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

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.

Efficient Synthesis of D‐Phenylalanine from L‐Phenylalanine via a Tri‐Enzymatic Cascade Pathway

“…A similar study combining PmLAAD whole‐cell with purified StDAPDH H227V and Burkholderia stabilis BsFDH yielded 13.2 g L −1 D‐Phe in 6 h, with 100 % conversion rate and >99 % ee [15] . And more recently, the three enzymes were assembled in one cell and the titer of D‐Phe was improved to 24.7 g L −1 in 24 h by 75 g L −1 whole‐cell catalyst and 15 mM NADP + [23] . A quick cost analysis of our final process from materials and catalyst showed that the cost for D‐Phe production is about $11450 per ton.…”

“…[15] And more recently, the three enzymes were assembled in one cell and the titer of D-Phe was improved to 24.7 g L À 1 in 24 h by 75 g L À 1 whole-cell catalyst and 15 mM NADP + . [23] A quick cost analysis of our final process from materials and catalyst showed that the cost for D-Phe production is about $11450 per ton. By contrast, the factory price for D-Phe is as high as $30500 per ton (Table S4).…”

Multienzymatic Cascades and Nanomaterial Scaffolding—A Potential Way Forward for the Efficient Biosynthesis of Novel Chemical Products

“…Additional three enzyme cascades exist geared toward production of non-natural products for the interested reader. [138][139][140][141][142] Notably, Otte et al developed a three enzyme cascade to produce the industrially relevant polymer building block azelaic acid. [142] Additionally, Engelmann et al developed a three enzyme cascade for the production of cinnamyl cinnamate where they demonstrated potential scale-up methods for the production of the highvalue aromatic ester.…”

Dual Detection of Temperature And Chiral Amino Acid Using Triphenylamine‐Based Fluorescent Probes

“…Enantiomers of chiral clinical drugs often exhibit different pharmacological activities or toxicological properties, owing to their different spatial structures [27] . L‐phenylalanine (L‐Phe), an aromatic amino acid, is revealed to be the structural unit of many natural products and drug molecules, [28] and can be used as a drug‐carrier loading drug to directly inject the focal zone and improve drug utilization [29] . Therefore, it is necessary to distinguish the effective enantiomers between different structures of chiral molecules.…”