Discovery and Engineering of a Novel Bacterial L-Aspartate α-Decarboxylase for Efficient Bioconversion

Abstract

L-aspartate α-decarboxylase (ADC) is a pyruvoyl-dependent decarboxylase that catalyzes the conversion of L-aspartate to β-alanine in the pantothenate pathway. The enzyme has been extensively used in the biosynthesis of β-alanine and D-pantothenic acid. However, the broad application of ADCs is hindered by low specific activity. To address this issue, we explored 412 sequences and discovered a novel ADC from Corynebacterium jeikeium (CjADC). CjADC exhibited specific activity of 10.7 U/mg and Km of 3.6 mM, which were better than the commonly used ADC from Bacillus subtilis. CjADC was then engineered leveraging structure-guided evolution and generated a mutant, C26V/I88M/Y90F/R3V. The specific activity of the mutant is 28.8 U/mg, which is the highest among the unknown ADCs. Furthermore, the mutant displayed lower Km than the wild-type enzyme. Moreover, we revealed that the introduced mutations increased the structural stability of the mutant by promoting the frequency of hydrogen-bond formation and creating a more hydrophobic region around the active center, thereby facilitating the binding of L-aspartate to the active center and stabilizing the substrate orientation. Finally, the whole-cell bioconversion showed that C26V/I88M/Y90F/R3V completely transformed 1-molar L-aspartate in 12 h and produced 88.6 g/L β-alanine. Our study not only identified a high-performance ADC but also established a research framework for rapidly screening novel enzymes using a protein database.