Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid.

Jiajie Chen; Meijuan XU; Taowei Yang; Xian Zhang; Minglong Shao; Huazhong LI; Zhiming Rao

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Rational design of the C-terminal Loop region of leucine dehydrogenase and cascade biosynthesis L-2-aminobutyric acid.

Author: Jiajie CHEN ¹ ; Meijuan XU ¹ ; Taowei YANG ¹ ; Xian ZHANG ¹ ; Minglong SHAO ¹ ; Huazhong LI ¹ ; Zhiming RAO ¹
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1. Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Publication Type:Journal Article
Keywords: L-2-aminobutyric acid; Loop region; leucine dehydrogenase; multi-enzyme cascade catalysis; whole cell transformation
MeSH: Aminobutyrates; Escherichia coli/genetics*; Leucine Dehydrogenase/genetics*; Threonine Dehydratase
From: Chinese Journal of Biotechnology 2021;37(12):4254-4265
CountryChina
Language:Chinese
Abstract: Leucine dehydrogenase (LDH) is the key rate-limiting enzyme in the production of L-2-aminobutyric acid (L-2-ABA). In this study, we modified the C-terminal Loop region of this enzyme to improve the specific enzyme activity and stability for efficient synthesis of L-2-ABA. Using molecular dynamics simulation of LDH, we analyzed the change of root mean square fluctuation (RMSF), rationally designed the Loop region with greatly fluctuated RMSF, and obtained a mutant EsLDHD2 with a specific enzyme activity 23.2% higher than that of the wild type. Since the rate of the threonine deaminase-catalyzed reaction converting L-threonine into 2-ketobutyrate was so fast, the multi-enzyme cascade catalysis system became unbalanced. Therefore, the LDH and the formate dehydrogenase were double copied in a new construct E. coli BL21/pACYCDuet-RM. Compared with E. coli BL21/pACYCDuet-RO, the molar conversion rate of L-2-ABA increased by 74.6%. The whole cell biotransformation conditions were optimized and the optimal pH, temperature and substrate concentration were 7.5, 35 °C and 80 g/L, respectively. Under these conditions, the molar conversion rate was higher than 99%. Finally, 80 g and 40 g L-threonine were consecutively fed into a 1 L reaction mixture under the optimal conversion conditions, producing 97.9 g L-2-ABA. Thus, this strategy provides a green and efficient synthesis of L-2-ABA, and has great industrial application potential.