Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from <i>Lactobacillus plantarum</i>.

Jiewen Xiao; Jin Han; Zhina Qiao; Guodong Zhang; Wujun Huang; Kai Qian; Meijuan XU; Xian Zhang; Taowei Yang; Zhiming Rao

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Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum.

Author: Jiewen XIAO ¹ ; Jin HAN ¹ ; Zhina QIAO ¹ ; Guodong ZHANG ² ; Wujun HUANG ² ; Kai QIAN ² ; Meijuan XU ¹ ; Xian ZHANG ¹ ; Taowei YANG ¹ ; Zhiming RAO ¹
Author Information

1. School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
2. Renren Health Industry Limited Company, Yichun 331200, Jiangxi, China.
Publication Type:Journal Article
Keywords: glutamic acid decarboxylase; pH transformation; surface charge; whole cell transformation; γ-aminobutyric acid
MeSH: Glutamate Decarboxylase/genetics*; Lactobacillus plantarum/genetics*; Catalysis; gamma-Aminobutyric Acid; Hydrogen-Ion Concentration; Glutamic Acid
From: Chinese Journal of Biotechnology 2023;39(6):2108-2125
CountryChina
Language:Chinese
Abstract: γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGAD^{S24R/D88R/Y309K} was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and Lpgad^{S24R/D88R/Y309K} genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD₆₀₀) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.