HK1232126B - Methods of using capsaicin synthase for the microbial production of capsaicinoids - Google Patents

Description

Method for microbial production of capsaicinoids using capsaicin synthase

CROSS-REFERENCE TO RELATED APPLICATIONS

The present disclosure is a PCT patent application entitled METHODS FOR MICROBIAL PRODUCTION OF CAPSAICINoids USING CAPSAICIN SYNTHASE. This application claims priority to U.S. Provisional Patent Application No. 61/928,803, filed January 17, 2014, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure generally relates to methods for the biosynthetic production of capsaicin and related capsaicinoids using, inter alia, acyl-CoA synthetases (ACS), aminotransferases (PAMTs), and capsaicin synthases (CSs).

Background Art

Chili peppers are the fruit of the Capsicum genus, a member of the Solanaceae family. Due to their pungent nature, chili peppers have been widely used as a food additive in spicy dishes. Capsaicinoids are the substances primarily responsible for the pungency of chili peppers, and as previously mentioned, their production is limited to the Capsicum genus. Capsaicinoids (CP, 8-methyl-N-vanillyl-trans-6-nonenamide) and dihydrocapsaicinoids (DHCP, 8-methyl-N-vanillylnonanamide) are the two main capsaicinoids responsible for approximately 90% of the pungency of chili peppers (Garcés-Claver et al., 2007).

Except being mainly used as food additive for spicy sensation and spicy condiments, capsaicinoids have many medicines and medical uses. It has been found that they play a series of physiological and pharmacological effects, including analgesia, anticancer, anti-inflammatory, antioxidant and anti-obesity activity, and are used as the main component of ointment, patch, oil and cream, and are intended to alleviate the pain caused by several diseases such as vasomotor rhinitis, osteoarthritis and rheumatoid arthritis (Aza-González et al., 2011). Capsaicinoids are also currently used as the main active ingredient in the self-protection spray (i.e., pepper spray) sold (Reilly et al., 2001). Recently, it has been reported that capsaicinoids reduce the plasma cholesterol of hamsters and improve endothelial function (Liang et al., 2013).

Capsaicinoids are thought to be synthesized by CS, an acyltransferase that transfers the 8-methylnonenoyl moiety from 8-methylnonenoyl-CoA to vanillylamine to form an amide conjugate (Figure 1). Vanillylamine is formed by the phenylpropanoid pathway, in which branched-chain fatty acids are derived from branched-chain amino acids, such as valine (Curry et al., 1999; Mazourek et al., 2009). The aminotransferase (pAMT) catalyzes the formation of vanillylamine from vanillin. Applicants have cloned pAMT from ghost chili pepper. Another substrate, 8-methylnonenoyl-CoA, is derived from 8-methyl-trans-6-nonenoic acid through the activity of acyl-CoA synthetase (ACS).

In the present disclosure, applicants have used the gene product of CS to produce capsaicinoids through microbial synthesis. Applicants are the first to achieve microbial production of capsaicinoids, particularly capsaicin. Furthermore, the present invention addresses a long-felt but unmet need in industry for the production of capsaicinoids through microbial biosynthesis.

Summary of the Invention

The present disclosure is a bioconversion method for producing capsaicinoids, comprising expressing a first gene product of CS/AT3/Pun1 in a mixture, providing a first substrate to the mixture, and collecting capsaicinoids.

Another aspect of the present disclosure is a bioconversion method for producing capsaicinoids, comprising expressing a first gene product of CS/AT3/Pun1 in a cell system, culturing the cell system in a culture medium, and collecting capsaicinoids.

Another aspect of the present disclosure is a bioconversion method for producing a plurality of capsaicinoids, comprising expressing a gene product of CS/AT3/Pun1 in a cell system, providing 8-methyl-6-nonenoyl-CoA, providing vanillylamine, culturing the cell system in a culture medium, and collecting a plurality of capsaicinoids, wherein the plurality of capsaicinoids comprises more than about 90% capsaicin and less than about 5% dihydrocapsaicin in terms of quantitative ratio or molar ratio.

Another aspect of the present disclosure is a bioconversion method for producing a plurality of capsaicinoids, comprising expressing a gene product of CS/AT3/Pun1 in a cell system, providing 8-methyl-6-nonanoyl-CoA, providing vanillylamine, culturing the cell system in a culture medium, and collecting a plurality of capsaicinoids, wherein the plurality of capsaicinoids comprises more than about 90% dihydrocapsaicin and less than about 5% capsaicin.

Another aspect of the present disclosure is a biosynthetic method for producing capsaicinoids, comprising expressing the gene product of CS/AT3/Pun1 in a cell system, providing fatty acid coenzyme A (the active form of fatty acid), providing vanillylamine, growing the cell system in a culture medium, and collecting capsaicinoids.

BRIEF DESCRIPTION OF THE DRAWINGS

For a better understanding of the present invention, reference may be made to the accompanying drawings, in which:

Figure 1 shows the capsaicinoid biosynthesis pathway. Adapted from Stewart et al. (2007).

Figure 2 shows HPLC profiles of products extracted from Escherichia coli (E. coli) BL21 cells overexpressing the ACS1 and CS/AT3/Pun1 genes after addition of substrates. 1: Capsaicin (CP) and 2: Dihydrocapsaicin (DHCP). (A) Mixture of CP and DHCP standards from Sigma; (B) Control with no substrate added; (C) Vanillylamine (VN) and 8-methyl-6-nonenoic acid (6E) added; (D) VN and 8-methylnonanoic acid (8M) added; (E) VN, 6E, and 8M added.

FIG3 shows the GC/MS spectra of capsaicin and dihydrocapsaicin standards obtained from Sigma (Catalog No. 360376 Sigma, a mixture of CP and DHCP).

Figure 4 shows GC/MS spectra of products from BL21 cultures overexpressing ACS1 and CS/AT3/Pun1, where different substrates (e.g., VN, 6E, and 8M) were added. GC/MS analysis was performed using a Shimadzu GC-2010 system coupled with a GCMS-QP2010S detector. Separation was performed using an Rtx-5MS column (0.25 µm thick; 30 µm long; 0.25 mm diameter). Injection temperature: 265°C; Injection mode: split; Oven temperature: 140°C. Temperature gradient: 0-1 min, 140°C; 1-11.25 min, 140°C-263°C, rate 12; 11.25-21.25 min, 263°C.

Figure 5 shows the MS of the products spiked with substrates (6E and 8M) compared to capsaicin (CP) and dihydrocapsaicin (DHCP) control spectra.

Figure 6 shows SDS-PAGE analysis of His-SUMO-Pun1 expression in BL21(DE3) cells. A, 20: Total protein upon IPTG induction; C, Soluble crude protein extract; E1 to E3, Fractions eluted from a Ni-NTA column. The molecular weight of Pun1 is approximately 49 kDa, and the molecular weight of the His-SUMO tag is approximately 12 kDa.

Figure 7 shows HPLC profiles of the products of the coupling reaction between ACS1 and Pun1 when VN and 6E were used as substrates. #1, putative CP.

FIG8 shows the in vitro formation of CP (peak #1 in FIG7 ) by the ACS1-Pun1 coupled enzyme system as analyzed by GC/MC.

Figure 9 shows HPLC analysis of the in vitro activity of Pun1 when using octanoyl-CoA or decanoyl-CoA as substrates: #1, putative N-vanillyloctylamide; #2, putative N-vanillyldecylamide.

Figure 10 shows GC/MS analysis of the in vitro activity of Pun1 when octanoyl-CoA or decanoyl-CoA was used as a substrate. #1, putative N-vanillyloctylamide; #2, putative N-vanillyldecylamide.

Figure 11 shows the MS spectra of peaks #1 and #2 in Figure 10. #1, N-vanillyloctanamide; #2, N-vanillyldecanamide.

Figure 12 shows the effect of culture medium on capsaicinoid production (CP) from BL21(DE3) cultures overexpressing pCDFDuet-ACS1 and pETite N-His SUMO-ghost Pun1, supplemented with 50 mg/L vanillylamine (VN) and 50 mg/L 8-methyl-6-nonenoic acid (6E). LB, LB medium; TB, TB medium; M9, M9 minimal medium. Experiments were performed in triplicate, and the average values were used to plot the graph.

While the present disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof are shown by way of example in the drawings and will be described in detail herein. However, it should be understood that the drawings and detailed description set forth herein are not intended to limit the disclosure to the specific embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and scope of the present disclosure as defined by the appended claims.

DETAILED DESCRIPTION

definition

Cell System

A cell system is any cell that provides for ectopic protein expression. This includes bacteria, yeast, plant cells, and animal cells. It includes both prokaryotic and eukaryotic cells. It also includes in vitro protein expression using cellular components (e.g., ribosomes).

Cultured cell systems

Cultivation includes providing a culture medium that enables cell proliferation and differentiation. It also includes providing raw materials so that cells or cell components can translate and produce recombinant proteins.

Protein expression

Protein production can occur after gene expression. It consists of the stages after DNA has been transcribed into messenger RNA (mRNA). The mRNA is then translated into polypeptide chains, which ultimately fold into proteins. DNA is present in cells through transfection, which is the process of intentionally introducing nucleic acids into cells. The term is commonly used in non-viral methods for eukaryotic cells. It can also refer to other methods and cell types, although other terms are preferred: "transformation" is more commonly used to describe the transfer of non-viral DNA in bacteria, non-animal eukaryotic cells (including plant cells). Transduction is often used to describe viral-mediated DNA transfer. For the present application, transformation, transduction, and viral infection are included under the definition of transfection. In addition, protein expression includes in vitro translation, in which extracellular organelles are used to express proteins.

Biotransformation

The term bioconversion, also known as biotransformation, refers to the use of living organisms (usually microorganisms such as bacteria and yeast) to carry out more expensive or non-biologically feasible chemical reactions. These organisms convert substances into chemically modified forms.

mixture

A mixture is a physical combination of two or more substances in which the properties of the substances are preserved and mixed in the form of a solution, suspension, or colloid.

gene products

Gene products are biochemical materials, either RNA or protein, that result from the expression of a gene.

The present invention discloses a bioconversion method for producing capsaicinoids, comprising expressing a first gene product of CS/AT3/Pun1 in a mixture, providing a first substrate to the mixture, and collecting capsaicinoids. The first gene product of CS/AT3/Pun1 is based on the DNA sequence of SEQ ID No. 1. In further disclosures, the first gene product of CS/AT3/Pun1 is based on a DNA sequence that is at least about 95% identical to SEQ ID No. 1. In another embodiment, the first gene product of CS/AT3/Pun1 is derived from a ghost pepper. Additionally, the first substrate is an active fatty acid selected from the group consisting of 8-methyl-6-nonenoyl-CoA, 8-methylnonanoyl-CoA, octanoyl-CoA, decanoyl-CoA, and combinations thereof.

Another disclosure includes providing a first substrate to the mixture, further comprising: expressing a second gene product of an ACS, particularly ACS1, in the mixture, and providing a second substrate. In one embodiment, the second gene product of ACS1 is derived from a red bell pepper. The second substrate is a fatty acid selected from the group consisting of 8-methyl-6-nonenoic acid, 8-methylnonanoic acid, octanoic acid, decanoic acid, and combinations thereof. Additionally, in another disclosure, expressing any gene occurs by in vitro translation. In another disclosure, expressing any gene further occurs by expressing the gene in a cell system. The cell system is based on a microorganism selected from the group consisting of bacteria, yeast, and combinations thereof. The expression product of any gene is purified as a recombinant protein. In a further disclosure, a third substrate, vanillylamine, is provided.

Another disclosure includes expressing a third gene product of pAMT in the mixture and providing a fourth substrate, vanillin. In one embodiment, the third gene product of pAMT is derived from a ghost pepper. In one disclosure, any gene is expressed by in vitro translation. In another disclosure, any gene is expressed in a cell system. The cell system is based on a microorganism selected from the group consisting of bacteria, yeast, and combinations thereof. The expression product of any gene can be purified as a recombinant protein.

Another disclosure includes expressing a first gene product of CS/AT3/Pun1 in a cell system, culturing the cell system in culture, and collecting a capsaicinoid. In one disclosure, the capsaicinoid is capsaicin. Another embodiment further includes providing 8-methyl-6-nonenoyl-CoA and providing vanillylamine. Providing 8-methyl-6-nonenoyl-CoA includes expressing a second gene product of ACS1 in the cell system and providing the substrate 8-methyl-6-nonenoic acid. Providing vanillylamine includes expressing a third gene product of pAMT in the cell system and providing the substrate vanillin. In another disclosure, the capsaicinoid is capsaicin. Alternatively or additionally, the capsaicinoid is dihydrocapsaicin. In the case of producing dihydrocapsaicin, the disclosure further includes providing 8-methyl-nonanoyl-CoA and providing vanillylamine. Providing 8-methyl-nonanoyl-CoA includes expressing a second gene product of an ACS (particularly ACS1) in the cell system and providing 8-methylnonanoic acid. The disclosure further includes expressing a third gene product of pAMT in a cell system and providing the substrate vanillin. The first gene product is expressed from CS/AT3/Pun1 cloned from a red bell pepper. In one embodiment, the gene product is expressed from CS/AT3/Pun1 that shares at least about 95% sequence identity with CS/AT3/Pun1 cloned from a red bell pepper. The cell system is selected from the group consisting of bacteria, yeast, and combinations thereof.

Another disclosure provides a bioconversion method for producing capsaicinoids, comprising expressing a CS/AT3/Pun1 gene product in a cell system, providing a fatty acid coenzyme A, providing vanillylamine, culturing the cell system in a culture medium, and collecting capsaicinoids. In one disclosure, the fatty acid coenzyme A is 8-methyl-6-nonenoyl-CoA, and the capsaicinoid comprises greater than about 90% capsaicinoid by volume. In another disclosure, the fatty acid coenzyme A is 8-methyl-nonanoyl-CoA, and the capsaicinoid comprises greater than about 90% dihydrocapsaicinoid by volume. In another disclosure, the fatty acid coenzyme A is octanoyl-CoA, and the capsaicinoid product is N-vanillyloctylamide, more specifically greater than about 90% N-vanillyloctylamide. In another disclosure, the fatty acid coenzyme A is decanoyl-CoA, and the capsaicinoid product is N-vanillyldecylamide, more specifically greater than about 90% N-vanillyldecylamide.

For the cell systems used in various embodiments, they are selected from the group consisting of bacteria, yeast, and combinations thereof. Any cell system capable of biosynthetic production is provided.

It has long been known that the pungency of peppers is controlled by the Pun1 locus, and the corresponding gene has been identified as AT3, encoding a putative acyltransferase (Stewart et al., 2005). AT3 is a member of the BAHD acyltransferase superfamily and has been proposed as the putative CS/AT3/Pun1 (Kim et al., 2009). However, the biochemical activity of the CS/AT3/Pun1 gene product has not been reported to date. This lack of evidence for biochemical activity stems primarily from the fact that the acyl-CoA substrate for the CS/AT3/Pun1 gene product is not commercially available, and recombinant expression of CS/AT3/Pun1 is difficult due to the extreme insolubility of the protein (Stewart et al., 2005). It has also been hypothesized that CS may belong to a family of acyltransferases other than the BAHD family (Stewart et al., 2005). Applicants are the first to demonstrate that the CS/AT3/Pun1 gene product possesses CS functionality in biotransformation reactions. Applicants have addressed the long felt but unmet need to produce capsaicinoids, particularly capsaicin, in a bioconversion process.

In addition, due to the widespread use of capsaicinoids in food, medicine, and defense materials (e.g., pepper spray), there has been an increasing demand for capsaicinoids. To date, chili peppers are the only natural source of capsaicinoids. However, the content of capsaicinoids in chili peppers is generally low and is affected by the environment and growing conditions. For example, it has been reported that each gram of chili pepper (dry weight) contains 0.22-20 mg of total capsaicinoids (Thomas et al., 1998). The insufficient supply of natural capsaicinoids leads to extremely high prices for natural capsaicinoids, for example, US$2,000-3,000/kg ( http://www.alibaba.com/product-gs/810894171/Natural Capsaicin Capsaicine Powder9716.html ?s=p ). Having another source of capsaicinoids that can meet demand has been a long-standing unmet need in the industry.

Genetically engineered microorganisms have become an increasingly important platform for producing pharmaceuticals, chemicals, and biofuels from renewable resources (Du et al., 2011). When used in food, these biotechnological products can be labeled "natural" in the food industry according to current regulations (Münch, 1997). A prerequisite for the development of microbial production platforms is the cloning and characterization of the corresponding genes in the biotransformation pathway. Due to the importance of capsaicinoids, there has been a long-standing interest in cloning genes encoding capsaicin synthases. For example, over 100 years ago, Webber reported that the PUN1 locus controls the pungency of peppers (Webber, 1911). The corresponding gene was cloned, encoding the gene product of AT3, an acyltransferase in the BAHD superfamily (Stewart et al., 2005). However, until recently, as demonstrated by the applicants, no biochemical activity had been attributed to this putative acyltransferase, and the claim that the gene product of CS/AT3/Pun1 is a putative capsaicin synthase is questionable. Furthermore, due to the lack of an acyl-CoA substrate for the CS/AT3/Pun1 gene product, the activity from the CS/AT3/Pun1 gene product could never be effectively captured in a biotransformation mechanism to produce capsaicin and other capsaicinoids. Subsequently, in another study, using an enzyme-to-gene approach, Prasad et al. (2006) reported the identification of csyl as a hidden capsaicin synthase gene. However, two years later, this work was withdrawn (Prasad et al., 2008), and the CS gene remains unconfirmed and undetermined. Therefore, not only has the biochemical characterization and confirmation of a true capsaicin synthase been a long-standing goal in the industry, but the development of a biotransformation mechanism to utilize the CS gene to produce capsaicin and other capsaicinoids has long been desired.

Based on the applicants' confirmation of the activity of the ACS1 gene product, they were able to produce acyl-CoA substrates, thus demonstrating that CS/PUN1/AT3 possesses CS activity both in vitro and in vivo. This represents the first example of heterologous biosynthesis of capsaicinoids, paving the way for the development and optimization of microbial fermentation methods for producing "natural" capsaicinoids. Furthermore, in developing this method, the applicants have demonstrated that by incorporating different fatty acid substrates, they can also produce different capsaicinoids that may not exist in nature.

Example 1

The CS/Pun1/AT3 gene product has CS activity in vivo.

Based on the applicants' recent discovery of ACS activity in peppers (Chen H, Wang H and Yu O, US 61/898944), the gene products of ACS1 and CS/AT3/Pun1 were overexpressed in E. coli BL21(DE3) cells. The applicants discovered that the gene product of ACS1 has the ability to activate fatty acids and produce high-energy forms of fatty acids by adding coenzyme A. Protein expression was induced by IPTG and the addition of vanillylamine (VN) and 8-methyl-6-nonenoic acid (6E)/8-methylnonanoic acid (8M) to produce the putative CP/DHCP ( FIG. 2 ). In nature (i.e., from peppers), capsaicin and dihydrocapsaicin are produced together, and in biosynthetic reactions, the applicants have discovered that they can control the production of capsaicin, dihydrocapsaicin, and other capsaicinoids by adding specific activated fatty acids (e.g., 6E-CoA, 8M-CoA, octanoyl-CoA, and decanoyl-CoA).

Clone CS/Pun1/AT3

Applicants are the first to biochemically demonstrate CS activity in a cellular system through the bioconversion of the CS/AT3/Pun1 gene product and substrates. Specifically, applicants demonstrated the ability to catalyze the conversion of active fatty acids into capsaicinoids. The CS/AT3/Pun1 gene was initially cloned into the pENTR/D_TOPO vector. Cloning of CS required the following primers: Primers 309-pentr-F: CACCATGGCTTTTGCATTACCATC and 309-pentr-R: TTAGGCAATGAACTCAAGGAG were used to amplify the CS/AT3/Pun1 gene from cDNA of green fruit of the ghost pepper (Piper nigrum). The resulting PCR product was cloned into the pENTR/D_TOPO vector and then exchanged into the pDEST17 vector (Invitrogen) via an LR reaction. The CS/AT3/Pun1 gene product was then expressed in a bacterial system (e.g., BL21(DE3)), and CP and DHCP were detected by providing the required substrates. HPLC was performed using a 3000 LC system (Thermo Scientific) using a 120Cl8 reverse-phase column (Thermo Scientific; 3μ, 150×3 mm). The mobile phase consisted of solvent A (0.1% trifluoroacetic acid) and solvent B (acetonitrile). The gradient elution steps were as follows: 0-5 min, 5% B; 5-9 min, 5-80% B linear gradient; 9-11 min, 80% B; 11-12 min, 5% B. The flow rate was 0.6 ml/min. A diode array detector collected data from 200 to 400 nm. Peak areas were measured at 280 nm for detection and quantification of substrate and product.

CP/DHCP identity confirmation

The CP/DHCP identity was confirmed by further GC/MS analysis. As shown in Figure 3 (GC/MS spectrum), Sigma's CP standard is actually a mixture of CP and DHCP in a 60:40 ratio. The retention times of CP and DHCP are 13.80 and 14.04 min, respectively. The MS library in the GC/MS machine contains standard spectra for CP and DHCP, which match the spectrum from the Sigma standard. As shown in Figure 4, adding 6E and 8M to the culture medium expressing the gene products of ACS1 and CS/AT3/Pun1 results in the generation of CP and DHCP, respectively. In side-by-side comparisons, the spectra of the products match very well with the spectra of the standards (Figure 5).

The CS/Pun1/AT3 gene product has CS activity in vitro.

To determine in vitro activity, the applicants amplified the CS/Pun1/AT3 gene from cDNA of green fruit of a ghost pepper using primers 309-sumo-F: CGC GAA CAG ATT GGA GGTGCTTTTGCATTACCATC and 309-sumo-R: GTG GCG GCC GCT CTA TTA TTAGGCAATGAACTCAAGGAG. The resulting PCR product was purified on a 1% agarose gel and combined with a linearized pETite N-His SUMO Kan expression vector (Lucigen, Middleton, WI). The DNA mixture was used to transform HI-control 10G chemically competent cells (Lucigen) by heat shock. The gene insert was then fully sequenced, and the sequence was consistent with the sequence of the Pun1 gene from Capsicum chinense (GenBank: AY819027).

SEQ ID No. 1: Sequence from Pepper CS/Pun1/AT3

ATGGCTTTGCATTACCATCATCACTTGTTTCAGTTTGTGACAAATCTTTTATCAAACCTTCCTCTCTCACCCCCTCTAAACTTAGATTTCACAAGCTATCTTTCATCGATCAATCTTTAAGTAATATGTATATCCCTTGTGCATTTTTTTACCCTAAAGTACAA CAAAGACTAGAAGACTCCAAAAATTCTGATGAGCTTTCCCATATAGCCCACTTGCTACAAACATCTCTATCACAAACTCTAGTCTCTTACTATCCTTATGCAGGAAAGTTGAAGGACAATGCTACTGTTGACTGTAACGATATGGGAGCTGAGTTCTTGAGTGTT CGAATAAAATGTTCCATGTCTGAAATTCTTGATCATCCTCATGCATCTCTTGCAGAGAGCATAGTTTTGCCCAAGGATTTGCCTTGGGCGAATAATTGTGAAGGTGGTAATTTGCTTGTAGTTCAAGTAAGTAAGTTTGATTGTGGGGGAATAGCCATCAGTGTA TGCTTTTCGCACAAGATTGGTGATGGTTGCTCTCTGCTTAATTTCCTTAATGATTGGTCTAGCGTTACTCGTGATCATACGACAACAGCTTTAGTTCCATCTCCTAGATTTGTAGGAGATTCTGTCTTCTCTACAAAAAAATATGGTTCTCTTATTACGCCACAAA TTTTGTCCGATCTCAACGAGTGCGTACAGAAAAGACTCATTTTCCTACAGATAAGTTAGATGCACTTCGAGCTAAGGTGGCAGAAGAATCAGGAGTAAAAAATCCAACAAGGGCAGAAGTTGTTAGCGCTCTTCTTTTCAAATGTGCAACAAAGGCATCATCAT CAATGCTACCATCAAAGTTGGTTCACTTCTTAAACATACGTACTATGATCAAACCTCGTCTACCACGAAATGCCATTGGAAATCTCTCGTCTATTTTCTCCATAGAAGCAACTAACATGCAGGACATGGAGTTGCCAACGTTGGTTCGTAATTTAAGGAAGGAAGT TGAGGTGGCATACAAGAAAGACCAAGTCGAACAAAATGAACTGATCCTAGAAGTAGTAGAATCAATGAGAGAAGGGAAACTGCCATTTGAAAATATGGATGGCTATGAGAATGTGTATACTTGCAGCAATCTTTGCAAATATCCGTACTACACTGTAGATTTTGG ATGGGGAAGACCTGAAAGAGTGTGTCTAGGAAATGGTCCCTCCAAGAATGCCTTCTTCTTGAAAGATTACAAAGCTGGGCAAGGCGTGGAGGCGCGGGTGATGTTGCACAAGCAACAAATGTCTGAATTTGAACGCAATGAGGAACTCCTTGAGTTCATTGCCTAA

Applicants used pETite N-His SUMO-ghost Pun1 to transform HI-control BL21(DE3) cells (Lucigen). His-SUMO-Pun1 expression was induced with 0.5 mM IPTG at 16°C for 20 hours. The fusion protein was purified using a Ni-NTA column ( FIG6 ). The Pun1 gene product has a molecular weight of approximately 49 kDa, and the size of the His-SUMO tag is approximately 12 kDa. The His-SUMO-CS fusion protein migrated close to the predicted size (approximately 61 kDa) on SDS-PAGE ( FIG6 ).

Applicants used an enzyme system coupled with ACS1 and CS/Pun1/AT3 to detect the activity of the CS/Pun1/AT3 gene product. The ACS1 gene product promotes the production of substrates for the CS/Pun1/AT3 gene product. The system consisted of 100 mM Tris pH 8.5, 5 mM ATP, 0.5 mM Coenzyme A, ₁₀ mM MgCl₂, 100 mg/L VN, and 1 mM 6E. The reaction was initiated by the simultaneous addition of purified SUMO-ACS1 and SUMO-Pun1. The reaction was continued for 1 hour and then terminated by the addition of acetic acid. The reaction products were first analyzed by HPLC ( FIG7 ). Compared to the control, two products were formed: one was 8-methyl-6-nonenoyl-CoA, previously confirmed by MS/MS (Chen H, Wang H, and Yu O, 2013), and the other product (#1) matched the retention time of CP.

To further confirm the identity of Peak #1, the ethyl acetate extract was dried over N₂ and derivatized with MSTFA (N-methyl-N-(trimethylsilyl)trifluoroacetamide - Sigma). The product was analyzed by GC/MS (Figure 8). As shown in Figure 8, the CP produced by the enzymatic reaction exhibited an identical MS spectrum to the CP standard.

In addition, the applicant also tested the activity using other substrates. The applicant purchased octanoyl-CoA and decanoyl-CoA from Sigma. When these acyl-CoAs were used together with VN as substrates, the corresponding capsaicinoids were formed (#1 and #2 for N-vanillyloctylamide and N-vanillyldecylamide, respectively), confirming the enzymatic activity of Pun1 (Figures 9 and 10). The enzyme product was extracted with ethyl acetate and dried under _N2 gas. The MSTFA derivatives were analyzed by GC/MS. Figure 11 shows the MS spectra of peaks #1 and #2 in Figure 10.

Example 2

clone

Use primers ACS1-Bgl 11-F: GAAGATCTATGGCAACAGATAAATTTA and ACS1-XhoI-R: CCGCTCGAGTCACTTGGTACCCTTGTAC to amplify the ACS1 gene from the pETite N-His SUMO-ghost ACS1 template PCR, and connect it to the MCS2 site of the pCDFDuet-1 carrier (Novagen). Use the gained plasmid pCDFDuet-ACS1 to transform competent Escherichia coli BL21 (DE3) cells. In the LB plate that contains 100mg/L spectinomycin, select the cells transformed. The BL21 (DE3) cell that contains pCDFDuet-ACS1 of gained is used for the second transformation of the pETite N-His SUMO-ghost Pun1 carrier. On the LB plate that contains the spectinomycin of 50mg/L kanamycin and 100mg/L, select transformants.

Different culture media

The effects of different culture media on CP (capsaicinoid) production were examined in BL21 (DE3) cultures co-overexpressing ACS1 and Pun1 with the addition of VN (vanillylamine) and 6E (8-methyl-6-nonenoic acid). Briefly, overnight cultures were used to inoculate liquid LB, TB, or M9 medium (2%) containing 50 mg/L kanamycin and 100 mg/L spectinomycin. The cultures were first grown at 37°C to an OD600 of 0.6 and cooled to 16°C. 1 mM IPTG was then added to induce expression of ACS1 and Pun1. After 1 h of incubation at 16°C, 50 mg/L VN and 50 mg/L 6E were added to the cultures, which were then continued to be incubated at 16°C. Samples were taken at 0, 18, 22, 26, 42, and 48 h after substrate addition. CP was extracted with ethyl acetate and analyzed by HPLC. Figure 12 shows that of the three tested media, TB was the best for producing CP from VN and 6E.

Identity and Similarity

Identity is the fraction of identical amino acids between a pair of sequences after sequence alignment (sequence alignment can be achieved using only sequence information, structural information, or some other information, but is typically based solely on sequence information), while similarity is a score assigned based on alignment using some similarity matrix. The similarity index can be any of BLOSUM62, PAM250, or GONNET, or any matrix used by those of ordinary skill in the art for protein sequence alignment.

Identity is the degree of correspondence between two subsequences (sequences without spaces between them). An identity of 25% or greater indicates functional similarity, while 18-25% indicates structural or functional similarity. Remember, two completely unrelated or random sequences (greater than 100 residues) can have greater than 20% identity. Similarity is how similar two sequences are when compared. This is determined by their identity.

As will be apparent from the foregoing description, certain aspects of the present disclosure are not limited to the specific details of the examples described herein, and it is therefore contemplated that other modifications and applications or their equivalents will occur to those skilled in the art. It is therefore intended that the claims cover all modifications and applications that do not depart from the spirit and scope of the present disclosure.

In addition, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods or materials similar or equivalent to those described herein can be used in practicing or testing this disclosure, preferred methods and materials are as described above.

Other aspects, objects, and advantages of the disclosure can be obtained from a study of the drawings, the disclosure, and the appended claims.

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A and Münch T. (1997) Microbial production of natural flavors. ASMNews 63:551-559.

Kim JS, Park M, Lee DJ, Kim BD. (2009) Characterization of putativecapsaicin synthase promoter activity. Mol Cells. 28:331-339.

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Prasad BC, Kumar V, Gururaj HB, Parlmalan R, Giridhar P, Ravishankar GA. (2006) Characterization of capsaicin synthase and identification of its gene(csy1) for pungency factor capsaicin in pepper(Capsicum sp.). Proc Natl AcadSci U S A. 103:13315-13320.

Prasad BC, Kumar V, Gururaj HB, Parlmalan R, Giridhar P, Ravishankar GA. (2008) Retracted by Prasad et al., Characterization of capsaicin synthase and identification of its gene(csy1) for pungency factor capsaicin in pepper(Capsicum sp.). Proc Natl Acad Sci U S A. 105:20558.

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Sequence Listing

<110> Conagan

<120> Method for microbial production of capsaicinoids using capsaicin synthase

<130> 515281.18

<140> PCT/US

<141> 2015-01-10

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 1323

<212> DNA

<213> Yellow Lantern Pepper

<400> 1

atggcttttg cattaccatc atcacttgtt tcagtttgtg acaaatcttt tatcaaacct 60

tcctctctca ccccctctaa acttagattt cacaagctat ctttcatcga tcaatcttta 120

agtaatatgt atatcccttg tgcatttttt taccctaaag tacaacaaag actagaagac 180

tccaaaaatt ctgatgagct ttcccatata gcccacttgc tacaaacatc tctatcacaa 240

actctagtct cttactatcc ttatgcagga aagttgaagg acaatgctac tgttgactgt 300

aacgatatgg gagctgagtt cttgagtgtt cgaataaaat gttccatgtc tgaaattctt 360

gatcatcctc atgcatctct tgcagagagc atagttttgc ccaaggattt gccttgggcg 420

aataattgtg aaggtggtaa tttgcttgta gttcaagtaa gtaagtttga ttgtggggga 480

atagccatca gtgtatgcttttcgcacaag attggtgatg gttgctctct gcttaatttc 540

cttaatgatt ggtctagcgt tactcgtgat catacgacaa cagctttagt tccatctcct 600

agatttgtag gagattctgt cttctctaca aaaaaatatg gttctcttat tacgccacaa 660

attttgtccg atctcaacga gtgcgtacag aaaagactca tttttcctac agataagtta 720

gatgcacttc gagctaaggt ggcagaagaa tcaggagtaa aaaatccaac aagggcagaa 780

gttgttagcg ctcttctttt caaatgtgca acaaaggcat catcatcaat gctaccatca 840

aagttggttc acttcttaaa catacgtact atgatcaaac ctcgtctacc acgaaatgcc 900

attggaaatc tctcgtctat tttctccata gaagcaacta acatgcagga catggagttg 960

ccaacgttgg ttcgtaattt aaggaaggaa gttgaggtgg catacaagaa agaccaagtc 1020

gaacaaaatg aactgatcct agaagtagta gaatcaatga gagaagggaa actgccattt 1080

gaaaatatgg atggctatga gaatgtgtat acttgcagca atctttgcaa atatccgtac 1140

tacactgtag attttggatg gggaagacct gaaagagtgt gtctaggaaa tggtccctcc 1200

aagaatgcct tcttcttgaa agattacaaa gctgggcaag gcgtggaggc gcgggtgatg 1260

ttgcacaagc aacaaatgtc tgaatttgaa cgcaatgagg aactccttga gttcattgcc 1320

taa 1323

<210> 2

<211> 24

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 1 is used for initial cloning of the CS/AT3/Pun1 gene into the pENTR/D_TOPO vector

<400> 2

caccatggct tttgcattac catc 24

<210> 3

<211> 21

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 1 is used for initial cloning of the CS/AT3/Pun1 gene into the pENTR/D_TOPO vector

<400> 3

ttaggcaatg aactcaagga g 21

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence

<220>

Primer <223> is used to clone the CS/Pun1/AT3 gene into the pETite N-His SUMO Kan expression vector

<400> 4

cgcgaacaga ttggaggtgc ttttgcatta ccatc 35

<210> 5

<211> 39

<212> DNA

<213> Artificial sequence

<220>

Primer <223> is used to clone the CS/Pun1/AT3 gene into the pETite N-His SUMO Kan expression vector

<400> 5

gtggcggccg ctctattatt aggcaatgaa ctcaaggag 39

<210> 6

<211> 27

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 1 is used to clone ACS1 into the pCDFDuet-1 vector

<400> 6

gaagatctat ggcaacagat aaattta 27

<210> 7

<211> 28

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 1 is used to clone ACS1 into the pCDFDuet-1 vector

<400> 7

ccgctcgagt cacttggtac ccttgtac 28

Claims (31)

1. A biotransformation method for producing capsaicin-like substances, comprising: The first gene product of CS/AT3/Pun1 is expressed in a mixture, wherein the expression of the first gene product of CS/AT3/Pun1 is based on a DNA sequence having at least 95% identity with SEQ ID No. 1; A first substrate is provided to the mixture, wherein the first substrate is an active fatty acid selected from the group consisting of 8-methyl-6-nonenoyl-CoA, 8-methylnonenoyl-CoA, octyl-CoA, decyl-CoA, and combinations thereof. A second substrate is provided, wherein the second substrate is a fatty acid selected from the group consisting of 8-methyl-6-nonenoic acid, 8-methylnonanoic acid, octanoic acid, decanoic acid, and combinations thereof; Provides a third substrate, vanillin; and Collect the capsaicin-like substances. 2. The biotransformation method for producing capsaicin-like substances according to claim 1, wherein the first gene product expressing CS/AT3/Pun1 is based on the DNA sequence SEQ ID No. 1. 3. The biotransformation method for producing capsaicin-like substances according to claim 1, wherein the first gene product expressing CS/AT3/Pun1 is derived from the red pepper. 4. The biotransformation method for producing capsaicin-like substances according to claim 1, wherein providing the first substrate to the mixture comprises a second gene product expressing ACS1 in the mixture. 5. The biotransformation method for producing capsaicin-like substances according to claim 4, wherein the second gene product expressing ACS1 is derived from the red pepper. 6. The biotransformation method for producing capsaicinoids according to claim 4, wherein expressing any gene further includes expressing the gene by in vitro translation. 7. The biotransformation method for producing capsaicin-like substances according to claim 4, wherein expressing any gene further includes expressing said gene in a cellular system. 8. The biotransformation method for producing capsaicin-like substances according to claim 7, wherein the cell system is based on microorganisms selected from the group consisting of bacteria, yeast, and combinations thereof. 9. The biotransformation method for producing capsaicin-like substances according to claim 6, wherein the expression product of any of the genes is purified into a recombinant protein. 10. The biotransformation method for producing capsaicinoids according to claim 1, wherein providing the third substrate vanillin further comprises expressing a third gene product of pAMT in the mixture, and providing a fourth substrate vanillin. 11. The biotransformation method for producing capsaicin-like substances according to claim 10, wherein the third gene product expressing said pAMT is derived from the red pepper. 12. The biotransformation method for producing capsaicinoids according to claim 10, wherein expressing any gene further includes expressing said gene by in vitro translation. 13. The biotransformation method for producing capsaicin-like substances according to claim 10, wherein expressing any gene further includes expressing said gene in a cellular system. 14. The biotransformation method for producing capsaicin-like substances according to claim 13, wherein the cell system is based on microorganisms selected from the group consisting of bacteria, yeast, and combinations thereof. 15. The biotransformation method for producing capsaicin-like substances according to claim 6, wherein the expression product of any of the genes is purified into a recombinant protein. 16. A biotransformation method for producing capsaicin-like substances, comprising: The first gene product of CS/AT3/Pun1 is expressed in a cell system, wherein the expression of the first gene product of CS/AT3/Pun1 is based on a DNA sequence having at least 95% identity with SEQ ID No. 1; Provides 8-methyl-6-nonenoyl coenzyme A; Provides vanillin; Cell systems cultured in culture media; and Collect the capsaicin-like substances. 17. The biotransformation method for producing capsaicin-like substances according to claim 16, wherein the capsaicin-like substances are capsaicin. 18. The biotransformation method for producing capsaicin-like substances according to claim 16, wherein the 8-methyl-6-nonenoyl coenzyme A is provided comprising: The second gene product of ACS1 is expressed in the cell system; and Provides 8-methyl-6-nonenoic acid. 19. The biotransformation method for producing capsaicin-like substances according to claim 16, wherein the vanillin is provided comprising: The third gene product of pAMT is expressed in the cell system; and Provides vanillin. 20. The biotransformation method for producing capsaicinoids according to claim 16, wherein the capsaicinoids are dihydrocapsaicin. 21. The biotransformation method for producing capsaicin-like substances according to claim 20, further comprising: Provides 8-methyl-nonanoyl coenzyme A; and Provides vanillin. 22. The biotransformation method for producing capsaicin-like substances according to claim 21, wherein the 8-methyl-6-nonanoyl coenzyme A provided comprises: The second gene product of ACS1 is expressed in the cell system; and Provides 8-methylnonanoic acid. 23. The biotransformation method for producing capsaicin-like substances according to claim 21, wherein the vanillin is provided comprising: The third gene product of pAMT is expressed in the cell system; and Provides vanillin. 24. The biotransformation method for producing capsaicin-like substances according to claim 16, wherein the gene product is expressed by CS/AT3/Pun1 cloned from the red pepper. 25. The biotransformation method for producing capsaicinoids according to claim 16, wherein the gene product is expressed by CS/AT3/Pun1, which shares at least 95% sequence identity with CS/AT3/Pun1 cloned from the red pepper. 26. The biotransformation method for producing capsaicin-like substances according to claim 16, wherein the cell system is selected from the group consisting of bacteria, yeast, and combinations thereof. 27. A biotransformation method for producing capsaicin-like substances, comprising: The gene product of CS/AT3/Pun1 is expressed in a cellular system, wherein the gene product of CS/AT3/Pun1 is based on a DNA sequence having at least 95% identity with SEQ ID No. 1; Provides fatty acid coenzyme A, which is selected from 8-methyl-6-nonenoyl coenzyme A, 8-methylnonenoyl coenzyme A, octyl coenzyme A, decyl coenzyme A and combinations thereof; Provides vanillin; The cell system was cultured in a culture medium; and Collect the capsaicin-like substances. 28. The biotransformation method for producing capsaicin-like substances according to claim 27, wherein the fatty acid coenzyme A is 8-methyl-6-nonenoyl coenzyme A; and the capsaicin-like substances are capsaicin in a quantity ratio of more than 90%. 29. The biotransformation method for producing capsaicin-like substances according to claim 27, wherein the fatty acid coenzyme A is 8-methyl-nonanoyl coenzyme A; and the capsaicin-like substances are dihydrocapsaicin in a quantity ratio of more than 90%. 30. The biotransformation method for producing capsaicin-like substances according to claim 27, wherein the fatty acid coenzyme A is octanoyl coenzyme A. 31. The biotransformation method for producing capsaicin-like substances according to claim 27, wherein the fatty acid coenzyme A is decanoyl coenzyme A.