Abstract
Abstract
Background
d-Amino acids are increasingly used as building blocks to produce pharmaceuticals and fine chemicals. However, establishing a universal biocatalyst for the general synthesis of d-amino acids from cheap and readily available precursors with few by-products is challenging. In this study, we developed an efficient in vivo biocatalysis system for the synthesis of d-amino acids from l-amino acids by the co-expression of membrane-associated l-amino acid deaminase obtained from Proteus mirabilis (LAAD), meso-diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and formate dehydrogenase obtained from Burkholderia stabilis (FDH), in recombinant Escherichia coli.
Results
To generate the in vivo cascade system, three strategies were evaluated to regulate enzyme expression levels, including single-plasmid co-expression, double-plasmid co-expression, and double-plasmid MBP-fused co-expression. The double-plasmid MBP-fused co-expression strain Escherichia coli pET-21b-MBP-laad/pET-28a-dapdh-fdh, exhibiting high catalytic efficiency, was selected. Under optimal conditions, 75 mg/mL of E. coli pET-21b-MBP-laad/pET-28a-dapdh-fdh whole-cell biocatalyst asymmetrically catalyzed the stereoinversion of 150 mM l-Phe to d-Phe, with quantitative yields of over 99% ee in 24 h, by the addition of 15 mM NADP+ and 300 mM ammonium formate. In addition, the whole-cell biocatalyst was used to successfully stereoinvert a variety of aromatic and aliphatic l-amino acids to their corresponding d-amino acids.
Conclusions
The newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of d-amino acids via stereoinversion.
Funder
National Natural Science Foundation of China
Program of Introducing Talents of Discipline to Universities
High-End Foreign Experts Recruitment Program
Program for Advanced Talents within Six Industries of Jiangsu Province
Postgraduate Research & Practice Innovation Program of Jiangsu Province
Program for the Key Laboratory of Enzymes of Suqian
National First-Class Discipline Program of Light Industry Technology and Engineering
Publisher
Springer Science and Business Media LLC
Subject
Applied Microbiology and Biotechnology,Bioengineering,Biotechnology
Cited by
8 articles.
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