Cloning of bacterial cellulose gene into Zymomonas Mobilis for cellulosic ethanol production

Abstract

Five cellulolytic bacteria viz, Enterobacter cloacae, Pseudomonas aeruginosa, Klebsiella pneumoniae, Pseudomonas fluorescens and Proteus mirabilis were isolated from the gut of phytophagous insects. Cellulase gene from the above five bacteria was cloned separately into E. coli using pET20b(+) plasmid and the cellulase gene containing plasmids such as, pET-cel-Ec, pET-cel-Pa, pET-cel-Pm, pET-cel-Pf and pET-cel-Kp were developed and the cellulase genes were characterized by restriction analysis and DNA sequencing. The cloned cellulase genes were subcloned in pKT 230 plasmid, a stable vector of Zymomonas mobilis, an ethanol fermenting bacterium and the recombinant pKT-cel-Ec, pKT-cel Pa, pKT-cel-Pm, pKT-cel-Pf and pKT-cel- Kp were developed and transformed into Z. mobilis. These recombinant Z. mobilis strains expressing bacterial cellulase gene were used for ethanol production using carboxymethyl cellulose, 4% NaOH pretreated bagasse, 6% NaOH pretreated rice straw and 3% HCl pretreated coirpith as substrates. All the recombinant Z. mobilis strains were found to ferment ethanol from pretreated cellulosic substrates but the recombinant Z. mobilis strain harboring cellulase gene cloned from E. cloacae (pKT-cel-Ec) produced ethanol 12% (using glucose), 5.5% (using CMC), 4% (using 4% NaOH pretreated bagasse), 3.5% (using 6% NaOH pretreated rice straw) and 3% (using 3% HCl pretreated coirpith). The recombinant Z. mobilis strain could be improved further by simultaneous expression of additional cellulase genes and the strains could be used for industrial level ethanol production.