Metabolic Engineering Of Escherichia Coli Towards Enhancement Ofacid Tolerance And Automatic Switch

Escherichia coli as an important model species has been widely applied to biological production of organic acids and pharmaceuticals. However, the organic acid fermentation is usually often subject to low productivity, yield and purity due to the formation of byproducts and the low tolerance of cells to acids. Organic acids are toxic to many microorganisms which serve as platform strains with economical importance. The reason behind the toxicity may be two-fold. One is the changing pH value which inhibits cell growth. Another may be metabolites which also affect cell metabolism. To improve the E. coli tolerance to acids, synthetic biology approaches were recruited to remodel E. coli to be an appropriate platform host for production of organic acids. Below are the main achievements in this study.(1) To determine which genes confer tolerance to propionic acid, different genes were overexpressed in E. coli BL21. Both pgi encoding glucose-6-phosphate isomerase and rpoS encoding RNA polymerase sigma factor showed high tolerance to propionic acid. Notably, the recombinant strain overexpressing rpoS grew actively under mild (pH at 5.12) or moderate (pH at 4.62) acid conditions. The minimum inhibitory concentration of propionic acid to E. coli (pET-rpoS) was 7.81 mg/mL, which is 2 fold higher compared with wild type. Additionally, under shake-flask conditions, the recombinant strain overexpressing rpoS showed faster growth relative to wild type. In view of above results, we reasoned that the mechanism underlying acid tolerance may be ascribed to transcription and glucose metabolism.(2) Acyl-homoserine lactone-dependent quorum sensing (QS) was first uncovered in Vibrio fischeri. QS controls the biosynthesis of numerous intra-and extracellular metabolites. Engineering predetermined functionality in living cells represents a focus in the boomingsynthetic biology. Here we constructed two plasmids for automatic switch, including pET-Pluxl-ldhA for producing lactic acid when cell density reaches a threshold, and a plasmid pUC-luxI-luxR for response to cell density. The two plasmids were transformed into E. coli BL21 ? ldhA whose lactate dehydrogenase was knocked out. This automatic switch allowed the strain automatic switch from growth state to production of lactic acid without addition of any inducer during the fermentation. Consequently, it could grow under lactic acid at 1.233 g/L, which is 1.72 fold higher than the control E. coli BL21 ? ldhA.(3) Since phosphoenolpyruvate carboxylase is a key enzyme in rTCA, its coding gene was cloned by PCR and ligated to vector pET-28a. To improve the growth of E. coli carring two plasmids, three genes including pncB, nadD and nadE were cloned into vector pUC19. These genes respectively encodenicotinate phosphoribosyltransferase, nicotinic acid mononucleotide adenylyltransferase and NAD synthetase in E. coli BL21. Compared with control strain, this recombinant E. coli overexpressing above three enzymes produced more NAD+, which in turn benefited cell growth. In addition, this recombinant E. coli produced 0.9 g/L succinic acid and 5.4 mg/L fumaric acid.