title: Improved production of α-ketoglutaric acid (α-KG) by a Bacillus subtilis whole-cell biocatalyst via engineering of L-amino acid deaminase and deletion of the α-KG utilization pathway.
abstract: We previously developed a novel one-step biotransformation process for the production of α-ketoglutarate (α-KG) from L-glutamic acid by a Bacillus subtilis whole-cell biocatalyst expressing an L-amino acid deaminase (pm1) of Proteus mirabilis. However, the biotransformation efficiency of this process was low owing to low substrate specificity and high α-KG degradation. In this study, we further improved α-KG production by protein engineering P. mirabilis pm1 and deleting the B. subtilis α-KG degradation pathway. We first performed three rounds of error-prone polymerase chain reaction and identified mutations at six sites (F110, A255, E349, R228, T249, and I352) that influence catalytic efficiency. We then performed site-saturation mutagenesis at these sites, and the mutant F110I/A255T/E349D/R228C/T249S/I352A increased the biotransformation ratio of L-glutamic acid from 31% to 83.25% and the α-KG titer from 4.65 g/L to 10.08 g/L. Next, the reaction kinetics and biochemical properties of the mutant were analyzed. The Michaelis constant for L-glutamic acid decreased from 49.21 mM to 23.58 mM, and the maximum rate of α-KG production increased from 22.82 μM min(-1) to 56.7 μM min(-1). Finally, the sucA gene, encoding α-ketodehydrogenase, was deleted to reduce α-KG degradation, increasing the α-KG titer from 10.08 g/L to 12.21 g/L. Protein engineering of P. mirabilis pm1 and deletion of the α-KG degradation pathway in B. subtilis improved α-KG production over that of previously developed processes.
authors: School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors: Gazi Sakir
authors: GS
authors: Hossain
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors: Jianghua
authors: J
authors: Li
authors: School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332, USA.
authors: Hyun-dong
authors: HD
authors: Shin
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China. Electronic address: longliu@jiangnan.edu.cn.
authors: Long
authors: L
authors: Liu
authors: School of Food Science and Technology, Jiangnan University, Wuxi 214122, China. Electronic address: mwang@jiangnan.edu.cn.
authors: Miao
authors: M
authors: Wang
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China; Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.
authors: Guocheng
authors: G
authors: Du
authors: National Engineering of Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China.
authors: Jian
authors: J
authors: Chen
mesh_terms: D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism*; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D007656:Ketoglutaric Acids / Q000032:analysis / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D011513:Proteus mirabilis / Q000201:enzymology / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D007656:Ketoglutaric Acids; D000619:Aminohydrolases
keywords: Error-prone PCR; Proteus mirabilis; Site-saturation mutagenesis; Whole-cell transformation; l-Amino acid deaminase; α-Ketoglutarate