Sunday, July 26, 2009
P101

Design of an Improved Host Platform for the Overexpression of Recombinant Proteins in E.coli

Chaitali Ghosh and K.J. Mukherjee. School of Biotechnology, Jawaharlal Nehru University, New Delhi, 110067, India

 An inverse metabolic engineering approach was adopted where we designed a screening strategy involving the selection of slow growing clones with heightened metabolic activity. A genomic library of E.coli was prepared in  E.coli . It was assumed that some of the clones would carry partial copies of the E.coli genes in the reverse orientation and hence produce antisense RNA. Upon expression these antisense RNAs would tend to down regulate the corresponding gene. Many colonies were screened and few clones were selected which showed a combination of heightened metabolic activity and slow growth phenotype. Sequencing was done to identify these fragments and one of these genes identified was ribB which had got cloned in the reverse orientation. RibB catalyzes biosynthesis of riboflavin.

Since oxygen availability and acetate production are the two major problems associated with HCDC we  constructed an E.coli host wherein the acetate gene has been knocked out and replaced by the vhb gene  leading to strain CG1. CG1 had been shown to grow well under hypoxic conditions, produced less acetate and expressed higher levels of recombinant protein. The effect of ribB knock out (by pRed/ ET recombineering) was checked with this CG1 to see whether this lead to additional improvement. The final modified host CG2 gave higher recombinant protein yields in fed batch cultures compared to the control.  Thus the inverse metabolic strategy allowed us to identify novel modifications which have an additive effect on host performance, thus leading to the design of a better platform for recombinant protein expression.


See more of Poster Session 1
See more of Posters

See more of The Annual Meeting and Exhibition 2009 (July 26 - 30, 2009)