Rationally Engineering the Cofactor Specificity of Lf SDR1 for Biocatalytic Synthesis of the Key Intermediate of Telotristat Ethyl

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.