AUTHOR=Muratovska Nina , Carlquist Magnus 

TITLE=Recombinant yeast for production of the pain receptor modulator nonivamide from vanillin

JOURNAL=Frontiers in Chemical Engineering

VOLUME=4

YEAR=2023

URL=https://www.frontiersin.org/journals/chemical-engineering/articles/10.3389/fceng.2022.1097215

DOI=10.3389/fceng.2022.1097215

ISSN=2673-2718

ABSTRACT=<p>We report on the development of a method based on recombinant yeast <italic>Saccharomyces cerevisiae</italic> to produce nonivamide, a capsaicinoid and potent agonist of the pain receptor TRPV1. Nonivamide was produced in a two-step batch process where yeast was i) grown aerobically on glucose and ii) used to produce nonivamide from vanillin and non-anoic acid by bioconversion. The yeast was engineered to express multiple copies of an amine transaminase from <italic>Chromobacterium violaceum</italic> (CvTA), along with an NADH-dependent alanine dehydrogenase from <italic>Bacillus subtilis</italic> (BsAlaDH) to enable efficient reductive amination of vanillin<italic>.</italic> Oxygen-limited conditions and the use of ethanol as a co-substrate to regenerate NADH were identified to favour amination over the formation of the by-products vanillic alcohol and vanillic acid. The native alcohol dehydrogenase ADH6 was deleted to further reduce the formation of vanillic alcohol. A two-enzyme system consisting of an <italic>N</italic>-acyltransferase from <italic>Capsicum annuum</italic> (CaAT), and a CoA ligase from <italic>Sphingomonas</italic> sp. Ibu-2 (IpfF) was co-expressed to produce the amide. This study provides proof of concept for yeast-based production of non-ivamide by combined transamination and amidation of vanillin.</p>