Inhibition of ackA and pta genes using two specific antisense RNAs reduced acetate accumulation in batch fermentation of E. coli BL21 (DE3)

Abstract

Expression of foreign proteins in E. coli is normally inhibited by exogenous production of acetate. To overcome<br />this problem, various strategies have been proposed and tested to reduce the extent of acetate accumulation.<br />Although these strategies can improve the outcome, the implementation of their proposed techniques<br />is not practical. Because to achieve optimal results, it requires extremely tight control conditions<br />and the actual cost is very high. Furthermore, a simple knockout mutation of the target metabolic pathway<br />would not be appropriate because the acetate pathway plays an important physiological role in E. coli. In this<br />study, we employed an antisense RNA strategy as an elaborated metabolic engineering tool to partially block<br />biosynthesis of two major acetate pathway enzymes, acetate kinase (ACK) and phosphotransacetylase<br />(PTA). The fragments of antisense cassette were cloned sequentially in pBluescriptsk+ and completed<br />cassette subcloned in pLT10T3. The function of this cassette was evaluated with RT-PCR and ACK and<br />PTA assay. The effect of cassette on cell physiology was monitored by determination of optical density, glucose consumption and acetate production. We found that the antisense method partially reduced mRNA levels of the target genes, lowered the concentration of acetate in culture media and increased growth rate<br />and final cell density in antisense-regulated strain. This strategy could provide us with a useful, inexpensive<br />and practical tool to achieve a large-scale protein production system.<br /><br />