary peak was also observed in the NphB-catalyzed sample, with the same m / z value as CBGA. It has been previously reported that this peak is a nonspecific OLA prenylation product at another position of CBGA, namely 2-O-geranyl dihydroxypentylbenzoic acid (Zirpel et al., J. Biotechnol., 2017, 259: 204-212). NphB was shown to be functional when expressed in yeast strain BY4741, so the low yield of CBGA depends on the supply of substrates OLA and GPP in the construct. Without being bound by theory, insufficient GPP production may be the most likely reason for the low yield of CBGA, because peaks corresponding to OLA were detected in all structures.

[0080] Methods for overexpressing monoterpenes derived from GPP in Saccharomyces cerevisiae are known in the art. There is a large number of published papers describing work done to increase monoterpene production in yeast because they are of high commercial value. One way to increase GPP production is to reduce the metabolic burden borne by the yeast host. It has been reported that by overexpressing four genes in the mevalonate pathway and the corresponding monoterpene synthase geraniol synthase, geraniol production was increased sevenfold compared to previous engineered yeast strains (Zhao et al.).

[0081] refer to Figure 6, isoprenoid diphosphate isomerase (IDI1) catalyzes the isomerization between DMAPP and IPP. This helps to increase the ratio of precursors that favor GPP production (Liu et al., J. Biotechnol., 2013, 168 (4): 446-451). HMG-CoA has been identified as a key rate-limiting step in the mevalonate pathway, and overexpression of a truncated version of the gene, tHMG1, can increase the supply of mevalonate in the pathway (Asadollahi et al., Biotechnol. Bioeng., 2010, 106 (1): 86-96; Scalcinati et al., Metab Eng, 2012. 14 (2): p. 91-103). UPC2 is a transcription factor involved in regulating sterol biosynthesis, and overexpression of the mutant, UPC2-1, promotes mevalonate production by enhancing aerobic uptake of sterols (Davies et al., Mol. Cell. Biol., 2005, 25(16):7375-7385).

[0082] The IDI1, tHMG1 and UPC2-1 genes, along with the previously described GPP synthase Erg20pWW, were assembled into Level 1 POTs using the YeastFab system described above and subsequently assembled into Level 2 assembly plasmids (pCKL / pMKL) expressing the auxotrophic marker LEU2. This allowed the cannabinoid and isoprenoid genes to remain expressed on separate plasmids to reduce the length of each plasmid that was assembled and transformed into the yeast strain.

[0083] A series of medium to strong promoters and terminators were specified using a leucine auxotrophic marker (LEU2) to overexpress the four GPP production genes maintained on the pCKL / pMKL plasmids, while a different set of promoters and terminators were specified for the enzymes in the cannabinoid genes, maintained on a separate plasmid, pCKU / pMKU. See Table 3 below.

[0084] Table 3. Selected promoters and terminators for (A) ORFs from the mevalonate pathway maintained on pCKL / pMKL and (B) cannabinoid enzymes maintained on pCKU / pMKU.

[0085]

[0086]

[0087] After the cannabinoid (pCKU / pMKU) and mevalonate genes were assembled, both plasmids were transformed into yeast strain BY4741 and grown in medium lacking uracil and leucine to select transformants. The same bioproduction procedures were performed on constructs expressing both plasmids. After organic extraction of the lysed cell pellets, trace amounts of CBGA were detected within the cells. Figure 7 The peak with an elution time of about 6-7 minutes and an m / z value of 359.2223 (ppm error of -1.34) corresponds to the CBGA standard.

[0088] To further increase the yield of CBGA produced by the yeast expression pathway, computational protein engineering was performed on wild-type NphB to improve its selectivity for producing CBGA rather than the byproduct 2-O-geranyldihydroxypentylbenzoic acid. Four mutants, V49W / Y288A (SEQ ID NO: 8), V49W / Y288P (SEQ ID NO: 9), V49W / Y288A / Q295F (SEQ ID NO: 10), and V49W / Y288P / Q295F (SEQ ID NO: 11), were generated that were highly specific in using the geranyl moiety in GPP to prenylate dihydroxypentylbenzoic acid at the correct position to produce CBGA. These mutants, which produced no byproducts at all while significantly increasing CBGA production, were subsequently integrated into the pathway and significantly increased the titer of CBGA in vivo.

[0089] Example 6: Diversification of Cannabinoids

[0090] Branch points in the cannabinoid biosynthetic pathway can be exploited to produce novel cannabinoid compounds. The universality of the acyl-CoA ligase and polyketide synthase (i.e., OLS) used in the above structures allows the use of different short- to medium-chain fatty acids (C3-C10) as substrates instead of hexanoic acid (C6) to produce dihydroxypentylbenzoic acid analogs. An example in cannabis is the production of the cannabinoids tetrahydrocannabinol (THCV) and cannabidivarin-based acid (CBDVA), both of which are produced by the same enzymes in the pathway. These small-volume produced cannabinoids use the polyketide precursor 2,4-dihydroxy-6-propyl-benzoic acid (DVA) instead of OLA. DVA has a C3 chain length on the aromatic ring C2 instead of the C5 chain found in OLA (see Figure 8 ), which is also produced by the CsAAE1 enzyme in cannabis, using butyrate (C4) as the starting unit instead of hexanoate (C6).

[0091] The substrate universality of OLS and OAC was tested by using different starting units (C3–C10) instead of hexanoic acid (C6). Fig. 9 shown.

[0092] Certain OLA analogs, namely (C3, C9, C10) are not produced in S. cerevisiae but are produced in E. coli using the same biosynthetic pathway constructs. Fig.10 This may be due to the presence of endogenous acyl-CoA ligase in E. coli, but not in S. cerevisiae, which is able to act on substrates such as propionate, nonanoic acid, and decanoic acid. Such an endogenous ligase can provide a primed acyl-CoA thioester as a substrate for the action of polyketide synthase OLS.

[0093] Example 7: Further diversification of cannabinoid production

[0094] Traditional precursor-directed combinatorial biosynthesis approaches require laborious screening of large substrate libraries to detect whether new products are formed. To shorten the time required to identify favorable substrates and reduce the resources consumed, computational methods such as docking are used as predictive models to identify substrates that can fit into the enzyme binding pocket and have good binding affinity to the active site. This helps screen thousands of substrate libraries in a high-throughput manner to produce a ranked list of the most favorable substrates for experimental testing.

[0095] From OLS The high-resolution structure was firstly obtained by docking the enzyme's natural substrates hexanoyl CoA and malonyl CoA as positive controls. OLS uses two substrates with different R-groups that enter the active site via the CoA moiety; one hexanoyl CoA starter unit reacts with three malonyl CoA extension units, which iteratively extend the starter unit by attaching the acetyl ketone group to the hexyl intermediate attached to Cys157 of OLS, as shown in Figure 2. Fig.11 and 12 shown.

[0096] To determine whether different initiator units can perform the same three elongation steps using elongation units such as malonyl-CoA, the final intermediate state still covalently attached to OLS, the triketide intermediate, 3,5-dioxodecanoyl-CoA (after elongation with two malonyl-CoAs) was docked using GOLD (Groom et al.; see Fig.12 , left). This allows describing the spatial constraints that regulate the reaction mechanism that performs multiple elongation steps. If the binding pocket can accommodate the last covalently attached intermediate state of the corresponding initiator unit, it should be able to perform the first few rounds of elongation.

[0097] The final malonyl-CoA unit was superimposed using a crystallized malonyl-CoA ligand, which was complexed with a chalcone synthase (CHS2) homologous protein from Medicago sativa (PDB code: 1CML) (Ferrer et al., Nat. Struct. Biol., 1999, 6(8): 775-784). The superimposed malonyl-CoA ligand ( Fig.12 The yellow ligand in the figure, right) and the docked 3,5-dioxododecanyl CoA intermediate ( Fig.12 The blue ligand in the figure was energy minimized in the OLS structure (Jacobson et al., J. Phys. Chem. B, 2002, 106(44): 11673-11680; Jacobson et al., Proteins, 2004, 55(2): 351-367; Zhao et al., Proteins, 2011, 79(10): 2920-2935; Zhu et al., Proteins, 2006, 65(2): 438-452; Zhu et al., J. Chem. Theory Comput., 2007, 3(6): 2108-2119). The last superposition step identified starting units that were likely to have larger R-groups and would result in steric clashes with the last malonyl-CoA ligand in the OLS binding pocket, thus preventing the last elongation step. These candidates were subsequently eliminated. Fig.12 As observed in the positive controls on the left and right, the position of the docked 3,5-dioxodecanoyl-CoA intermediate from the hexanoyl-CoA initiator and the energy-minimized position of the malonyl-CoA both place the two substrates in a state where they can jointly undergo the final extension reaction.

[0098] As mentioned above, a library of 70 acyl-CoA starter units (Go et al.) and 12 different acyl-CoA extender units (Go et al., ACS Catalystation, 2015, 5(7): 4033-4042) is available. Overall, this corresponds to a total of 840 combinations of starter and extender units that can serve as substrates for type III polyketide synthases such as OLS. They are used as a substrate library and docked into the OLS structure. The output of the docking results is sorted accordingly and compared with the experimental results to check the accuracy of the prediction model. Experimental testing of the OLS substrate universality will generate dihydroxypentylbenzoic acid analogs with different chain lengths or R-groups, which can then be used to prepare cannabinoids with different R-groups on the C2 backbone of the dihydroxypentylbenzoic acid cannabinoid (see Figure 8 ).

[0099] Example 8: Protein Engineering

[0100] Protein engineering is a long-standing field of engineering enzymes to achieve specific desired properties. However, without good selection methods, the vast sequence space requires a predictive model to select mutants that display product diversity.

[0101] As described above, the NphB mutants were designed to be highly selective and to produce CBGA without any byproducts. Different NphB mutants were designed to allow the prenyltransferase to accept different substrates, thereby producing different desired cannabinoid products.

[0102] The final cyclization step in the cannabinoid biosynthetic pathway cyclizes the C10 chain previously transferred from GPP (see Figure 1 ). CBGA analogs are produced with longer prenyl chain lengths and will serve as precursors to a greater diversity of structures due to the longer chain lengths available. The C15 chain length analog of GPP, farnesyl pyrophosphate (FPP), and the C20 chain length analog geranyl pyrophosphate (GGPP) are used as suitable substitutes for GPP to produce CBGA analogs with C15 or C20 prenyl chains. Using the same computational protein engineering approach, specific mutations on the NphB enzyme can be identified that provide better affinity for longer FPP and GPP. These mutations can be tested and subsequently generated into CBGA analogs.

[0103] A homologous library of cannabinoid synthases was screened against CBGA analogue producing strains to test the production of various cannabinoid chemical structures and subsequently their biological activity.

[0104] Other Implementations

[0105] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0106] According to the above description, those skilled in the art can easily determine the basic characteristics of the present invention, and without departing from the spirit and scope of the present invention, various changes and modifications can be made to the present invention to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims. Sequence Listing <110> National University of Singapore <120> Sustainable production of cannabinoids from simple precursors using Saccharomyces cerevisiae <130> 218005-0109PCT <150> US 62 / 914,058 <151> 2019-10-11 <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 542 <212> PRT <213> Nicotiana tabacum <220> <221> misc_feature <223> 4-Coumarate-CoA ligase 2 <400> 1 Met Glu Lys Asp Thr Lys Gln Val Asp Ile Ile Phe Arg Ser Lys Leu 1 5 10 15 Pro Asp Ile Tyr Ile Pro Asn His Leu Pro Leu His Ser Tyr Cys Phe 20 25 30 Glu Asn Ile Ser Glu Phe Ser Ser Arg Pro Cys Leu Ile Asn Gly Ala 35 40 45 Asn Lys Gln Ile Tyr Thr Tyr Ala Asp Val Glu Leu Asn Ser Arg Lys 50 55 60 Val Ala Ala Gly Leu His Lys Gln Gly Ile Gln Pro Lys Asp Thr Ile 65 70 75 80 Met Ile Leu Leu Pro Asn Ser Pro Glu Phe Val Phe Ala Phe Ile Gly 85 90 95 Ala Ser Tyr Leu Gly Ala Ile Ser Thr Met Ala Asn Pro Leu Phe Thr 100 105 110 Pro Ala Glu Val Val Lys Gln Ala Lys Ala Ser Ser Ala Lys Ile Ile 115 120 125 Val Thr Gln Ala Cys His Val Asn Lys Val Lys Asp Tyr Ala Phe Glu 130 135 140 Asn Asp Val Lys Ile Ile Cys Ile Asp Ser Ala Pro Glu Gly Cys Leu 145 150 155 160 His Phe Ser Val Leu Thr Gln Ala Asn Glu His Asp Ile Pro Glu Val 165 170 175 Glu Ile Gln Pro Asp Asp Val Val Ala Leu Pro Tyr Ser Ser Gly Thr 180 185 190 Thr Gly Leu Pro Lys Gly Val Met Leu Thr His Lys Gly Leu Val Thr 195 200 205 Ser Val Ala Gln Gln Val Asp Gly Glu Asn Pro Asn Leu Tyr Ile His 210 215 220 Ser Glu Asp Val Met Leu Cys Val Leu Pro Leu Phe His Ile Tyr Ser 225 230 235 240 Leu Asn Ser Val Leu Leu Cys Gly Leu Arg Val Gly Ala Ala Ile Leu 245 250 255 Ile Met Gln Lys Phe Asp Ile Val Ser Phe Leu Glu Leu Ile Gln Ser 260 265 270 Tyr Lys Val Thr Ile Gly Pro Phe Val Pro Pro Ile Val Leu Ala Ile 275 280 285 Ala Lys Ser Pro Met Val Asp Asp Tyr Asp Leu Ser Ser Val Arg Thr 290 295 300 Val Met Ser Gly Ala Ala Pro Leu Gly Lys Glu Leu Glu Asp Thr Val 305 310 315 320 Arg Ala Lys Phe Pro Asn Ala Lys Leu Gly Gln Gly Tyr Gly Met Thr 325 330 335 Glu Ala Gly Pro Val Leu Ala Met Cys Leu Ala Phe Ala Lys Glu Pro 340 345 350 Phe Glu Ile Lys Ser Gly Ala Cys Gly Thr Val Val Arg Asn Ala Glu 355 360 365 Met Lys Ile Val Asp Pro Lys Thr Gly Asn Ser Leu Pro Arg Asn Gln 370 375 380 Ser Gly Glu Ile Cys Ile Arg Gly Asp Gln Ile Met Lys Gly Tyr Leu 385 390 395 400 Asn Asp Pro Glu Ala Thr Ala Arg Thr Ile Asp Lys Glu Gly Trp Leu 405 410 415 Tyr Thr Gly Asp Ile Gly Tyr Ile Asp Asp Asp Asp Glu Leu Phe Ile 420 425 430 Val Asp Arg Leu Lys Glu Leu Ile Lys Tyr Lys Gly Phe Gln Val Ala 435 440 445 Pro Ala Glu Leu Glu Ala Leu Leu Leu Asn His Pro Asn Ile Ser Asp 450 455 460 Ala Ala Val Val Pro Met Lys Asp Glu Gln Ala Gly Glu Val Pro Val 465 470 475 480 Ala Phe Val Val Arg Ser Asn Gly Ser Thr Ile Thr Glu Asp Glu Val 485 490 495 Lys Asp Phe Ile Ser Lys Gln Val Ile Phe Tyr Lys Arg Ile Lys Arg 500 505 510 Val Phe Phe Val Asp Ala Ile Pro Lys Ser Pro Ser Gly Lys Ile Leu 515 520 525 Arg Lys Asp Leu Arg Ala Lys Leu Ala Ala Gly Leu Pro Asn 530 535 540 <210> 2 <211> 524 <212> PRT <213> Rhodopseudomonas palustris <220> <221> misc_feature <223> Benzoate CoA ligase <400> 2 Met Asn Ala Ala Ala Val Thr Pro Pro Pro Glu Lys Phe Asn Phe Ala 1 5 10 15 Glu His Leu Leu Gln Thr Asn Arg Val Arg Pro Asp Lys Thr Ala Phe 20 25 30 Val Asp Asp Ile Ser Ser Leu Ser Phe Ala Gln Leu Glu Ala Gln Thr 35 40 45 Arg Gln Leu Ala Ala Ala Leu Arg Ala Ile Gly Val Lys Arg Glu Glu 50 55 60 Arg Val Leu Leu Leu Met Leu Asp Gly Thr Asp Trp Pro Val Ala Phe 65 70 75 80 Leu Gly Ala Ile Tyr Ala Gly Ile Val Pro Val Ala Val Asn Thr Leu 85 90 95 Leu Thr Ala Asp Asp Tyr Ala Tyr Met Leu Glu His Ser Arg Ala Gln 100 105 110 Ala Val Leu Val Ser Gly Ala Leu His Pro Val Leu Lys Ala Ala Leu 115 120 125 Thr Lys Ser Asp His Glu Val Gln Arg Val Ile Val Ser Arg Pro Ala 130 135 140 Ala Pro Leu Glu Pro Gly Glu Val Asp Phe Ala Glu Phe Val Gly Ala 145 150 155 160 His Ala Pro Leu Glu Lys Pro Ala Ala Thr Gln Ala Asp Asp Pro Ala 165 170 175 Phe Trp Leu Tyr Ser Ser Gly Ser Thr Gly Arg Pro Lys Gly Val Val 180 185 190 His Thr His Ala Asn Pro Tyr Trp Thr Ser Glu Leu Tyr Gly Arg Asn 195 200 205 Thr Leu His Leu Arg Glu Asp Asp Val Cys Phe Ser Ala Ala Lys Leu 210 215 220 Phe Phe Ala Tyr Gly Leu Gly Asn Ala Leu Thr Phe Pro Met Thr Val 225 230 235 240 Gly Ala Thr Thr Leu Leu Met Gly Glu Arg Pro Thr Pro Asp Ala Val 245 250 255 Phe Lys Arg Trp Leu Gly Gly Val Gly Gly Val Lys Pro Thr Val Phe 260 265 270 Tyr Gly Ala Pro Thr Gly Tyr Ala Gly Met Leu Ala Ala Pro Asn Leu 275 280 285 Pro Ser Arg Asp Gln Val Ala Leu Arg Leu Ala Ser Ser Ala Gly Glu 290 295 300 Ala Leu Pro Ala Glu Ile Gly Gln Arg Phe Gln Arg His Phe Gly Leu 305 310 315 320 Asp Ile Val Asp Gly Ile Gly Ser Thr Glu Met Leu His Ile Phe Leu 325 330 335 Ser Asn Leu Pro Asp Arg Val Arg Tyr Gly Thr Thr Gly Trp Pro Val 340 345 350 Pro Gly Tyr Gln Ile Glu Leu Arg Gly Asp Gly Gly Gly Pro Val Ala 355 360 365 Asp Gly Glu Pro Gly Asp Leu Tyr Ile His Gly Pro Ser Ser Ala Thr 370 375 380 Met Tyr Trp Gly Asn Arg Ala Lys Ser Arg Asp Thr Phe Gln Gly Gly 385 390 395 400 Trp Thr Lys Ser Gly Asp Lys Tyr Val Arg Asn Asp Asp Gly Ser Tyr 405 410 415 Thr Tyr Ala Gly Arg Thr Asp Asp Met Leu Lys Val Ser Gly Ile Tyr 420 425 430 Val Ser Pro Phe Glu Ile Glu Ala Thr Leu Val Gln His Pro Gly Val 435 440 445 Leu Glu Ala Ala Val Val Gly Val Ala Asp Glu His Gly Leu Thr Lys 450 455 460 Pro Lys Ala Tyr Val Val Pro Arg Pro Gly Gln Thr Leu Ser Glu Thr 465 470 475 480 Glu Leu Lys Thr Phe Ile Lys Asp Arg Leu Ala Pro Tyr Lys Tyr Pro 485 490 495 Arg Ser Thr Val Phe Val Ala Glu Leu Pro Lys Thr Ala Thr Gly Lys 500 505 510 Ile Gln Arg Phe Lys Leu Arg Glu Gly Val Leu Gly 515 520 <210> 3 <211> 448 <212> PRT <213> Streptomyces coelicolor <220> <221> misc_feature <223> phenylacetate-CoA ligase <400> 3 Met Ser Ser Glu Pro Thr Thr Gly Thr Ala Pro Ala Pro Arg Arg Gly 1 5 10 15 Glu Pro Leu Pro His Asp Leu Leu Asp Asp Ala Glu Arg Leu Ser Arg 20 25 30 Glu Gln Leu Arg Glu Leu Gln Leu Asp Arg Leu Arg Ala Thr Leu Arg 35 40 45 His Ala Tyr Asp Asn Val Glu Leu Tyr Arg Lys Lys Phe Asp Ala Ala 50 55 60 Gly Val Thr Pro Asp Asp Cys Arg Ser Leu Ala Asp Leu Ser Arg Phe 65 70 75 80 Pro Phe Thr Thr Lys Ala Asp Leu Arg Asp Thr Tyr Pro Phe Gly Met 85 90 95 Phe Ala Val Pro Met Ala Asp Val Arg Arg Val His Ala Ser Ser Gly 100 105 110 Thr Thr Gly Arg Ala Thr Val Val Gly Tyr Thr Glu Asn Asp Leu Ser 115 120 125 Met Trp Ala Asp Val Val Ala Arg Ser Ile Arg Ala Ala Gly Gly Arg 130 135 140 Pro Gly His Lys Val His Ile Ser Tyr Gly Tyr Gly Leu Phe Thr Gly 145 150 155 160 Gly Leu Gly Ala His Tyr Gly Ala Glu Arg Ala Gly Cys Thr Val Ile 165 170 175 Pro Ala Ser Gly Gly Met Thr Ala Arg Gln Val Gln Ile Ile Gln Asp 180 185 190 Phe Arg Pro Glu Ile Ile Met Val Thr Pro Ser Tyr Met Leu Thr Leu 195 200 205 Leu Asp Glu Phe Glu Arg Gln Gly Val Asp Pro Arg Thr Ser Ser Leu 210 215 220 Gln Val Gly Ile Phe Gly Ala Glu Pro Trp Thr Glu Glu Met Arg Arg 225 230 235 240 Glu Ile Glu Glu Arg Met Asp Ile His Ala Val Asp Ile Tyr Gly Leu 245 250 255 Ser Glu Val Ile Gly Pro Gly Val Ala Gln Glu Cys Val Glu Thr Lys 260 265 270 Asp Gly Leu His Ile Trp Glu Asp His Phe Tyr Pro Glu Val Val Asp 275 280 285 Pro Leu Thr Asp Ala Val Leu Pro Gly Gly Glu Glu Gly Glu Ile Val 290 295 300 Phe Thr Ser Leu Thr Lys Glu Ala Leu Pro Val Ile Arg Tyr Arg Thr 305 310 315 320 Arg Asp Leu Thr Arg Leu Leu Pro Gly Thr Ala Arg Pro Ala Phe Arg 325 330 335 Arg Met Arg Lys Val Thr Gly Arg Cys Asp Asp Met Ile Ile Leu Arg 340 345 350 Gly Val Asn Val Phe Pro Thr Gln Val Glu Glu Ile Val Leu Arg Thr 355 360 365 Pro Gly Val Ala Pro His Phe Gln Met Arg Leu Thr Glu Arg Gly Arg 370 375 380 Met Asp His Met Thr Val Arg Val Glu Ala Arg Pro Asp Ala Ala Pro 385 390 395 400 Glu Gln Arg Asp Ala Ala Ala Arg Ala Ile Ala Gln Gly Val Lys Asp 405 410 415 Gly Val Gly Val Thr Val Glu Val Glu Val Val Glu Pro Glu Thr Leu 420 425 430 Glu Arg Ser Leu Gly Lys Ile Arg Arg Val Trp Asp Gln Arg Gly Ala 435 440 445 <210> 4 <211> 543 <212> PRT <213> Cannabis <220> <221> misc_feature <223> Oxalyl coenzyme A <400> 4 Met Glu Lys Ser Gly Tyr Gly Arg Asp Gly Ile Tyr Arg Ser Leu Arg 1 5 10 15 Pro Pro Leu His Leu Pro Asn Asn Asn Asn Leu Ser Met Val Ser Phe 20 25 30 Leu Phe Arg Asn Ser Ser Ser Tyr Pro Gln Lys Pro Ala Leu Ile Asp 35 40 45 Ser Glu Thr Asn Gln Ile Leu Ser Phe Ser His Phe Lys Ser Thr Val 50 55 60 Ile Lys Val Ser His Gly Phe Leu Asn Leu Gly Ile Lys Lys Asn Asp 65 70 75 80 Val Val Leu Ile Tyr Ala Pro Asn Ser Ile His Phe Pro Val Cys Phe 85 90 95 Leu Gly Ile Ile Ala Ser Gly Ala Ile Ala Thr Thr Ser Asn Pro Leu 100 105 110 Tyr Thr Val Ser Glu Leu Ser Lys Gln Val Lys Asp Ser Asn Pro Lys 115 120 125 Leu Ile Ile Thr Val Pro Gln Leu Leu Glu Lys Val Lys Gly Phe Asn 130 135 140 Leu Pro Thr Ile Leu Ile Gly Pro Asp Ser Glu Gln Glu Ser Ser Ser 145 150 155 160 Asp Lys Val Met Thr Phe Asn Asp Leu Val Asn Leu Gly Gly Ser Ser 165 170 175 Gly Ser Glu Phe Pro Ile Val Asp Asp Phe Lys Gln Ser Asp Thr Ala 180 185 190 Ala Leu Leu Tyr Ser Ser Gly Thr Thr Gly Met Ser Lys Gly Val Val 195 200 205 Leu Thr His Lys Asn Phe Ile Ala Ser Ser Leu Met Val Thr Met Glu 210 215 220 Gln Asp Leu Val Gly Glu Met Asp Asn Val Phe Leu Cys Phe Leu Pro 225 230 235 240 Met Phe His Val Phe Gly Leu Ala Ile Ile Thr Tyr Ala Gln Leu Gln 245 250 255 Arg Gly Asn Thr Val Ile Ser Met Ala Arg Phe Asp Leu Glu Lys Met 260 265 270 Leu Lys Asp Val Glu Lys Tyr Lys Val Thr His Leu Trp Val Val Pro 275 280 285 Pro Val Ile Leu Ala Leu Ser Lys Asn Ser Met Val Lys Lys Phe Asn 290 295 300 Leu Ser Ser Ile Lys Tyr Ile Gly Ser Gly Ala Ala Pro Leu Gly Lys 305 310 315 320 Asp Leu Met Glu Glu Cys Ser Lys Val Val Pro Tyr Gly Ile Val Ala 325 330 335 Gln Gly Tyr Gly Met Thr Glu Thr Cys Gly Ile Val Ser Met Glu Asp 340 345 350 Ile Arg Gly Gly Lys Arg Asn Ser Gly Ser Ala Gly Met Leu Ala Ser 355 360 365 Gly Val Glu Ala Gln Ile Val Ser Val Asp Thr Leu Lys Pro Leu Pro 370 375 380 Pro Asn Gln Leu Gly Glu Ile Trp Val Lys Gly Pro Asn Met Met Gln 385 390 395 400 Gly Tyr Phe Asn Asn Pro Gln Ala Thr Lys Leu Thr Ile Asp Lys Lys 405 410 415 Gly Trp Val His Thr Gly Asp Leu Gly Tyr Phe Asp Glu Asp Gly His 420 425 430 Leu Tyr Val Val Asp Arg Ile Lys Glu Leu Ile Lys Tyr Lys Gly Phe 435 440 445 Gln Val Ala Pro Ala Glu Leu Glu Gly Leu Leu Val Ser His Pro Glu 450 455 460 Ile Leu Asp Ala Val Val Ile Pro Phe Pro Asp Ala Glu Ala Gly Glu 465 470 475 480 Val Pro Val Ala Tyr Val Val Arg Ser Pro Asn Ser Ser Leu Thr Glu 485 490 495 Asn Asp Val Lys Lys Phe Ile Ala Gly Gln Val Ala Ser Phe Lys Arg 500 505 510 Leu Arg Lys Val Thr Phe Ile Asn Ser Val Pro Lys Ser Ala Ser Gly 515 520 525 Lys Ile Leu Arg Arg Glu Leu Ile Gln Lys Val Arg Ser Asn Met 530 535 540 <210> 5 <211> 504 <212> PRT <213> Rhizobium trifolii <220> <221> misc_feature <223> malonyl-CoA synthetase <400> 5 Met Ser Asn His Leu Phe Asp Ala Met Arg Ala Ala Ala Pro Gly Asn 1 5 10 15 Ala Pro Phe Ile Arg Ile Asp Asn Thr Arg Thr Trp Thr Tyr Asp Asp 20 25 30 Ala Phe Ala Leu Ser Gly Arg Ile Ala Ser Ala Met Asp Ala Leu Gly 35 40 45 Ile Arg Pro Gly Asp Arg Val Ala Val Gln Val Glu Lys Ser Ala Glu 50 55 60 Ala Leu Ile Leu Tyr Leu Ala Cys Leu Arg Ser Gly Ala Val Tyr Leu 65 70 75 80 Pro Leu Asn Thr Ala Tyr Thr Leu Ala Glu Leu Asp Tyr Phe Ile Gly 85 90 95 Asp Ala Glu Pro Arg Leu Val Val Val Ala Ser Ser Ala Arg Ala Gly 100 105 110 Val Glu Thr Ile Ala Lys Pro Arg Gly Ala Ile Val Glu Thr Leu Asp 115 120 125 Ala Ala Gly Ser Gly Ser Leu Leu Asp Leu Ala Arg Asp Glu Pro Ala 130 135 140 Asp Phe Val Asp Ala Ser Arg Ser Ala Asp Asp Leu Ala Ala Ile Leu 145 150 155 160 Tyr Thr Ser Gly Thr Thr Gly Arg Ser Lys Gly Ala Met Leu Thr His 165 170 175 Gly Asn Leu Leu Ser Asn Ala Leu Thr Leu Arg Asp Phe Trp Arg Val 180 185 190 Thr Ala Gly Asp Arg Leu Ile His Ala Leu Pro Ile Phe His Thr His 195 200 205 Gly Leu Phe Val Ala Thr Asn Val Thr Leu Leu Ala Gly Ala Ser Met 210 215 220 Phe Leu Leu Ser Lys Phe Asp Pro Glu Glu Ile Leu Ser Leu Met Pro 225 230 235 240 Gln Ala Thr Met Leu Met Gly Val Pro Thr Phe Tyr Val Arg Leu Leu 245 250 255 Gln Ser Pro Arg Leu Asp Lys Gln Ala Val Ala Asn Ile Arg Leu Phe 260 265 270 Ile Ser Gly Ser Ala Pro Leu Leu Ala Glu Thr His Thr Glu Phe Gln 275 280 285 Ala Arg Thr Gly His Ala Ile Leu Glu Arg Tyr Gly Met Thr Glu Thr 290 295 300 Asn Met Asn Thr Ser Asn Pro Tyr Glu Gly Lys Arg Ile Ala Gly Thr 305 310 315 320 Val Gly Phe Pro Leu Pro Asp Val Thr Val Arg Val Thr Asp Pro Ala 325 330 335 Thr Gly Leu Ala Leu Pro Pro Glu Gln Thr Gly Met Ile Glu Ile Lys 340 345 350 Gly Pro Asn Val Phe Lys Gly Tyr Trp Arg Met Pro Glu Lys Thr Ala 355 360 365 Ala Glu Phe Thr Ala Asp Gly Phe Phe Ile Ser Gly Asp Leu Gly Lys 370 375 380 Ile Asp Arg Asp Gly Tyr Val His Ile Val Gly Arg Gly Lys Asp Leu 385 390 395 400 Val Ile Ser Gly Gly Tyr Asn Ile Tyr Pro Lys Glu Val Glu Gly Glu 405 410 415 Ile Asp Gln Ile Glu Gly Val Val Glu Ser Ala Val Ile Gly Val Pro 420 425 430 His Pro Asp Phe Gly Glu Gly Val Thr Ala Val Val Val Arg Lys Pro 435 440 445 Gly Ala Ala Leu Asp Glu Lys Ala Ile Val Ser Ala Leu Gln Asp Arg 450 455 460 Leu Ala Arg Tyr Lys Gln Pro Lys Arg Ile Ile Phe Ala Glu Asp Leu 465 470 475 480 Pro Arg Asn Thr Met Gly Lys Val Gln Lys Asn Ile Leu Arg Gln Gln 485 490 495 Tyr Ala Asp Leu Tyr Thr Arg Thr 500 <210> 6 <211> 385 <212> PRT <213> Cannabis <220> <221> misc_feature <223> Olivol synthase <400> 6 Met Asn His Leu Arg Ala Glu Gly Pro Ala Ser Val Leu Ala Ile Gly 1 5 10 15 Thr Ala Asn Pro Glu Asn Ile Leu Leu Gln Asp Glu Phe Pro Asp Tyr 20 25 30 Tyr Phe Arg Val Thr Lys Ser Glu His Met Thr Gln Leu Lys Glu Lys 35 40 45 Phe Arg Lys Ile Cys Asp Lys Ser Met Ile Arg Lys Arg Asn Cys Phe 50 55 60 Leu Asn Glu Glu His Leu Lys Gln Asn Pro Arg Leu Val Glu His Glu 65 70 75 80 Met Gln Thr Leu Asp Ala Arg Gln Asp Met Leu Val Val Glu Val Pro 85 90 95 Lys Leu Gly Lys Asp Ala Cys Ala Lys Ala Ile Lys Glu Trp Gly Gln 100 105 110 Pro Lys Ser Lys Ile Thr His Leu Ile Phe Thr Ser Ala Ser Thr Thr 115 120 125 Asp Met Pro Gly Ala Asp Tyr His Cys Ala Lys Leu Leu Gly Leu Ser 130 135 140 Pro Ser Val Lys Arg Val Met Met Tyr Gln Leu Gly Cys Tyr Gly Gly 145 150 155 160 Gly Thr Val Leu Arg Ile Ala Lys Asp Ile Ala Glu Asn Asn Lys Gly 165 170 175 Ala Arg Val Leu Ala Val Cys Cys Asp Ile Met Ala Cys Leu Phe Arg 180 185 190 Gly Pro Ser Glu Ser Asp Leu Glu Leu Leu Val Gly Gln Ala Ile Phe 195 200 205 Gly Asp Gly Ala Ala Ala Val Ile Val Gly Ala Glu Pro Asp Glu Ser 210 215 220 Val Gly Glu Arg Pro Ile Phe Glu Leu Val Ser Thr Gly Gln Thr Ile 225 230 235 240 Leu Pro Asn Ser Glu Gly Thr Ile Gly Gly His Ile Arg Glu Ala Gly 245 250 255 Leu Ile Phe Asp Leu His Lys Asp Val Pro Met Leu Ile Ser Asn Asn 260 265 270 Ile Glu Lys Cys Leu Ile Glu Ala Phe Thr Pro Ile Gly Ile Ser Asp 275 280 285 Trp Asn Ser Ile Phe Trp Ile Thr His Pro Gly Gly Lys Ala Ile Leu 290 295 300 Asp Lys Val Glu Glu Lys Leu His Leu Lys Ser Asp Lys Phe Val Asp 305 310 315 320 Ser Arg His Val Leu Ser Glu His Gly Asn Met Ser Ser Ser Thr Val 325 330 335 Leu Phe Val Met Asp Glu Leu Arg Lys Arg Ser Leu Glu Glu Gly Lys 340 345 350 Ser Thr Thr Gly Asp Gly Phe Glu Trp Gly Val Leu Phe Gly Phe Gly 355 360 365 Pro Gly Leu Thr Val Glu Arg Val Val Val Arg Ser Val Pro Ile Lys 370 375 380 Tyr 385 <210> 7 <211> 101 <212> PRT <213> Cannabis <220> <221> misc_feature <223> Dihydroxypentylbenzoate cyclase <400> 7 Met Ala Val Lys His Leu Ile Val Leu Lys Phe Lys Asp Glu Ile Thr 1 5 10 15 Glu Ala Gln Lys Glu Glu Phe Phe Lys Thr Tyr Val Asn Leu Val Asn 20 25 30 Ile Ile Pro Ala Met Lys Asp Val Tyr Trp Gly Lys Asp Val Thr Gln 35 40 45 Lys Asn Lys Glu Glu Gly Tyr Thr His Ile Val Glu Val Thr Phe Glu 50 55 60 Ser Val Glu Thr Ile Gln Asp Tyr Ile Ile His Pro Ala His Val Gly 65 70 75 80 Phe Gly Asp Val Tyr Arg Ser Phe Trp Glu Lys Leu Leu Ile Phe Asp 85 90 95 Tyr Thr Pro Arg Lys 100 <210> 8 <211> 307 <212> PRT <213> Artificial Sequence <220> <223> Streptomyces CL190 NphB V49W, Y288A <400> 8 Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu 1 5 10 15 Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro 20 25 30 Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val 35 40 45 Trp Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser 50 55 60 Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu 65 70 75 80 Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala 85 90 95 Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu 100 105 110 Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn 115 120 125 Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala 130 135 140 Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val 145 150 155 160 Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe 165 170 175 Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu 180 185 190 Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe 195 200 205 Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly 210 215 220 Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu 225 230 235 240 Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala 245 250 255 Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr 260 265 270 Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Ala 275 280 285 Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser 290 295 300 Leu Glu Asp 305 <210> 9 <211> 307 <212> PRT <213> Artificial Sequence <220> <223> Streptomyces CL190 NphB V49W, Y288P <400> 9 Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu 1 5 10 15 Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro 20 25 30 Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val 35 40 45 Trp Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser 50 55 60 Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu 65 70 75 80 Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala 85 90 95 Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu 100 105 110 Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn 115 120 125 Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala 130 135 140 Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val 145 150 155 160 Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe 165 170 175 Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu 180 185 190 Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe 195 200 205 Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly 210 215 220 Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu 225 230 235 240 Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala 245 250 255 Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr 260 265 270 Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Pro 275 280 285 Tyr His Ile Thr Asp Val Gln Arg Gly Leu Leu Lys Ala Phe Asp Ser 290 295 300 Leu Glu Asp 305 <210> 10 <211> 307 <212> PRT <213> Artificial Sequence <220> <223> Streptomyces CL190 NphB V49W, Y288A, Q295F <400> 10 Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu 1 5 10 15 Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro 20 25 30 Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val 35 40 45 Trp Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser 50 55 60 Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu 65 70 75 80 Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala 85 90 95 Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu 100 105 110 Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn 115 120 125 Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala 130 135 140 Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val 145 150 155 160 Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe 165 170 175 Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu 180 185 190 Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe 195 200 205 Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly 210 215 220 Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu 225 230 235 240 Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala 245 250 255 Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr 260 265 270 Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Ala 275 280 285 Tyr His Ile Thr Asp Val Phe Arg Gly Leu Leu Lys Ala Phe Asp Ser 290 295 300 Leu Glu Asp 305 <210> 11 <211> 307 <212> PRT <213> Artificial Sequence <220> <223> Streptomyces CL190 NphB V49W, Y288P, Q295F <400> 11 Met Ser Glu Ala Ala Asp Val Glu Arg Val Tyr Ala Ala Met Glu Glu 1 5 10 15 Ala Ala Gly Leu Leu Gly Val Ala Cys Ala Arg Asp Lys Ile Tyr Pro 20 25 30 Leu Leu Ser Thr Phe Gln Asp Thr Leu Val Glu Gly Gly Ser Val Val 35 40 45 Trp Phe Ser Met Ala Ser Gly Arg His Ser Thr Glu Leu Asp Phe Ser 50 55 60 Ile Ser Val Pro Thr Ser His Gly Asp Pro Tyr Ala Thr Val Val Glu 65 70 75 80 Lys Gly Leu Phe Pro Ala Thr Gly His Pro Val Asp Asp Leu Leu Ala 85 90 95 Asp Thr Gln Lys His Leu Pro Val Ser Met Phe Ala Ile Asp Gly Glu 100 105 110 Val Thr Gly Gly Phe Lys Lys Thr Tyr Ala Phe Phe Pro Thr Asp Asn 115 120 125 Met Pro Gly Val Ala Glu Leu Ser Ala Ile Pro Ser Met Pro Pro Ala 130 135 140 Val Ala Glu Asn Ala Glu Leu Phe Ala Arg Tyr Gly Leu Asp Lys Val 145 150 155 160 Gln Met Thr Ser Met Asp Tyr Lys Lys Arg Gln Val Asn Leu Tyr Phe 165 170 175 Ser Glu Leu Ser Ala Gln Thr Leu Glu Ala Glu Ser Val Leu Ala Leu 180 185 190 Val Arg Glu Leu Gly Leu His Val Pro Asn Glu Leu Gly Leu Lys Phe 195 200 205 Cys Lys Arg Ser Phe Ser Val Tyr Pro Thr Leu Asn Trp Glu Thr Gly 210 215 220 Lys Ile Asp Arg Leu Cys Phe Ala Val Ile Ser Asn Asp Pro Thr Leu 225 230 235 240 Val Pro Ser Ser Asp Glu Gly Asp Ile Glu Lys Phe His Asn Tyr Ala 245 250 255 Thr Lys Ala Pro Tyr Ala Tyr Val Gly Glu Lys Arg Thr Leu Val Tyr 260 265 270 Gly Leu Thr Leu Ser Pro Lys Glu Glu Tyr Tyr Lys Leu Gly Ala Pro 275 280 285 Tyr His Ile Thr Asp Val Phe Arg Gly Leu Leu Lys Ala Phe Asp Ser 290 295 300 Leu Glu Asp 305 <210> 12 <211> 538 <212> PRT <213> artificial sequence <220> <223> VSP- CBDA fusion <400> 12 Met Phe Ser Leu Lys Ala Leu Leu Pro Leu Ala Leu Leu Leu Val Ser 1 5 10 15 Ala Asn Gln Val Ala Ala Asn Pro Arg Glu Asn Phe Leu Lys Cys Phe 20 25 30 Ser Gln Tyr Ile Pro Asn Asn Ala Thr Asn Leu Lys Leu Val Tyr Thr 35 40 45 Gln Asn Asn Pro Leu Tyr Met Ser Val Leu Asn Ser Thr Ile His Asn 50 55 60 Leu Arg Phe Thr Ser Asp Thr Thr Pro Lys Pro Leu Val Ile Val Thr 65 70 75 80 Pro Ser His Val Ser His Ile Gln Gly Thr Ile Leu Cys Ser Lys Lys 85 90 95 Val Gly Leu Gln Ile Arg Thr Arg Ser Gly Gly His Asp Ser Glu Gly 100 105 110 Met Ser Tyr Ile Ser Gln Val Pro Phe Val Ile Val Asp Leu Arg Asn 115 120 125 Met Arg Ser Ile Lys Ile Asp Val His Ser Gln Thr Ala Trp Val Glu 130 135 140 Ala Gly Ala Thr Leu Gly Glu Val Tyr Tyr Trp Val Asn Glu Lys Asn 145 150 155 160 Glu Asn Leu Ser Leu Ala Ala Gly Tyr Cys Pro Thr Val Cys Ala Gly 165 170 175 Gly His Phe Gly Gly Gly Gly Tyr Gly Pro Leu Met Arg Asn Tyr Gly 180 185 190 Leu Ala Ala Asp Asn Ile Ile Asp Ala His Leu Val Asn Val His Gly 195 200 205 Lys Val Leu Asp Arg Lys Ser Met Gly Glu Asp Leu Phe Trp Ala Leu 210 215 220 Arg Gly Gly Gly Ala Glu Ser Phe Gly Ile Ile Val Ala Trp Lys Ile 225 230 235 240 Arg Leu Val Ala Val Pro Lys Ser Thr Met Phe Ser Val Lys Lys Ile 245 250 255 Met Glu Ile His Glu Leu Val Lys Leu Val Asn Lys Trp Gln Asn Ile 260 265 270 Ala Tyr Lys Tyr Asp Lys Asp Leu Leu Leu Met Thr His Phe Ile Thr 275 280 285 Arg Asn Ile Thr Asp Asn Gln Gly Lys Asn Lys Thr Ala Ile His Thr 290 295 300 Tyr Phe Ser Ser Val Phe Leu Gly Gly Val Asp Ser Leu Val Asp Leu 305 310 315 320 Met Asn Lys Ser Phe Pro Glu Leu Gly Ile Lys Lys Thr Asp Cys Arg 325 330 335 Gln Leu Ser Trp Ile Asp Thr Ile Ile Phe Tyr Ser Gly Val Val Asn 340 345 350 Tyr Asp Thr Asp Asn Phe Asn Lys Glu Ile Leu Leu Asp Arg Ser Ala 355 360 365 Gly Gln Asn Gly Ala Phe Lys Ile Lys Leu Asp Tyr Val Lys Lys Pro 370 375 380 Ile Pro Glu Ser Val Phe Val Gln Ile Leu Glu Lys Leu Tyr Glu Glu 385 390 395 400 Asp Ile Gly Ala Gly Met Tyr Ala Leu Tyr Pro Tyr Gly Gly Ile Met 405 410 415 Asp Glu Ile Ser Glu Ser Ala Ile Pro Phe Pro His Arg Ala Gly Ile 420 425 430 Leu Tyr Glu Leu Trp Tyr Ile Cys Ser Trp Glu Lys Gln Glu Asp Asn 435 440 445 Glu Lys His Leu Asn Trp Ile Arg Asn Ile Tyr Asn Phe Met Thr Pro 450 455 460 Tyr Val Ser Lys Asn Pro Arg Leu Ala Tyr Leu Asn Tyr Arg Asp Leu 465 470 475 480 Asp Ile Gly Ile Asn Asp Pro Lys Asn Pro Asn Asn Tyr Thr Gln Ala 485 490 495 Arg Ile Trp Gly Glu Lys Tyr Phe Gly Lys Asn Phe Asp Arg Leu Val 500 505 510 Lys Val Lys Thr Leu Val Asp Pro Asn Asn Phe Phe Arg Asn Glu Gln 515 520 525 Ser Ile Pro Pro Leu Pro Arg His Arg His 530 535

Claims

1. A recombinant cell of Saccharomyces cerevisiae comprising in its genome a plurality of nucleic acids, each nucleic acid encoding a cannabinoid biosynthetic pathway gene, wherein a cannabinoid is produced by said recombinant cell in the presence of a cannabinoid precursor substrate, and at least one of said cannabinoid biosynthetic pathway genes is from an organism other than Cannabis; wherein the plurality of nucleic acids encode NphB prenyltransferase, Coenzyme A (CoA) ligase, Malonyl CoA synthetase (MCS), Oleoleic acid synthase (OLS) and dihydroxypentylbenzoate cyclase (OAC); Wherein the NphB isopentenyl transferase is selected from SEQ ID NO: 8-11.

2. The recombinant cell of claim 1, wherein the CoA ligase is selected from Nicotiana tabacum ( Nicotiana tabacum ) 4-Coumaryl-CoA ligase, Rhodopseudomonas palustris ( Rhodopseudomonas palstri )benzoate-CoA ligase and Streptomyces coelicolor ( Streptomyces coelicolor ) Phenylacetate-CoA ligase.

3. The recombinant cell of claim 2, wherein the CoA ligase consists of any one of the amino acid sequences in SEQ ID NOs: 1-3.

4. The recombinant cell of claim 1, wherein the MCS consists of the amino acid sequence of SEQ ID NO:

5.

5. A method for producing cannabinoids, the method comprising contacting the recombinant cell of claim 1 with a cannabinoid precursor substrate and culturing the recombinant cell.

6. The method of claim 5, wherein the CoA ligase is selected from Nicotiana tabacum ( Nicotiana tabacum ) 4-Coumaryl-CoA ligase, Rhodopseudomonas palustris ( Rhodopseudomonas palstri )benzoate-CoA ligase and Streptomyces coelicolor ( Streptomyces coelicolor ) Phenylacetate-CoA ligase.

7. The method of claim 6, wherein the CoA ligase consists of any one of the amino acid sequences of SEQ ID NOs: 1-3.

8. The method of claim 5, wherein the MCS consists of the amino acid sequence of SEQ ID NO:

5.

9. An isopentenyl transferase, wherein the amino acid sequence of the isopentenyl transferase is any one of SEQ ID NOs: 8-11.

10. A nucleic acid encoding an isopentenyl transferase, wherein the amino acid sequence of the isopentenyl transferase is any one of SEQ ID NOs: 8-11.

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