Ethanol limits E. coli growth kinetics and does not increase rChiC protein yield

Abstract

Chitin is an abundant polysaccharide which is an integral structural component in exoskeletons of plant-harming insects. Chitin can be degraded into its carbon and nitrogen-based components to be recycled by chitinase secreting organisms. Pseudomonas aeruginosa is a chitinolytic soil microbe known to express chitinase C (ChiC). ChiC has been a target for biopesticide design as ChiC has the potential to kill insects without the polluting effects of traditional chemical pesticides. Previous researchers have successfully cloned chiC from P. aeruginosa PAO1 into the pET-28a vector to generate a recombinant ChiC (rChiC) expression plasmid, pM3CRYY. This paper aims to optimize the yield of rChiC in Escherichia coli BL21(DE3) by inducing a heat shock response through ethanol supplementation. Growth curves generated with E. coli grown under 1%, 3% and 5% ethanol conditions revealed that ethanol decreases growth parameters. Then, SDS-PAGE analysis of pM3CRYY transformed E. coli BL21 (DE3) whole cell lysates showed increased rChiC expression under 1% ethanol conditions, but not under 3% or 5% ethanol conditions. Furthermore, western blot analysis probing for DnaK, which is a known heat-shock associated molecular chaperone, revealed that DnaK is only upregulated when induced with 5% ethanol. Although our findings suggest 1% ethanol increases rChiC yield, further research should be done on understanding its mechanism, optimizing growth conditions and harvesting times, and probing for other heat shock proteins. Altogether, this study offers insight into the use of ethanol as a heat-shock inducing agent in E.coli BL21 (DE3) that could potentially be used to optimize rChiC protein yield in large-scale production for industrial use.