Identifying genomic targets for protein over-expression by "omics" analysis of Quiescent Escherichia coli cultures

A cellular stress response is triggered upon induction of recombinant protein expression which feedback inhibits both growth as well as protein synthesis. In order to separate these two effects, it was decided to study "quiescent cultures" which continue to be metabolically active and express recombinant proteins even after growth cessation. The idea was to identify and up-regulate genes which are responsible for protein synthesis in the absence of growth. This would ensure that, even if growth were adversely affected post induction, there would be no attendant reduction in the protein expression capability of the cells. This strategy allowed us to design host strains, which did not grow better post induction but had significantly higher levels of protein expression. A quiescent Escherichia coli culture, which is able to sustain recombinant protein expression in the absence of growth, was analyzed by transcriptomic and proteomic profiling. Many genes involved in carbon utilization, biosynthesis of building blocks and stress protection were found to be up-regulated in the quiescent phase. Analysis of the global regulators showed that fis, which tends to get down-regulated as the cells enter stationary phase, remained up-regulated throughout the non-growing quiescent phase. The downstream genes regulated by fis like carB, fadB, nrfA, narH and queA were also up-regulated in the quiescent phase which could be the reason behind the higher metabolic activity and protein expression ability of these non-growing cells. To test this hypothesis, we co-expressed fis in a control culture expressing recombinant L-asparaginase and observed a significantly higher buildup of L-asparaginase in the culture medium. This work represents an important breakthrough in the design of a superior host platform where a gene not directly associated with protein synthesis was used to generate a phenotype having higher protein expression capability. Many alternative gene targets were also identified which may have beneficial effects on expression ability.