title: Modular optimization of multi-gene pathways for fumarate production.
abstract: Microbial fumarate production from renewable feedstock is a promising and sustainable alternative to petroleum-based chemical synthesis. Here, we report a modular engineering approach that systematically removed metabolic pathway bottlenecks and led to significant titer improvements in a multi-gene fumarate metabolic pathway. On the basis of central pathway architecture, yeast fumarate biosynthesis was re-cast into three modules: reduction module, oxidation module, and byproduct module. We targeted reduction module and oxidation module to the cytoplasm and the mitochondria, respectively. Combinatorially tuning pathway efficiency by constructing protein fusions RoMDH-P160A and KGD2-SUCLG2 and optimizing metabolic balance by controlling genes RoPYC, RoMDH-P160A, KGD2-SUCLG2 and SDH1 expression strengths led to significantly improved fumarate production (20.46 g/L). In byproduct module, synthetizing DNA-guided scaffolds and designing sRNA switchs enabled further production improvement up to 33.13 g/L. These results suggest that modular pathway engineering can systematically optimize biosynthesis pathways to enable an efficient production of fumarate.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, 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, 1800 Lihu Road, 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: Pan
authors.initials: P
authors.lastname: Zhu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, 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. Electronic address: mingll@jiangnan.edu.cn.
authors.forename: Liming
authors.initials: L
authors.lastname: Liu
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005650:Fumarates / Q000302:isolation & purification / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D024861:Genetic Enhancement / Q000379:methods; D060847:Metabolic Engineering / Q000379:methods*; D064688:Metabolic Flux Analysis / Q000379:methods; D005810:Multigene Family / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*
chemical_list: D029968:Escherichia coli Proteins; D005650:Fumarates; D005656:Fungal Proteins; C032005:fumaric acid
keywords: Fumarate; Modular pathway engineering; Multi-gene pathway; Synthetic biology