| Summary: | As a life-essential coenzyme, nicotinamide adenine dinucleotide (NAD<sup>+</sup>) has been explored for more than a century. In <i>Saccharomyces</i>, the natural NAD<sup>+</sup><i>de novo</i> 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 <i>Escherichia coli</i> previously was constructed in <i>Saccharomyces</i> as a short detour for <i>de novo</i> NAD<sup>+</sup> biosynthesis. After the functional expressions of the C3N genes were identified in <i>Saccharomyces cerevisiae</i> BY4741 by in vitro enzymatic assays, the C3N module was introduced into an NAD<sup>+</sup> auxotrophic <i>S. cerevisiae</i> strain BY01, in which the <i>BNA2</i> gene encoding tryptophan 2,3-dioxygenase was inactivated. The efficient NAD<sup>+</sup> 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 <i>S. cerevisiae</i> BY4741, which improved the cellular NAD(H) level considerably. Consequently, this study proved that the <i>de novo</i> NAD<sup>+</sup> 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.
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