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Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

ABSTRACT: d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B₅. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for the deracemization of dl-pantolactone, using l-pantolactone dehydrogenase from Amycolatopsis methanolica (AmeLPLDH), conjugated polyketone reductase from Zygosaccharomyces parabailii (ZpaCPR), and glucose dehydrogenase from Bacillus subtilis (BsGDH). The AmeLPLDH was used to catalyze the dehydrogenated l-pantolactone into ketopantolactone; the ZpaCPR was used to further catalyze the ketopantolactone into d-pantolactone; and glucose dehydrogenase together with glucose fulfilled the function of coenzyme regeneration. All three enzymes were co-expressed in E. coli strain BL21(DE3), which served as the whole-cell biocatalyst. Under optimized conditions, 36 h deracemization of 1.25 M dl-pantolactone d-pantolactone led to an e.e._p value of 98.6%, corresponding to productivity of 107.7 g/(l·d).

SUBMITTER: Jin L

PROVIDER: S-EPMC10384591 | biostudies-literature | 2023 Jul

REPOSITORIES: biostudies-literature

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Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

Jin Lijun L Liu Xun X Wang Tairan T Wang Yi Y Zhou Xueting X Mao Wangwei W Zhang Yinjun Y Wang Zhao Z Sun Jie J Ying Xiangxian X

Molecules (Basel, Switzerland) 20230710 14

d-pantolactone is an intermediate in the synthesis of d-pantothenic acid, which is known as vitamin B<sub>5</sub>. The commercial synthesis of d-pantolactone is carried out through the selective resolution of dl-pantolactone catalyzed by lactone hydrolase. In contrast to a kinetic resolution approach, the deracemization of dl-pantolactone is a simpler, greener, and more sustainable way to obtain d-pantolactone with high optical purity. Herein, an efficient three-enzyme cascade was developed for ...[more]

PMID: 37513182

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Project description:Pyrrolidone is a high value-added monomer and an important active drug intermediate. However, the efficient enzymatic synthesis of pyrrolidone remains a challenge. Here, we developed and reconstructed a three-enzyme cascade pathway using Escherichia coli BL21(DE3) for the production of pyrrolidone from l-glutamate (l-Glu). The carnitine-CoA ligase from Escherichia coli (EcCaiC) at a low expression level and with a low activity is regarded as the rate-limiting enzyme. Here, we obtained the best EcCaiCF380M/N430D double mutant with a kcat/Km value 1.5 times higher than that of the wild type via mechanism-based protein engineering. For this, we (i) eliminated the steric hindrance of the loop ring to improve the precatalytic conformation of the adenylation intermediate and (ii) fixed the hinge region to stabilize the closed conformation of the enzyme. Furthermore, ribosome-binding site (RBS) optimization led to an increase in the expression level of EcCaiCF380M/N430D, which was then cloned into the plasmid pET-EcCaiCF380M/N430D-DegoPPK2. Finally, under optimal induction and transformation conditions, 16.62 g/L of pyrrolidone was generated from 30 g/L l-Glu (batch feeding) within 24 h with a molar conversion rate of 95.2% and the highest productivity ever obtained, to our knowledge (0.69 g/L/h). Our findings demonstrate a strategy that is potentially attractive for the industrial production of pyrrolidone. IMPORTANCE This study developed a three-enzyme cascade pathway for the production of pyrrolidone from l-Glu. The catalytic efficiency of carnitine CoA ligase from Escherichia coli (EcCaiC) was improved by mechanism-based protein engineering, and the titer of pyrrolidone was further increased by ribosome-binding site (RBS), induction conditions, and conversion conditions optimization. Finally, we efficiently produced pyrrolidone by one pot in vivo with 95.2% conversion and 0.69 g/L/h productivity. Our study provides a new possibility for the industrial production of enzymatic synthesis of pyrrolidone.

Dataset Information

Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

Publications

Multi-Enzymatic Cascade for Efficient Deracemization of dl-Pantolactone into d-Pantolactone.

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