title: Mutagenesis of conserved active site residues of dihydrolipoamide succinyltransferase enhances the accumulation of α-ketoglutarate in Yarrowia lipolytica.
abstract: α-Ketoglutarate (α-KG) is an important intermediate in the tricarboxylic acid cycle and has broad applications. The mitochondrial ketoglutarate dehydrogenase (KGDH) complex catalyzes the oxidation of α-KG to succinyl-CoA. Disruption of KGDH, which may enhance the accumulation of α-KG theoretically, was found to be lethal to obligate aerobic cells. In this study, individual overexpression of dihydrolipoamide succinyltransferase (DLST), which serves as the inner core of KGDH, decreased overall activity of the enzyme complex. Furthermore, two conserved active site residues of DLST, His419, and Asp423 were identified. In order to determine whether these residues are engaged in enzyme reaction or not, these two conserved residues were individually mutated. Analysis of the kinetic parameters of the enzyme variants provided evidence that the catalytic reaction of DLST depended on residues His419 and Asp423. Overexpression of mutated DLST not only impaired balanced assembly of KGDH, but also disrupted the catalytic integrity of the enzyme complex. Replacement of the Asp423 residue by glutamate increased extracellular α-KG by 40 % to 50 g L(-1) in mutant strain. These observations uncovered catalytic roles of two conserved active site residues of DLST and provided clues for effective metabolic strategies for rational carbon flux control for the enhanced production of α-KG and related bioproducts.
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Hongwei
authors.initials: H
authors.lastname: Guo
authors.affiliation: UMR1238 Microbiologie et Génétique Moléculaire, INRA/CNRS/AgroPan's Tech, CBAI, BP 01, 78850, Thiverval-Grignon, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Madzak
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China.
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhoujw1982@jiangnan.edu.cn.|Synergetic Innovation Center of Food Safety and Nutrition, 1800 Lihu Road, Wuxi, 214122, Jiangsu, China. zhoujw1982@jiangnan.edu.cn.
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhou
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000217:Acyltransferases / Q000235:genetics* / Q000378:metabolism; D057486:Carbon Cycle; D020134:Catalytic Domain / Q000235:genetics*; D002952:Citric Acid Cycle; D007655:Ketoglutarate Dehydrogenase Complex / Q000235:genetics / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism*; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
chemical_list: D000214:Acyl Coenzyme A; D007656:Ketoglutaric Acids; C012046:succinyl-coenzyme A; D007655:Ketoglutarate Dehydrogenase Complex; D000217:Acyltransferases; C021515:dihydrolipoamide succinyltransferase
keywords: Ketoglutarate dehydrogenase complex; Non-conventional yeast; Organic acids; Site-directed mutagenesis