A synergistic approach of co-expression of catabolite repressor activator (cra) gene and deletion of acetate pathway to enhance the production of recombinant L-Asparaginase-II in Escherichia coli

Abstract

Escherichia coli, a Gram-negative bacteria is extensively utilized as a microbial platform for the recombinant protein production. Various modifications have been introduced to E. coli strains to maximise the production of different types of recombinant proteins. However, a limited amount of research is dedicated to the manipulation of the central carbon metabolic pathways of E. coli. The synthesis of proteins relies on the utilization of carbon and energy precursors from the central carbon metabolic pathways. The high demand for precursors and energy in the biosynthesis of heterologous proteins prompts the cells to readjust their anabolic and catabolic reactions. This readjustment gives rise to another issue, namely the acetate accumulation, which leads to a decline in biomass and production of recombinant protein. Therefore, an improved engineering strategy would involve the manipulation of global regulators and gene sets, encompassing multiple pathways, to enhance carbon assimilation while avoiding the formation of by-products such as acetate. Thus, a synergistic approach has been proposed to boost the productivity of recombinant L-Asparaginase-II, which involves the co-expression of the Catabolite repressor activator (cra) gene, a global regulator of central carbon metabolism, and the deletion of acetate biosynthesis genes. This approach has demonstrated a significant increase in the yield of L-Asparaginase-II in both E. coli BW25113ΔpoxB and BW25113Δpta cultures, where cra is co-expressed using a plasmid-based system. A pelB signal sequence leads to extracellular secretion of recombinant L-Asparaginase-II and it was observed that the productivity of E. coli BW25113ΔpoxB and BW25113Δpta cultures was 129% and 66% higher than wild type BW25113 strains.

Keywords

Recombinant protein / Co-expression / Cra / Acetate / Asparaginase

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