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Title: CLONING OF CELLOBIOSE PHOSPHOENOLPYRUVATE-DEPENDENT PHOSPHOTRANSFERASE GENES: FUNCTIONAL EXPRESSION IN RECOMBINANT ESCHERICHIA COLI AND IDENTIFICATION OF A PUTATIVE BINDING REGION FOR DISACCHARIDES

Author
item LAI, XIAOKUANG - UNIV OF FLORIDA
item DAVIS, F - UNIV OF FLORIDA
item Hespell, Robert
item INGRAM, LONNIE - UNIV OF FLORIDA

Submitted to: Journal of Applied & Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/4/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: About one-half of the weight of plant materials such as grasses, straws, or woods is in the form of cellulose. Recent interest in the development of bioprocesses for conversion of plant material into fuel ethanol has focused on microbial enzymes that break down cellulose to various sugar products. In this report, several microorganisms were examined for the presence of specific genes needed for rapid uptake of one of the sugars. Genes from one bacterial species were isolated and characterized. These genes have unique properties useful for understanding sugar uptake mechanisms. In the future, we may be able to transfer these and other genes to genetically engineered microorganisms so that they can ferment a variety of sugars to fuel ethanol.

Technical Abstract: Genomic libraries from nine cellobiose-metabolizing bacteria were screened for cellobiose utilization. Positive clones were recovered from six libraries, all of which encode phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) proteins. Clones from Bacillus subtilis, Butyrivibrio fibrisolvens, and Klebsiella oxytoca allowed the growth of recombinant Escherichia coli in cellobiose-M9 minimal medium. The K. oxytoca clone, pLOI1906, exhibited an unusually broad substrate range (cellobiose, arbutin, salicin, and methylumbelliferyl derivatives of glucose, cellobiose, mannose, and xylose) and was sequenced. The insert in this plasmid encoded the carboxy-terminal region of a putative regulatory protein, cellobiose permease (single polypeptide), and phospho-beta-glucosidase, which appear to form an operon (casRAB). Subclones allowed both casA and casB to be expressed independently, as evidenced by in vitro complementation. An analysis of the translated sequences from the EIIC domains of cellobiose, aryl-beta-glucoside, and other disaccharide permeases allowed the identification of a 50-amino-acid conserved region. A disaccharide consensus sequence is proposed for the most conserved segment (13 amino acids), which may represent part of the EIIC active site for binding and phosphorylation.