title: Activation of glycerol metabolic pathway by evolutionary engineering of Rhizopus oryzae to strengthen the fumaric acid biosynthesis from crude glycerol.
abstract: Rhizopus oryzae is strictly inhibited by biodiesel-based by-product crude glycerol, which results in low fumaric acid production. In this study, evolutionary engineering was employed to activate the glycerol utilization pathway for fumaric acid production. An evolved strain G80 was selected, which could tolerate and utilize high concentrations of crude glycerol to produce 14.9g/L fumaric acid with a yield of 0.248g/g glycerol. Key enzymes activity analysis revealed that the evolved strain displayed a significant upregulation in glycerol dissimilation, pyruvate consumption and reductive tricarboxylic acid pathways, compared with the parent strain. Subsequently, intracellular metabolic profiling analysis showed that amino acid biosynthesis, tricarboxylic acid cycle, fatty acid and stress response metabolites accounted for metabolic difference between two strains. Moreover, a glycerol fed-batch strategy was optimized to obtain the highest fumaric acid production of 25.5g/L, significantly increased by 20.9-fold than that of the parent strain of 1.2g/L.
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China; Tianjin Key Laboratory of Microbial Functional Genomics, Tianjin 300457, PR China. Electronic address: huangdi@nankai.edu.cn.
authors.forename: Di
authors.initials: D
authors.lastname: Huang
authors.affiliation: TEDA Institute of Biological Sciences and Biotechnology, Nankai University, TEDA, Tianjin 300457, PR China; Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Tianjin 300071, PR China.
authors.forename: Ru
authors.initials: R
authors.lastname: Wang
authors.affiliation: Key Laboratory of System Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, PR China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Du
authors.affiliation: Key Laboratory of System Bioengineering, Ministry of Education, Tianjin University, Tianjin 300072, PR China.
authors.forename: Guanyi
authors.initials: G
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing 100190, PR China; University of Chinese Academy of Sciences, Beijing 100039, PR China.
authors.forename: Menglei
authors.initials: M
authors.lastname: Xia
mesh_terms: D056804:Biofuels; D005075:Biological Evolution; D005285:Fermentation; D005650:Fumarates / Q000378:metabolism*; D005990:Glycerol / Q000378:metabolism*; D053858:Metabolic Networks and Pathways / Q000235:genetics; D055442:Metabolome; D012233:Rhizopus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
chemical_list: D056804:Biofuels; D005650:Fumarates; C032005:fumaric acid; D005990:Glycerol
keywords: Crude glycerol; Evolutionary engineering; Fumaric acid; Intracellular metabolic profiling; Rhizopus oryzae