title: Pathway construction and metabolic engineering for fermentative production of ectoine in Escherichia coli.
abstract: Ectoine is a protective agent and stabilizer whose synthesis pathway exclusively exists in select moderate halophiles. A novel established process called "bacterial milking" efficiently synthesized ectoine in moderate halophiles, however, this method places high demands on equipment and is cost prohibitive. In this study, we constructed an ectoine producing strain by introducing the ectoine synthesis pathway into Escherichia coli and improved its production capacity. Firstly, the ectABC gene cluster from Halomonas elongata was introduced into E. coli W3110 and the resultant strain synthesized 4.9g/L ectoine without high osmolarity. Subsequently, thrA encoding the bifunctional aspartokinase/homoserine dehydrogenase was deleted to weaken the competitive l-threonine branch, resulting in an increase of ectoine titer by 109%. Furthermore, a feedback resistant lysC from Corynebacterium glutamicum encoding the aspartate kinase was introduced to complement the enzymatic activity deficiency caused by thrA deletion and a 9% increase of ectoine titer was obtained. Finally, the promoter of ppc that encodes phosphoenolpyruvate carboxylase was replaced by a trc promoter, and iclR, a glyoxylate shunt transcriptional repressor gene, was deleted. The oxaloacetate pool, was thus reinforced and ectoine titer increased by 21%. The final engineered strain ECT05 (pTrcECT, pSTVLysC-CG) produced 25.1g/L ectoine by fed-batch fermentation in low salt concentration with glucose as a carbon source. The specific ectoine production and productivity was 0.8g/g DCW and 0.84gL(-)(1)h(-)(1) respectively. The overall ectoine yield was 0.11g/g of glucose.
authors.affiliation: National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
authors.forename: Yike
authors.initials: Y
authors.lastname: Ning
authors.affiliation: National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
authors.forename: Xuejiao
authors.initials: X
authors.lastname: Wu
authors.affiliation: National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
authors.forename: Chenglin
authors.initials: C
authors.lastname: Zhang
authors.affiliation: National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China.
authors.forename: Qingyang
authors.initials: Q
authors.lastname: Xu
authors.affiliation: National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China. Electronic address: ningch@tust.edu.cn.
authors.forename: Ning
authors.initials: N
authors.lastname: Chen
authors.affiliation: National and Local United Engineering Lab of Metabolic Control Fermentation Technology, Tianjin University of Science and Technology, Tianjin 300457, PR China; Tianjin Engineering Lab of Efficient and Green Amino Acid Manufacture, Tianjin University of Science and Technology, Tianjin 300457, PR China; College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, PR China. Electronic address: xixianxie@tust.edu.cn.
authors.forename: Xixian
authors.initials: X
authors.lastname: Xie
mesh_terms: D000599:Amino Acids, Diamino / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D053898:Biosynthetic Pathways / Q000502:physiology*; D004926:Escherichia coli / Q000502:physiology*; D005285:Fermentation / Q000502:physiology*; D024861:Genetic Enhancement / Q000379:methods*; D005947:Glucose / Q000378:metabolism*; D060847:Metabolic Engineering / Q000379:methods*; D053858:Metabolic Networks and Pathways / Q000502:physiology; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D000599:Amino Acids, Diamino; D011994:Recombinant Proteins; C045628:ectoine; D005947:Glucose
keywords: Aspartokinase; Ectoine; Escherichia coli; Glyoxylate shunt; Phosphoenolpyruvate carboxylase