Efficient biosynthesis of D/L-alanine in the recombinant Escherichia coli BL21(DE3) by biobrick approach

Alanine is the most abundant chiral amino acid that exists into the D-alanine or L-alanine forms with diverse applications in the biomedical, pharmaceutical, plastics, and food industries. D/L-alanine production can be carried out through chemical, microbial fermentation, and biocatalytic methods and not much effective due to complicated processes or purification issues and is still challenging to achieve a higher yield. In the present study, biobrick method was utilized for efficient production of D/L-alanine in the recombinant <i>Escherichia coli</i> BL21(DE3) with tandem three-gene co-expression plasmid. Firstly, the co-expression plasmid pET-22bNS-DadX-Ald-Gdh containing three genes, alanine dehydrogenase (<i>ald)</i>, alanine racemase (<i>dadX)</i>, and glucose dehydrogenase (<i>gdh)</i> from <i>Bacillus pseudofirmus</i> OF4 were successfully constructed and introduced into the <i>E. coli</i> BL21(DE3) strain. Then, under optimized conditions in the whole-cell biocatalytic reaction [20 mM Na₂CO₃-NaHCO₃ (pH 10.1), 200 mM D-glucose, 200 mM sodium pyruvate, and 200 mM ammonium chloride], the concentration of D-alanine and L-alanine reached the maximum value (6.48 g/L and 7.05 g/L) after 3.0 h reaction time at 37°C under 180 rpm rotation. Meanwhile, promoter replacement experiments and Western blot analysis revealed that the expression level of protein OF4Ald had a significant effect on the production of D/L-alanine, indicating that alanine dehydrogenase might be the rate-limiting enzyme for D/L-alanine synthesis. This study provides a simple, feasible, and efficient biosynthesis process of D/L-alanine, which could explore emerging applications for large-scale production of industrial bioproducts.