Identification, characterization, and application of an NADPH-dependent carbonyl reductase in Saccharomyces cerevisiae AS2.346 for improving production of (13R,17S)-ethyl secol

Levonorgestrel is currently the most widely used contraceptive, with advantages that include rapid oral absorption and high bioavailability. Among the various synthetic methods employed in its production, <i>Saccharomyces cerevisiae</i> AS2.346 was first introduced by the Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, in 1976, successfully catalyzing the conversion of ethyl secodione to (13R,17S)-ethyl secol. Presently, <i>Saccharomyces cerevisiae</i> AS2.346 whole-cell catalysis is still used in levonorgestrel industrial production. To identify the enzyme responsible for catalyzing the conversion of ethyl secodione to (13R,17S)-ethyl secol, we isolated two genes, As<i>sdr1</i> and As<i>sdr2</i>, both encoding carbonyl reductases. The biochemical characterization of AsSDR1 and AsSDR2 showed that they had high activity for ethyl secodione. Functional analysis demonstrated that the deletion of As<i>sdr</i>1 almost completely abolished the production of (13R,17S)-ethyl secol, while the deletion of As<i>sdr</i>2 had little to no effect on the production, indicating that As<i>sdr1</i> plays a pivotal role in the biosynthesis of (13R,17S)-ethyl secol. The reason for the difference in the function of AsSDR1 and AsSDR2 <i>in vivo</i> may be attributed to their different cellular localizations, with AsSDR1 located outside the mitochondria and AsSDR2 in the mitochondria. A metabolomics analysis of As<i>sdr</i>1 knockout strains revealed that AsSDR1 may act as a pterin reductase. Overexpression of the newly isolated gene As<i>sdr</i>1 led to an average 47.88 % higher (13R,17S)-ethyl secol yield compared to that of the wild-type strain. Furthermore, engineering the metabolism of the NADPH cofactor by overexpressing the truncated gene <i>pos</i>5Δ17 encoding mitochondrial NADH kinase produced a 111.25 % higher (13R,17S)-ethyl secol yield compared to that of the wild-type strain. These findings not only elucidate the key enzymatic players involved in the synthesis of (13R,17S)-ethyl secol but also provide a framework for optimizing the industrial biotransformation process for levonorgestrel production.