In our previous study, one-step pyruvate and d-alanine production from d,l-alanine by a whole-cell biocatalyst Escherichia coli expressing l-amino acid deaminase (Pm1) derived from Proteus mirabilis was investigated. However, due to the low catalytic efficiency of Pm1, the pyruvate titer was relatively low. Here, semi-rational design based on site-directed saturation mutagenesis was carried out to improve the catalytic efficiency of Pm1. A novel high-throughput screening (HTS) method for pyruvate based on 2,4-dinitrophenylhydrazine indicator was then established. The catalytic efficiency (kcat/Km) of the mutant V437I screened out by this method was 1.88 times higher than wild type. Next, to improve the growth of the engineered strain BLK07, the genes encoding for Xpk and Fbp were integrated into its genome to construct non-oxidative glycolysis (NOG) pathway. Finally, the CRISPR/Cas9 system was used to integrate the N6-pm1-V437I gene into the genome of BLK07. Pyruvic acid titer of the plasmid-free strain reached 42.20 g/L with an l-alanine conversion rate of 77.62% and a d-alanine resolution of 82.4%. This work would accelerate the industrial production of pyruvate and d-alanine by biocatalysis, and the HTS method established here could be used to screen other Pm1 mutants with high pyruvate titers.
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This work was financially supported by the Key Research and Development Program of China (2018YFA0900504), the National Natural Science Foundation of China (31870069, 32021005) and the Fundamental Research Funds for the Central Universities (USRP52019A, JUSRP121010, JUSRP221013).
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Liu, K., Yu, H., Sun, G. et al. Semi-rational design of L-amino acid deaminase for production of pyruvate and d-alanine by Escherichia coli whole-cell biocatalyst. Amino Acids 53, 1361–1371 (2021). https://doi.org/10.1007/s00726-021-03067-8
- L-amino acid deaminase
- Whole-cell biocatalysis
- Semi-rational design
- High-throughput screening
- Genome engineering