Constracting expression vector with The ability to down regulate the Acctate Prodution with Antisence RNA in E.Coli

Subject Areas : Biotechnology and Applied Microbiology journal

Received: 2017-11-05 Accepted : 2017-11-05 Published : 2013-02-19

Keywords: antisense RNA, acetate, recombinant protein, Escherichia coli, ACK, PTA,

Abstract :

A problem with the use of Escherichia coli to produce foreign proteins is that although endogenously produced acetate is physiologically indispensable, it inhibits protein expression. Here we employed an antisense RNA strategy as an elaborate metabolic engineering tool to partially block biosynthesis of two major acetate pathway enzymes, phosphotransacetylase (PTA) and acetate kinase (ACK). A recombinant plasmid containing antisense genes targeting both of pta and ackA were constructed. We used RT-PCR technique and enzyme assay for measurement of mRNA transcription level and enzyme activity. It has been found that the antisense method partially reduced mRNA levels of target enzyme genes and specific activity of their proteins. Observation of these effects demonstrating that, the antisense strategy can be applied successfully to practical recombinant protein production processes.