Engineering the C3N Pathway as a Short Detour for <i>De Novo</i> NAD⁺ Biosynthesis in <i>Saccharomyces cerevisiae</i>

Abstract

As a life-essential coenzyme, nicotinamide adenine dinucleotide (NAD+) has been explored for more than a century. In Saccharomyces, the natural NAD+de novo biosynthetic pathway initiating from tryptophan has been well elucidated. To bypass this stringently controlled natural pathway in yeast, an economical C3N pathway that was developed in Escherichia coli previously was constructed in Saccharomyces as a short detour for de novo NAD+ biosynthesis. After the functional expressions of the C3N genes were identified in Saccharomyces cerevisiae BY4741 by in vitro enzymatic assays, the C3N module was introduced into an NAD+ auxotrophic S. cerevisiae strain BY01, in which the BNA2 gene encoding tryptophan 2,3-dioxygenase was inactivated. The efficient NAD+ synthesis via the C3N pathway was confirmed by both plate assays and fermentation analysis. The applicability of the C3N pathway in cofactor engineering was tested by introducing it into S. cerevisiae BY4741, which improved the cellular NAD(H) level considerably. Consequently, this study proved that the de novo NAD+ biosynthetic pathway can be replaced by an artificial pathway in yeast, which paves a way to design more promising schemes in eukaryotes for rational manipulation of the cellular NAD(H) levels.

Keywords

• C3N pathway
• NAD⁺ <i>de novo</i> biosynthesis
• <i>Saccharomyces cerevisiae</i>
• eukaryotes