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

Document Type: Research Paper

Authors

1 Biochemistry Group, Faculty of Biological Sciences, Tarbiat Modares University, P.O. Box 1411-5111, Tehran, I.R. Iran.

2 Biochemical Engineering Group, Biotechnology Research Center, P.O. Box 15857-1774, Tehran, I.R.Iran.

3 Neuroscience Research Center, Shahid Beheshti University of Medical Science, P.O. Box 19615-1178, Tehran, I.R. Iran.

Abstract

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

Keywords