Cloning and expression of a β-glucosidase gene from Xanthomonas albilineans in Escherichia coli and Zymomonas mobilis

Abstract

A pKT230 gene bank of the genome from the cellulolytic bacterium, Xanthomonas albilineans, was screened for the ability to convert Escherichia coli to cellobiose utilization. The β-glucosidase enzyme from one such clone which harboured the plasmid, pNSW904, and which grew efficiently on cellobiose was partially characterised. Transfer of the β-glucosidase gene to Zymomonas mobilis strains ZM6 and ZM6100 was achieved by subcloning the β-glucosidase gene onto the small broad-host-range plasmid, pRK404, followed by three-way mating involving the helper plasmid, pRK2013. The recombinant strains from ZM6 and ZM6100 were designated ZM6901 and ZM6902, respectively. β-Glucosidase was produced by both recombinant strains. The enzyme levels in ZM6901 and ZM6902 were respectively 7.5% and 10% of those expressed in Escherichia coli. The time course of enzymatic hydrolysis of cellobiose was followed using thin layer chromatography and showed that cellobiose was consumed and glucose was formed simultaneously by cell extracts of the recombinant Zymomonas strains and the glucose produced was then further metabolized in ZM6901. Gas chromatography of crude cell extracts of ZM6901 indicated that after three days incubation 13.3 mM ethanol could be produced from 5 mM cellobiose. Intact cells of ZM6901 were capable of producing 132 mM ethanol from 110 mM cellobiose after 11 days.