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:: Volume 22, Issue 3 (Summer 2020) ::
Koomesh 2020, 22(3): 505-517 Back to browse issues page
Effects of ackA, pta and poxB inhibition by antisense RNA on acetate excretion and recombinant beta interferon expression in Escherichia coli
Mohammad Hossein Morowvat
Abstract:   (1203 Views)
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.
Keywords: Escherichia Coli, Recombinant Proteins, Acetic Acid, Antisense Technology, Interferon Beta
Full-Text [PDF 1172 kb]   (148 Downloads)    
Type of Study: Research | Subject: General
Received: 2019/07/5 | Accepted: 2019/11/24 | Published: 2020/05/13
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Morowvat M H. Effects of ackA, pta and poxB inhibition by antisense RNA on acetate excretion and recombinant beta interferon expression in Escherichia coli. Koomesh. 2020; 22 (3) :505-517
URL: http://koomeshjournal.semums.ac.ir/article-1-5766-en.html


Volume 22, Issue 3 (Summer 2020) Back to browse issues page
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