Effects of cascaded vgb promoters on poly(hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli grown micro-aerobically

Appl Microbiol Biotechnol. 2014 Dec;98(24):10013-21. doi: 10.1007/s00253-014-6059-y. Epub 2014 Sep 13.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aerobiosis
  • Anaerobiosis
  • Bacterial Outer Membrane Proteins / genetics
  • Bacterial Proteins / genetics*
  • Escherichia coli / genetics
  • Escherichia coli / growth & development*
  • Escherichia coli / metabolism*
  • Escherichia coli Proteins / genetics
  • Gene Deletion
  • Hemeproteins / genetics*
  • Hydroxybutyrates / metabolism*
  • Metabolic Engineering*
  • Polyesters / metabolism*
  • Promoter Regions, Genetic
  • Repressor Proteins / genetics
  • Vitreoscilla / genetics

Substances

  • Bacterial Outer Membrane Proteins
  • Bacterial Proteins
  • Escherichia coli Proteins
  • Hemeproteins
  • Hydroxybutyrates
  • Polyesters
  • Repressor Proteins
  • Vgb protein, Bacteria
  • arcA protein, E coli
  • poly-beta-hydroxybutyrate