Effects of ackA, pta and poxB inhibition by antisense RNA on acetate excretion and recombinant beta interferon expression in Escherichia coli

Abstract

Introduction: Escherichia coli (E.coli) is one of the most widely used hosts for the production of recombinant proteins. The main problem in getting high product yields and productivity is the accumulation of acetic acid (acetate) as an unwanted metabolic by-product. In this study, an antisense-based strategy as a metabolic engineering approach was employed to hamper the acetate excretion problem. Materials and Methods: A recombinant plasmid containing the encoding genes for human recombinant interferon beta (rhINF-β) and three antisense oligonucleotides against acetate kinase, phosphotransacetylase and pyruvate oxidase B was designed. The effects of recombinant plasmid on the cell physiology, rhINF-β production and acetate excretion were studied in E. coli. Results: The mRNA levels of the targeted enzymes were lowered in antisense-regulated cells compared to the control cells. The concentration of acetate in culture media was decreased due to the constructed plasmid. The expression of antisense RNA did not affect the cell growth, negatively. Besides, the rhINF-β production was enhanced in antisense-regulated strain compared to the control plasmid without antisense genes. Conclusion: Application of an antisense strategy on the acetate pathway was successful in metabolically engineering E. coli. This enhancement of production yield by antisense technology suggests that this strategy may be successfully applied to high cell density fermentations of E. coli expression system to overexpress other recombinant proteins.