Development of an NAD(H)‐Driven Biocatalytic System for Asymmetric Synthesis of Chiral Amino Acids

Abstract

Chiral amino acids are extensively applied in the pharmaceutical, food, cosmetic, and agricultural industries. As a representative example, l‐phosphinothricin (l‐PPT, a chiral non‐natural amino acid) is a broad‐spectrum herbicide. An NAD(H)‐driven biocatalytic system for the asymmetric synthesis of chiral amino acids (focused on l‐PPT) with high efficiency and low cost is highly desired. The key for the development of such biocatalytic system is to obtain an NADH‐dependent biocatalyst with high catalytic performance toward l‐PPT pro‐ketone PPO. Herein, an engineered glutamate dehydrogenase from Lysinibacillus composti (LcGluDH) with desired activity was obtained by gene mining and protein engineering. In silico analyses suggested that the volume of substrate‐binding pocket was substantially enlarged from 330.5 Å3 to 409.6 Å3. The stability of LcGluDH was increased (Tm value increased from 47.3 °C to 65.3 °C) by introducing positively charged amino acids or aromatic amino acids at position 375. The desired biocatalytic system was constructed by coupling the engineered LcGluDH and an NAD+‐dependent FDH. Through this biocatalytic system, the batch production of l‐PPT exhibited high space‐time yield (207.3 g ⋅ L−1 ⋅ day−1) with strict stereoselectivity (ee of l‐PPT>99%). Furthermore, eight other chiral amino acids were synthesised by the developed NAD(H)‐driven biocatalytic system with high ee values.

Development of an aminotransferase‐driven biocatalytic cascade for deracemization of d,l‐phosphinothricin

“…Owing to the excellent enantioselectivity, lower costs and high space–time yield, biosynthesis of l ‐PPT has been considered as an effective alternative method to traditional chemical synthesis (Bachar et al, 2022; Gomm & O'Reilly, 2018; Grogan, 2018; Hepworth et al, 2017). Moreover, asymmetric synthesis of l ‐PPT from PPO with the presence of amino acid dehydrogenase (AADH, EC 1.4.1.X) or aminotransferase (transaminase, TA, EC 2.6.1.X) was preferred (Cheng et al, 2020, 2022; Jia et al, 2019; Jin et al, 2022; Zhou et al, 2020). At present, the large‐scale production of PPO is still a challenge due to the complexity of the processes and the large number of by‐products that are generated, resulting in high environmental impact and low yields.…”

A light-controlled biocatalytic system for precise regulation of enzymatic decarboxylation

“…3c). Moreover, 6.9 g desired R isomers (L-PPT) were extracted and purified from the one-pot system according to our previously developed process 16,17 In contrast, under continuous illumination the conversion of rac-PPT reached 44%, but the ee value of product was only 71%.…”

“…3c). Moreover, 6.9 g desired r isomers ( l -PPT) were extracted and purified from the one-pot system according to our previously developed process 16,17 and characterized by polarimetry ([a]D 25 = +16.9 ( c = 1, H 2 O), MS (IT-TOF (ESI): m / z = 182.0577, calcd. 182.0577) and NMR analysis (Fig.…”

Switching the Cofactor Preference of Formate Dehydrogenase to Develop an NADPH-Dependent Biocatalytic System for Synthesizing Chiral Amino Acids

“…The formate dehydrogenase (FDH)-based cofactor regeneration system supplies reducing power for redox biocatalytic systems and has been widely applied for the biosynthesis of fine chemicals such as chiral amino acids. − The system is considered as an ideal reducing agent for the regeneration of NAD­(P)H and is suitable for scale-up applications owing to several advantages. − First, formate oxidation is practically irreversible, and the byproduct CO 2 is nontoxic and can be easily expelled from the biocatalytic system. Second, formate is a small molecule that can easily cross microbial cell membranes, making it accessible within cellular compartments.…”