The Effect Of Over-expressing Anti-micAs On The Tolerance Of E.coli To Ethanol And Pullulanase Production

Escherichia coli is frequently influenced by many stresses in the process of recombinant exogenous protein expression and established various stress signaling systems that sensing and responding to specific stimuli,resulting in a number of viable cells entering the“viable but nonculturable”(VBNC) state, which in turn limits E.coli capacity to function as a cell factory. Recently it has been identified that many small non-coding RNAs(sRNA) have an important role in regulating mRNA translation by influencing the stability and translation efficiency of mRNA, which can cause inhibition or enhancemnet of target genes expression.In this research, in order to block sigma E-mediated cell lysis signal pathway, we have and studied the effect of overexpressing anti-micAs on the tolerance of E.coli to ethanol, Pullulanase production and the trans-coded RNA interference efficiency, by using artificial trans-coded sRNAs of micA(anti-micAs), bprO and omU to silence micA. The main results obtained are as follows:(1) Compared to the control, the over-expression of micA significantly decreased OmpA expression and E.coli viability. and resulted in the colony forming units decreasing by about five orders of magnitude, but had slight effect on the biomass. E.coli stressed by ethanol and over-expression of micA showed aberrant growth, lower viability and cell lysis.(2) Anti-micAs bprO and omU increase the expression levels of OmpA by blocking the inhibition of MicA on OmpA translation, then resulting in increasing of the biomass of E.coli and the colony-forming units. Moreover, the higher the OmpA protein content, the stronger the cell viability, while OmpA levels in E.coli are controlled within a certain range.(3) 1.8% final ethanol concentration had a significantly impact on of E.coli growth. The overexpression of bprO and omU enhanced the tolerance of E.coli to ethanol during the exponential and stabtionary phase.(4) Overexpressing bpr O and omU improved both soluble Pullulanase yield and Pullulanase activity of per OD600 by increasing the quantity of CFU.(5) Compared to bprO, the effect of omU on E.coli physiology was more obvious, indicating that adding nucleotide sequence forming suitable structure to sRNA 5’ end will enhance its stability and efficiency of interference, and protect itself from degradation.This study provides a new method for strain improvement using RNA interference, theoretical and practical basis for the high-levels expression of recombinant heterologous protein, and the referable guidance for designing artifical trans-coded RNA.