Capsaicin biosynthesis in baker’s yeast Saccharomyces cerevisiae

Abstract

Background: Capsaicinoids are the compounds responsible for the pungent taste in the chili pepper genus capsicum. They are potent agonists of TRPV-receptors have large potential to be used as pharmaceutical agents for the treatment of various disease conditions associated to the peripheral and central nervous systems. Capsaicinoids are synthesized by the acylation of the aromatic compound vanillylamine with a branched-chain fatty acid-CoA by the enzyme capsaicin synthase (CS).

Methods: In this study, we constructed Saccharomyces cerevisiae yeast strains over-expressing two genes encoding CS and Acyl-coA synthetase (ACS), which are involved in the capsaicinoid biosynthesis pathway. In vivo enzyme activity of CS was investigated using whole cell bioconversion of vanillylamine or vanillyl alcohol and nonanoic acid as substrates for production of the capsaicin analog nonivamide.

Results: We found that over-expression of these enzymes had no inhibitory effect on cell growth, since there was no significant differences in maximum growth rate (µmax = 0,354 and 0,356) between the control strain and the recombinant strain, respectively. Moreover, LC-MS/MS analysis of fermentation extracts showed that the capsaicin analogues nonivamide and vanillyl nonanoate were indeed formed, thereby demonstrating that the enzymes were active in the engineered yeast.

Conclusion: CS and ACS were active in vivo and capable to catalyze the condensation of vanillyl amine and vanillyl alcohol with nonanoic acid into the products nonivamide and vanillyl nonanoate, respectively. This is the first example of engineered yeast as production host for capsaicinoids and capsinoids.

Popular Abstract

Chili pepper is a plant of the genus Capsicum, belongs to the nightshade family Solanaceae. Its fruit has been used widely as a food additive due to its desirable pungent nature. Capsaicinoids are the compounds responsible for the pungent taste in the hot pepper genus capsicum and both capsaicin (8-methyl-N-vanillyl-trans-6-nonenamide) and dihydro-capsaicin (8-methyl-N-vanillylnonenamide) are the most predominant, making up to around 90% of all capsaicinoids. Capsaicinoids are widely used for industrial and medical purposes. They are used in the human diet for their distinct pungent taste and bioactivities like thermogenesis. In addition, they are potent agonists of TRPV-receptors, and have large potential to be used as pharmaceutical agents for the treatment of various disease conditions associated to the peripheral and central nervous systems. The biosynthetic route to capsaicinoids in pungent cultivars of chili pepper is achieved through combination of two metabolic pathways:
1. Phenylalanine conversion to vanillylamine. The last step in this pathway is the conversion of vanillin to vanillylamine through transamination catalyzed by putative aminotransferase (pAMT).
2. Valine conversion to 8-methyl-6-nonenyl-CoA acid through the fatty acid metabolism where Acyl-coA synthase plays vital role.
The condensation reaction of vanillylamine with 8-methyl-6-nonenoyl-CoA is catalyzed by capsaicinoid synthase (CS) to form capsaicin.
Currently, capsaicin is produced naturally by chili pepper plant or chemically using chlorinated fatty acid and amines. However, these ways of production have many limitations where, natural production of capsaicin, by chili paper, is highly affected by different environmental and growth conditions. In addition, problems linked to chemical production of capsaicin such as using of high temperature and toxicity of used reagents and solvents.
Recently the demands to capsaicin and it analogous has been increased due to the wide uses of them in food and medicines. Thus, new source of capsaicin is needed to meet the industry demands, and therefore, recombinant yeast can be a very promising alternate platform to produce capsaicin and its analogues. Production in recombinant yeast offers the possibility to obtain capsaicinoids in simple and cheap fermentation process. Furthermore, biosynthesis in yeast may also make it possible to build novel yeast-specific capsaicinoids with potentially beneficial properties. So far there are no reported examples of recombinant production of capsaicinoids in a yeast in the literature.
In this project, we have introduced genes coding for capsaicin synthase (CS) and acyl-coa syntethase (ACS) from chili pepper into S. cerevisiae yeast, and our results show that these genes have no negative impact on cell growth. After that, we investigated the activity of the recombinant yeast cells as whole cell biocatalysts for the conversion of vanillyl amine or vanillyl alcohol and nonanoic acid to the capsaicin analogs nonivamide, and vanillyl nonanoate. The products were extracted from bulk liquid by ethyl acetate and analyzed by using Liquid chromatography–mass spectrometry (LC-MS). The LC-MS results showed that the constructed yeast strains were able to produce the desired compounds, demonstrating that the recombinant chili pepper enzymes were indeed active in yeast. Taken together, we have been able to produce capsaicinoids by recombinant yeast strains for first time.

Supervisor: Magnus Carlquist
Degree Project in Molecular Biology 30 credits
Department of Biology, Lund University

• author: Youssef, Khaled
• supervisor: Magnus Carlquist
• organization: Degree Projects in Molecular Biology
• course: MOBY01 20171
• year: 2017
• type: H1 - Master's Degree (One Year)
• subject: Biology and Life Sciences
• language: English
• id: 8926545
• date added to LUP: 2017-09-29 09:38:01
• date last changed: 2018-01-16 11:31:18