title: Genome engineering Escherichia coli for L-DOPA overproduction from glucose.
abstract: Genome engineering has become a powerful tool for creating useful strains in research and industry. In this study, we applied singleplex and multiplex genome engineering approaches to construct an E. coli strain for the production of L-DOPA from glucose. We first used the singleplex genome engineering approach to create an L-DOPA-producing strain, E. coli DOPA-1, by deleting transcriptional regulators (tyrosine repressor tyrR and carbon storage regulator A csrA), altering glucose transport from the phosphotransferase system (PTS) to ATP-dependent uptake and the phosphorylation system overexpressing galactose permease gene (galP) and glucokinase gene (glk), knocking out glucose-6-phosphate dehydrogenase gene (zwf) and prephenate dehydratase and its leader peptide genes (pheLA) and integrating the fusion protein chimera of the downstream pathway of chorismate. Then, multiplex automated genome engineering (MAGE) based on 23 targets was used to further improve L-DOPA production. The resulting strain, E. coli DOPA-30N, produced 8.67 g/L of L-DOPA in 60 h in a 5 L fed-batch fermentation. This titer is the highest achieved in metabolically engineered E. coli having PHAH activity from glucose.
authors.affiliation: Biotechnology Research Center and Biomedical Center, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Wei
authors.affiliation: Biotechnology Research Center and Biomedical Center, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Bi-Yan
authors.initials: BY
authors.lastname: Cheng
authors.affiliation: Biotechnology Research Center and Biomedical Center, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China.
authors.forename: Jian-Zhong
authors.initials: JZ
authors.lastname: Liu
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D019149:Bioreactors / Q000382:microbiology*; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D005941:Glucokinase / Q000235:genetics; D005947:Glucose / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics; D007980:Levodopa / Q000096:biosynthesis* / Q000235:genetics; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis / Q000235:genetics; D033902:Periplasmic Binding Proteins / Q000096:biosynthesis / Q000235:genetics; D011302:Prephenate Dehydratase / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; C093587:CsrA protein, E coli; D029968:Escherichia coli Proteins; D009004:Monosaccharide Transport Proteins; D033902:Periplasmic Binding Proteins; D016601:RNA-Binding Proteins; D012097:Repressor Proteins; C069256:TyrR protein, E coli; C023550:galactose-binding protein; D007980:Levodopa; D005954:Glucosephosphate Dehydrogenase; C096446:GlkA protein, Bacteria; D005941:Glucokinase; D011302:Prephenate Dehydratase; D005947:Glucose