ISOLATION AND MOLECULAR CHARACTERIZATION OF HIGH-PERFORMANCE CELLOBIOSE-FERMENTING SPONTANEOUS MUTANTS OF ETHANOLOGENIC ESCHERICHIA-COLI KO11 CONTAINING THE KLEBSIELLA-OXYTOCA CASAB OPERON

Escherichia coli KO11 was previously constructed to produce ethanol fr om acid hydrolysates of hemicellulose (pentoses and hexoses) by the ch romosomal integration of Zymomonas mobilis genes encoding pyruvate dec arboxylase (pdc) and alcohol dehydrogenase (adhB). Klebsiella oxytoca P2 was constructed in an analogous fashion for the simultaneous saccha rification and fermentation of cellulose and contains PTS enzymes for cellobiose. In this study, KO11 was further engineered for the ferment ation of cellulose by adding the K. oxytoca casAB genes encoding Enzym e IIcellobiose and phospho-beta-glucosidase. Although the two K. oxyto ca genes were well expressed in cloning hosts such as DH5 alpha, both were expressed poorly in E. coli KO11, a derivative of E. coli B. Spon taneous mutants which exhibited more than 15-fold-higher specific acti vities for cellobiose metabolism were isolated. The mutations of these mutants resided in the plasmid rather than the host. Three mutants we re characterized by sequence analysis. All contained similar internal deletions which eliminated the casAB promoter and operator regions and placed the lacZ Shine-Dalgarno region immediately upstream from the c asA Shine-Dalgarno region. KO11 harboring mutant plasmids (pLOI1908, p LOI1909, or pLOI1910) rapidly fermented cellobiose to ethanol, and the yield was more than 90% of the theoretical yield. Two of these strain s were used with commercial cellulase to ferment mixed-caste office pa per to ethanol.