Biotransformation and chiral resolution of d,l ‐alanine into pyruvate and d‐alanine with a whole‐cell biocatalyst expressing l‐amino acid deaminase

Pyruvate production by whole ‐cell biocatalysis AbstractPyruvate is an important pharmaceutical intermediate and is widely used in food, nutraceuticals, and pharmaceuticals. However, high environmental pollution caused by chemical synthesis or complex separation process of microbial fermentation methods constrain the supply of pyruvate. Here, one ‐step pyruvate andd‐alanine production fromd,l‐alanine by whole‐cell biocatalysis was investigated. First,l‐amino acid deaminase (Pm1) fromProteus mirabilis was expressed inEscherichia coli, resulting in pyruvate titer of 12.01  g/L. Then, N‐terminal coding sequences were introduced to the 5′‐end of thepm1 gene to enhance the expression of Pm1 and the pyruvate titer increased to 15.13  g/L. Next, product utilization by the biocatalyst was prevented by knocking out the pyruvate uptake transporters (cstA,btsT) and the pyruvate metabolic pathway genespps,poxB,pflB,ldhA, andaceEF using CRISPR/Cas9, yielding 30.88  g/L pyruvate titer. Finally, by optimizing the reaction conditions, the pyruvate titer was further enhanced to 43.50 g/L in 8 H with a 79.99%l‐alanine conversion rate; meanwhile, the resolution ofd‐alanine reached 84.0%. This work developed a whole‐cell biocatalystE. coli strain for high ‐yield, high‐efficiency, and low‐pollution pyruvate andd‐alanine production, which has great potential for the commercial application in the future.
Source: Biotechnology and Applied Biochemistry - Category: Biochemistry Authors: Tags: Original Article Source Type: research