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

Abstract

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 shortchain dehydrogenases/reductases (SDRs). Combined with bioinformatics analyses and in silico analyses, a small and smart mutant library (Mu1-Mu3) of LfSDR1 was rationally designed and constructed. Thus, the excellent NADH-dependent recombinant LfSDR1-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 PpFDH 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 À 1 •d À 1 and a 98.8 % ee value.

Recent advances in structure‐based enzyme engineering for functional reconstruction

“…The cofactor specificity reversal‐structural analysis and library design (CSR‐SALAD) strategy was developed to accelerate the engineering of cofactor specificity of enzymes (Figure 5a) (Cahn et al, 2017). The strategy has been proven to be effective in altering the cofactor preference of several NADPH‐dependent enzymes, including the glyoxylate reductase from Arabidopsis thaliana (Cahn et al, 2017), the imine reductase from Myxococcus stipitatus (Borlinghaus & Nestl, 2018), and the SDR from L. fermentum (Li et al, 2022). Xu's group recently proposed the cofactor specificity reversal‐small‐and‐smart library design (CSR‐SaSLiD) strategy by focusing on comparing the difference in the cofactor binding pocket of NADH‐ and NADPH‐dependent oxidoreductases (Figure 5b), which has been successfully applied in altering the cofactor specificity of some 7β‐hydroxysteroid dehydrogenases (7β‐HSDHs).…”

Design of a cofactor self‐sufficient whole‐cell biocatalyst for enzymatic asymmetric reduction via engineered metabolic pathways and multi‐enzyme cascade

“…[16] Although this strategy can improve the rate of NAD(P)H regeneration, it is still necessary to add a certain stoichiometric oxidized cofactor exogenously, which increases the process costs and complicates the workup upon reaction completion. Altering cofactor preference by enzyme modification based on structural biotechnology, [17][18][19][20][21] such as turning NADPH-dependent to NADH-dependent, can reduce redox cofactor costs. However, changing the cofactor specificity from the NADPH preference to the NADH preference through protein engineering is difficult to achieve.…”