title: Engineering rTCA pathway and C4-dicarboxylate transporter for L-malic acid production.
abstract: L-Malic acid is an important component of a vast array of food additives, antioxidants, disincrustants, pharmaceuticals, and cosmetics. Here, we presented a pathway optimization strategy and a transporter modification approach to reconstruct the L-malic acid biosynthesis pathway and transport system, respectively. First, pyruvate carboxylase (pyc) and malate dehydrogenase (mdh) from Aspergillus flavus and Rhizopus oryzae were combinatorially overexpressed to construct the reductive tricarboxylic acid (rTCA) pathway for L-malic acid biosynthesis. Second, the L-malic acid transporter (Spmae) from Schizosaccharomyces pombe was engineered by removing the ubiquitination motification to enhance the L-malic acid efflux system. Finally, the L-malic acid pathway was optimized by controlling gene expression levels, and the final L-malic acid concentration, yield, and productivity were up to 30.25 g L-1, 0.30 g g-1, and 0.32 g L-1h-1in the resulting strain W4209 with CaCO3as a neutralizing agent, respectively. In addition, these corresponding parameters of pyruvic acid remained at 30.75 g L-1, 0.31 g g-1, and 0.32 g L-1h-1, respectively. The metabolic engineering strategy used here will be useful for efficient production of L-malic acid and other chemicals.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, 214122, China.
authors.forename: Xiulai
authors.initials: X
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, 214122, China.
authors.forename: Yuancai
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, 214122, China.
authors.forename: Xiaoxiang
authors.initials: X
authors.lastname: Dong
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, 214122, China.
authors.forename: Guipeng
authors.initials: G
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. mingll@jiangnan.edu.cn.|Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi, 214122, China. mingll@jiangnan.edu.cn.|Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi, 214122, China. mingll@jiangnan.edu.cn.
authors.forename: Liming
authors.initials: L
authors.lastname: Liu
mesh_terms: D001231:Aspergillus flavus / Q000201:enzymology; D053898:Biosynthetic Pathways / Q000235:genetics*; D002952:Citric Acid Cycle / Q000235:genetics* / Q000502:physiology; D029382:Dicarboxylic Acid Transporters / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000235:genetics; D008293:Malates / Q000032:analysis / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D011766:Pyruvate Carboxylase / Q000235:genetics; D019289:Pyruvic Acid / Q000032:analysis / Q000378:metabolism; D012233:Rhizopus / Q000201:enzymology; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012568:Schizosaccharomyces / Q000235:genetics; D054875:Ubiquitination / Q000235:genetics
chemical_list: D029382:Dicarboxylic Acid Transporters; D008293:Malates; C030298:malic acid; D019289:Pyruvic Acid; D008291:Malate Dehydrogenase; D011766:Pyruvate Carboxylase
keywords: C4-Dicarboxylate transporter; L-Malic acid; Pathway optimization; Reductive TCA pathway; Saccharomyces cerevisiae