β-Alanine production by L-aspartate-α-decarboxylase from Corynebacterium glutamicum and variants with reduced substrate inhibition

• A flux screening method was established to screen for ADC mutants that alleviated substrate inhibition. • Protein expression, kinetic parameters and substrate inhibition of the mutants confirmed that the mutants had high substrate affinities and enzymatic reaction rates. • The three-dimensional structure of ADC R12V and ADC Q17A was used to analyze the possible causes of the mutant's mitigation of substrate inhibition. β-Alanine is an important precursor in the production of food additives, pharmaceuticals, and nitrogen-containing chemicals. Biological production of β-alanine may be successful with enzyme l-aspartate-α-decarboxylase (ADC). However, substrate inhibition of ADC limits the application of this enzyme. In this study, an error-prone PCR was conducted using the gene encoding ADC enzyme from Corynebacterium glutamicum as a template in order to establish a mutant library and a screening method for high-yielding β-alanine strains with mutant enzymes. Two mutants with high activity, panD-56 (ADC R12V ) and panD-134 (ADC Q17A ), were obtained from the library of 2000 mutated enzymes. Kinetic analysis also indicated that ADC R12V and ADC Q17A had higher substrate affinities and enzymatic reaction rates than the wild enzyme. Finally, the characteristics of the strain panD-56 were evaluated and the yield and productivity were found to reach 0.65 g/g and 1.31 g/L/h with 40 g/L L-aspartic acid. When the concentration of L-aspartic acid reached 100 g/L, the yield was still 0.45 g/g, which was 125% higher than the wild type thus showing less substrate inhabitation compared to the wild type and showing strong potential for industrial biocatalytic production of β-alanine.