Engineering endoglucanase-secreting strains of ethanologenic Klebsiella oxytoca P2

Recombinant Klebsiella oxytoca P2 was developed as a biocatalyst for the si multaneous saccharification and fermentation (SSF) of cellulose by chromoso mally integrating Zymomonas mobilis genes (pdc, adhB) encoding the ethanol pathway. This strain contains the native ability to transport and metaboliz e cellobiose, eliminating the need to supplement with beta-glucosidase duri ng SSF, To increase the utility of this biocatalyst, we have now chromosoma lly integrated the celZ gene encoding the primary endoglucanase from Erwini a chrysanthemi, This gene was expressed at high levels by replacing the nat ive promoter with a surrogate promoter derived from Z. mobilis DNA. With th e addition of out genes encoding the type II protein secretion system from E. chrysanthemi, over half of the active endoglucanase (EGZ) was secreted i nto the extracellular environment. The two most active strains, SZ2(pCPP200 6) and SZ6(pCPP2006), produced approximately 24 000 IU L-1 of CMCase activi ty, equivalent to 5% of total cellular protein. Recombinant EGZ partially d epolymerized acid-swollen cellulose and allowed the production of small amo unts of ethanol by SZ6(pCPP2006) without the addition of fungal cellulase. However, additional endoglucanase activities will be required to complete t he depolymerization of cellulose into small soluble products which can be e fficiently metabolized to ethanol.