Highly selective synthesis of d-amino acids via stereoinversion of corresponding counterpart by an in vivo cascade cell factory

Dan-Ping Zhang,An-Wen Fan,Lun-Jie Wu,Yan Xu,Yao Nie,Xiao-Ran Jing

doi:10.1186/s12934-020-01506-x

Dan-Ping Zhang, An-Wen Fan + Show 4 more

Open Access

https://doi.org/10.1186/s12934-020-01506-x

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Abstract

Backgroundd-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.ResultsTo 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.ConclusionsThe newly constructed in vivo cascade biocatalysis system was effective for the highly selective synthesis of d-amino acids via stereoinversion.

Highlights

Background dAmino acids, as chiral auxiliaries and chiral synthons in organic synthesis, play important roles in the production of pharmaceuticals and fine chemicals [1,2,3]
As shown in Scheme 1, in our study, the designed in vivo cascade route was mainly composed of two modules: an oxidative deamination module catalyzed by l-amino acid deaminase and a reductive amination module catalyzed by d-amino acid dehydrogenase and formate dehydrogenase
In this study, l-amino acid deaminase obtained from Proteus mirabilis (LAAD), diaminopimelate dehydrogenases obtained from Symbiobacterium thermophilum (DAPDH), and FDH were assembled in a multi-enzyme cascade system in vivo

Summary

Introduction

Background dAmino acids, as chiral auxiliaries and chiral synthons in organic synthesis, play important roles in the production of pharmaceuticals and fine chemicals [1,2,3]. A number of enzymatic approaches have been used to produce d-amino acids, including the synthesis of d-amino acids from d-hydantoin catalyzed by d-hydantoinase coupled with d-carbamoylase [10], asymmetric reductive amination of an α-keto acid by d-amino acid dehydrogenase or d-amino acid aminotransferase [1, 11], hydrolysis of an N-acyl-d-amino acid by N-acyl-d-amino acid amidohydrolase [12, 13], and the kinetic resolution of a racemic mixture by l-amino acid oxidase [7] These methods usually require specific substrates that are generally expensive and not commercially available. There are only few reports about the applicability of this approach [3]

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