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, Saccharomyces cerevisiae 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, Saccharomyces cerevisiae 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, Assdr1 and Assdr2, 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 Assdr1 almost completely abolished the production of (13R,17S)-ethyl secol, while the deletion of Assdr2 had little to no effect on the production, indicating that Assdr1 plays a pivotal role in the biosynthesis of (13R,17S)-ethyl secol. The reason for the difference in the function of AsSDR1 and AsSDR2 in vivo may be attributed to their different cellular localizations, with AsSDR1 located outside the mitochondria and AsSDR2 in the mitochondria. A metabolomics analysis of Assdr1 knockout strains revealed that AsSDR1 may act as a pterin reductase. Overexpression of the newly isolated gene Assdr1 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 pos5Δ17 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.