1-31: Effect of promoter strength on heterologous pyruvate carboxylase gene expression and succinate yield by metabolic engineered E.coli

Succinate produced by fermentation represents a potential route to the production of commodity chemicals from renewable feed stocks. Succinate has drawn much interest because it has been used as a precursor of numerous chemicals including pharmaceuticals and biodegradable polymers. Various metabolic engineering strategies have been studied using different bacterial strains to improve the succinate productivity and yield. In our laboratory, a recombinant E.coli strain, SBS550MG, was created by deactivating adhE, ldhA, ack-pta and iclR genes. Additionally, the strain carries a plasmid, pHL413, encoding pyruvate carboxylase (PYC) from Lactococcus lactis to improve succinate production. To further improve the PYC gene expression and thereby to increase the succinate yield, we studied the PYC expression under the control of Plac, Ptac, Ptrc and PYC native promoter using different copy number plasmids. As compared to pHL413 plasmid, the PYC expression level dropped significantly under Plac, Ptac, Ptrc and native promoter alone, which resulted in decrease in succinate yield. These results prompted us to modify the native PYC promoter by site directed mutagenesis of -10, -35, and spacer regions between -10 to -35 and -36 to -44. We created 9 variants of the native promoter and measured PYC activity as well succinate yield by performing shake flask experiments. MIII variant of native promoter showed a 20% increase in PYC activity and improved succinate yield.