title: Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically.
abstract: Micro-aeration is a situation that will be encountered in bacterial cell growth especially when the saturated dissolved oxygen level cannot match the demand from cells grown to a high density. Therefore, it is desirable to separate aerobic growth and micro-aerobic product formation into two stages using methods including anaerobic or micro-aerobic promoters that are inducible under low aeration intensity. Eleven potential low aeration-inducible promoters were cloned and studied for their induction strengths under micro-aerobic conditions. Of them, Vitreoscilla hemoglobin promoter (P vgb ) was found to be the strongest among all 11 promoters. At the same time, six E. coli hosts harboring poly(R-3-hydroxybutyrate) (PHB) synthesis operon phaCAB were compared for their ability to accumulate poly(hydroxyalkanoates) (PHA). E. coli S17-1 was demonstrated to be the best host achieving a 70 % (mass fraction) PHB in the cell dry weigh (CDW) after 48 h under micro-aerobic growth. Cascaded P vgb repeats (P nvgb ) were investigated for enhanced expression level under micro-aerobic growth. The highest PHA production was obtained when a promoter containing eight cascaded P vgb repeats (P 8vgb ) was used, 5.37 g/l CDW containing 90 % PHB was obtained from recombinant in E. coli S17-1. Cells grew further to 6.30 g/l CDW containing 91 % PHB when oxygen-responsive transcription factor ArcA (arcA) was deleted in the same recombinant E. coli S17-1. This study revealed that vgb promoter containing cascaded P vgb repeats (P 8vgb ) is useful for product formation under low aeration intensity.
authors.affiliation: MOE Key Lab of Bioinformatics, Department of Biological Science and Biotechnology, School of Life Science, Tsinghua-Peking Center for Life Sciences, Tsinghua University, 100084, Beijing, China.
authors.forename: Hong
authors.initials: H
authors.lastname: Wu
authors.forename: Huan
authors.initials: H
authors.lastname: Wang
authors.forename: Jinchun
authors.initials: J
authors.lastname: Chen
authors.forename: Guo-Qiang
authors.initials: GQ
authors.lastname: Chen
mesh_terms: D000332:Aerobiosis; D000693:Anaerobiosis; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D017353:Gene Deletion; D006420:Hemeproteins / Q000235:genetics*; D006885:Hydroxybutyrates / Q000378:metabolism*; D060847:Metabolic Engineering; D011091:Polyesters / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics; D020589:Vitreoscilla / Q000235:genetics; D000094122:Polyhydroxybutyrates
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D006420:Hemeproteins; D006885:Hydroxybutyrates; D011091:Polyesters; D012097:Repressor Proteins; C100771:Vgb protein, Bacteria; C041610:arcA protein, E coli; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates