Investigation of bacterial cellulose production in genetically modified Escherichia coli

Abstract

Cellulose is the major biopolymer on earth with tremendous economic importance as it has been utilised in a multitude of industrial applications including tissueengineering products, composite materials and electronics. It exhibits outstanding physical and mechanical properties when compared to plant-based cellulose. Although A. xylinum is the most efficient producer of bacterial cellulose (BC), its long doubling time results in insufficient yields and high cost. In this study, a novel and functional BC production system was developed by recombinant DNA technology. The simultaneous expression of bacterial cellulose synthase operon (bcsABCD) and its upstream operon (cmcax and ccpAx) was achieved by pBCS and pCDF, respectively. Three different Escherichia coli strains were utilised as host microorganisms: E. coli BL21 (DE3), E. coli HMS174 (DE3) and E. coli C41 (DE3). It was verified that bcsABCD and the upstream operon were successfully cloned and expressed in E. coli strains. Fermentation of genetically modified (GM) strains was conducted at various IPTG concentrations (0.025, 0.05, 0.1, 0.2, 0.5 and 1.0 mM) and various temperatures (22, 30, and 37 °C). BC production was achieved by genetically modified E. coli HMS174 (DE3) in the presence of 0.025 mM IPTG at 22°C. GM E. coli C41 (DE3) accomplished the production when IPTG supplement was lower than 0.2 mM at 22°C or 30°C. The products were characterised by SEM and FTIR, which exhibited that morphology of product was stain-specific. Finally a dynamic mathematical model was developed to design a fed-batch system capturing characteristics incorporating acetate inhibition and cell death, which allowed predicting glucose consumption, acetate production and induction time for batch cultures, resulted in a volumetric productivity of 1.7 mg/L.h. In conclusion, this thesis reports the development of a novel BC production system by creating valuable cellulose-producing E. coli strains, resulting in a reproducible and stable recombinant expression system for potential improvement of BC.