Abstract:[Background] When Escherichia coli K-12 mutant S17-3 carries high copy number plasmid pBHR68 expressing the gene cluster for synthesis of poly-3-hydroxybutyrate (PHB), it presents some special physiological features, such as high-density growth, low pH tolerance, and high yield of colonic acid (CA) at low pH. [Objective] To systematically explore the molecular mechanism of high-density growth that related to strain (E. coli S17-3) and plasmid (pBHR68), and to reveal the coupling mechanism between the high-density growth and the anabolism of PHB or CA. [Methods] We dissected the plasmid composition and the gene structure for CA synthesis pathway that might have caused high-density growth, searched for key mutations by multiple genome alignment analysis. The transcriptomic data of E. coli S17-3 and its growth performances in different media were scrutinized, the functions of the verified genes were analyzed by gene knockout. [Results] The high-density growth of E. coli S17-3 was related to the overexpression of the whole gene cluster for PHB synthesis, as well as the multi-site mutations within rhsA; RcsA may play as a key factor that not only regulates the production of CA but also mediates the channeling of carbon flow during E. coli S17-3's growth; The knockout of key enzymes in CA operon led to the increase of biomass at low pH; In addition, the high-density growth of E. coli S17-3/pBHR68 may also be related to lactose metabolism, since lacZ null mutation abolished the capability of CA synthesis as well as the high-density growth. [Conclusion] In this study, we analyzed the factors that may link to the high-density growth of E. coli S17-3, and found out some important clues which laid a research foundation for studies to remold E. coli S17-3 as a chassis cell for production of oligosaccharides.
