Abstract
Pyruvate 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 and d-alanine production from d,l-alanine by whole-cell biocatalysis was investigated. First, l-amino acid deaminase (Pm1) from Proteus mirabilis was expressed in Escherichia coli, resulting in pyruvate titer of 12.01g/L. Then, N-terminal coding sequences were introduced to the 5'-end of the pm1 gene to enhance the expression of Pm1 and the pyruvate titer increased to 15.13g/L. Next, product utilization by the biocatalyst was prevented by knocking out the pyruvate uptake transporters (cstA, btsT) and the pyruvate metabolic pathway genes pps, poxB, pflB, ldhA, and aceEF using CRISPR/Cas9, yielding 30.88g/L pyruvate titer. Finally, by optimizing the reaction conditions, the pyruvate titer was further enhanced to 43.50g/L in 8H with a 79.99% l-alanine conversion rate; meanwhile, the resolution of d-alanine reached 84.0%. This work developed a whole-cell biocatalyst E. coli strain for high-yield, high-efficiency, and low-pollution pyruvate and d-alanine production, which has great potential for the commercial application in the future.
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