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Rationally Engineering the Cofactor Specificity of LfSDR1 for Biocatalytic Synthesis of the Key Intermediate of Telotristat Ethyl

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Switching cofactor preference of oxidoreductases from NADPH to NADH by rational engineering, replacing the expensive cofactor NADP + with the cheap cofactor NAD + , is a focus of attention in the industrial application of oxidoreductases. This study focuses on the reversal of cofactor preference for short-chain dehydrogenases/reductases (SDRs). Combined with bioinformatics analyses and in silico analyses, a small and smart mutant library (Mu1-Mu3) of Lf SDR1 was rationally designed and constructed. Thus, the excellent NADH-dependent recombinant Lf SDR1-V186A/G92V/E141L/G38D/T15A variant (Mu2) was obtained. Meanwhile, novel enzymatic processes for synthesis of the key intermediates [( R )- 2 and ( S )- 4 ] of telotristat ethyl and crizotinib were successfully created, which mainly relied on Mu2 coupled with an FDH-catalyzed cofactor regeneration system. A co-expressed E. coli whole-cell biocatalyst containing the genes of Mu2 and Pp FDH was developed to reduce ketones 1 and 3 . Finally, ketone 1 was almost completely converted into the product ( R )- 2 with a space-time yield of 115.7 g·L -1 ·d -1 and a 98.8% ee value.

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