No abstract

Previous studies have shown an unexpectedly high nutrient requirement for efficient ethanol production by ethanologenic recombinants of Escherichia coli B such as LY01 which contain chromosomally integrated Zymomonas mobilis genes (pdc,adhB) encoding the ethanol pathway. The basis for this requirement has been identified as a media-dependent effect on the expression of the Z. mobilis genes rather than a nutritional limitation. Ethanol production was substantially increased without additional nutrients simply by increasing the level of pyruvate decarboxylase activity. This was accomplished by adding a multicopy plasmid containing pdc alone (but not adhB alone) to strain LY01, and by adding multicopy plasmids which express pdc and adhB from strong promoters. New strong promoters were isolated from random fragments of Z. mobilis DNA and characterized but were not used to construct integrated biocatalysts. These promoters contained regions resembling recognition sites for 3 different E. coli sigma factors: sigma(70), sigma(38), and sigma(28). The most effective plasmid-based promoters for fermentation were recognized by multiple sigma factors, expressed both pdc and adhB at high levels, and produced ethanol efficiently while allowing up to 80% reduction in complex nutrients as compared to LY01. The ability to utilize multiple sigma factors may be advantageous to maintain the high levels of PDC and ADH needed for efficient ethanol production throughout batch fermentation. From this work, we propose that the activation of biosynthetic genes in nutrient-poor media creates a biosynthetic burden that reduces the expression of chromosomal pdc and adhB by competing for transcriptional and translational machinery. This reduced expression can be viewed as analogous to the effect of plasmids (plasmid burden) on the expression of native chromosomal genes.

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-␤-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-␤-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.

The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals. One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation. By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental ␤-glucosidase was previously eliminated. In the current study, this approach has been extended by adding genes encoding endoglucanase activities. Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression. Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth. During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2. Most of the beneficial contribution was attributed to CelY rather than to CelZ. These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations.The production of fuels and chemicals from cellulosic substrates using microbial biocatalysts offers the potential to reduce the use of fossil fuels and improve the environment (11,18,22,(29)(30)(31). However, the low activity of cellulase enzymes (15, 30) and the resulting cost of hydrolysis represent major barriers for the use of lignocellulosic feedstocks for fuels, bulk chemicals, and plastics (15,29,30,41). The enzymatic hydrolysis of cellulose has been extensively studied but remains poorly understood (2,3,5,21,32). Hydrolysis results from the combined action of at least three classes of ␤-1,4-glucanase activities (2, 5, 26, 28): endoglucanases, exoglucanases, and cellobiases that complete the hydrolysis of soluble products (from two to six glucosyl residues) to monomeric glucose. Cellobiose and soluble cellobiosides are potent competitive inhibitors of endo-and exoglucanases that must be removed to prevent autoinhibition. Fungi such as Trichoderma reesei (19, 27, 39) secrete soluble ␤-1,4-glucosidases (cellobiase) to complete the hydrolysis process. In some bacteria, however, hydrolysis of soluble cellobiosides is completed intracellularly. Cellobiose and cellotriose are actively transported by a ␤-glucoside-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) (18,20,40). Intracellular cellobiose-phosphate is subsequently hydrolyzed to glucose and glucose-6-phosphate by a cytoplasmic phospho-␤-glucosidase for immediate entry into glycolysis.One approach to reduce the costs of cellulase enzymes for bioprocessing is to deve...