AUTHOR=Chen Binbin , Foo Jee Loon , Ling Hua , Chang Matthew Wook 

TITLE=Mechanism-Driven Metabolic Engineering for Bio-Based Production of Free R-Lipoic Acid in Saccharomyces cerevisiae Mitochondria

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=8

YEAR=2020

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2020.00965

DOI=10.3389/fbioe.2020.00965

ISSN=2296-4185

ABSTRACT=<p>Lipoic acid is a valuable organosulfur compound used as an antioxidant for dietary supplementation, and potentially anti-diabetic and anti-cancer. Currently, lipoic acid is obtained mainly through chemical synthesis, which requires toxic reagents and organic solvents, thus causing environmental issues. Moreover, chemically synthesized lipoic acid is conventionally a racemic mixture. To obtain enantiomerically pure <italic>R</italic>-lipoic acid, which has superior bioactivity than the <italic>S</italic> form, chiral resolution and asymmetric synthesis methods require additional reagents and solvents, and often lead to wastage of <italic>S</italic>-lipoic acid or precursors with undesired chirality. Toward sustainable production of <italic>R</italic>-lipoic acid, we aim to develop a synthetic biology-based method using engineered yeast. Here, we deepened mechanistic understanding of lipoic acid biosynthesis and protein lipoylation in the model yeast <italic>Saccharomyces cerevisiae</italic> to facilitate metabolic engineering of the microbe for producing free <italic>R</italic>-lipoic acid. In brief, we studied the biosynthesis and confirmed the availability of protein-bound lipoate in yeast cells through LC-MS/MS. We then characterized <italic>in vitro</italic> the activity of a lipoamidase from <italic>Enterococcus faecalis</italic> for releasing free <italic>R</italic>-lipoic acid from lipoate-modified yeast proteins. Overexpression of the lipoamidase in yeast mitochondria enabled <italic>de novo</italic> free <italic>R</italic>-lipoic acid production in vivo. By overexpressing pathway enzymes and regenerating the cofactor, the production titer was increased ∼2.9-fold. This study represents the first report of free <italic>R</italic>-lipoic acid biosynthesis in <italic>S. cerevisiae</italic>. We envision that these results could provide insights into lipoic acid biosynthesis in eukaryotic cells and drive development of sustainable <italic>R</italic>-lipoic acid production.</p>