AUTHOR=Naeem Muhammad , Hao Shimiao , Chu Mengqiu , Zhang Xuan , Huang Xinyan , Wang Jiaying , He Guangzheng , Zhao Baohua , Ju Jiansong 

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

JOURNAL=Frontiers in Bioengineering and Biotechnology

VOLUME=12

YEAR=2024

URL=https://www.frontiersin.org/journals/bioengineering-and-biotechnology/articles/10.3389/fbioe.2024.1421167

DOI=10.3389/fbioe.2024.1421167

ISSN=2296-4185

ABSTRACT=<p>Alanine is the most abundant chiral amino acid that exists into the <sc>D</sc>-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 <italic>Escherichia coli</italic> BL21(DE3) with tandem three-gene co-expression plasmid. Firstly, the co-expression plasmid pET-22bNS-DadX-Ald-Gdh containing three genes, alanine dehydrogenase (<italic>ald)</italic>, alanine racemase (<italic>dadX)</italic>, and glucose dehydrogenase (<italic>gdh)</italic> from <italic>Bacillus pseudofirmus</italic> OF4 were successfully constructed and introduced into the <italic>E. coli</italic> BL21(DE3) strain. Then, under optimized conditions in the whole-cell biocatalytic reaction [20 mM Na<sub>2</sub>CO<sub>3</sub>-NaHCO<sub>3</sub> (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.</p>