Co-fermentation of xylose and cellobiose by an engineered Saccharomyces cerevisiae.

We have integrated and coordinately expressed in Saccharomyces cerevisiae a xylose isomerase and cellobiose phosphorylase from Ruminococcus flavefaciens that enables fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. The native xylose isomerase was active in cell-free extracts from yeast transformants containing a single integrated copy of the gene. We improved the activity of the enzyme and its affinity for xylose by modifications to the 5'-end of the gene, site-directed mutagenesis, and codon optimization. The improved enzyme, designated RfCO*, demonstrated a 4.8-fold increase in activity compared to the native xylose isomerase, with a K(m) for xylose of 66.7 mM and a specific activity of 1.41 µmol/min/mg. In comparison, the native xylose isomerase was found to have a K(m) for xylose of 117.1 mM and a specific activity of 0.29 µmol/min/mg. The coordinate over-expression of RfCO* along with cellobiose phosphorylase, cellobiose transporters, the endogenous genes GAL2 and XKS1, and disruption of the native PHO13 and GRE3 genes allowed the fermentation of glucose, xylose, and cellobiose under completely anaerobic conditions. Interestingly, this strain was unable to utilize xylose or cellobiose as a sole carbon source for growth under anaerobic conditions, thus minimizing yield loss to biomass formation and maximizing ethanol yield during their fermentation.

Biosynthesis and Regulation of the Branched-Chain Amino Acids†.

This review focuses on more recent studies concerning the systems biology of branched-chain amino acid biosynthesis, that is, the pathway-specific and global metabolic and genetic regulatory networks that enable the cell to adjust branched-chain amino acid synthesis rates to changing nutritional and environmental conditions. It begins with an overview of the enzymatic steps and metabolic regulatory mechanisms of the pathways and descriptions of the genetic regulatory mechanisms of the individual operons of the isoleucine-leucine-valine (ilv) regulon. This is followed by more-detailed discussions of recent evidence that global control mechanisms that coordinate the expression of the operons of this regulon with one another and the growth conditions of the cell are mediated by changes in DNA supercoiling that occur in response to changes in cellular energy charge levels that, in turn, are modulated by nutrient and environmental signals. Since the parallel pathways for isoleucine and valine biosynthesis are catalyzed by a single set of enzymes, and because the AHAS-catalyzed reaction is the first step specific for valine biosynthesis but the second step of isoleucine biosynthesis, valine inhibition of a single enzyme for this enzymatic step might compromise the cell for isoleucine or result in the accumulation of toxic intermediates. The operon-specific regulatory mechanisms of the operons of the ilv regulon are discussed in the review followed by a consideration and brief review of global regulatory proteins such as integration host factor (IHF), Lrp, and CAP (CRP) that affect the expression of these operons.

Global gene expression profiling in Escherichia coli K12: effects of oxygen availability and ArcA.

The ArcAB two-component system of Escherichia coli regulates the aerobic/anaerobic expression of genes that encode respiratory proteins whose synthesis is coordinated during aerobic/anaerobic cell growth. A genomic study of E. coli was undertaken to identify other potential targets of oxygen and ArcA regulation. A group of 175 genes generated from this study and our previous study on oxygen regulation (Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, G. W., and Gunsalus, R. P. (2003) J. Biol. Chem. 278, 29837-29855), called our gold standard gene set, have p values <0.00013 and a posterior probability of differential expression value of 0.99. These 175 genes clustered into eight expression patterns and represent genes involved in a large number of cell processes, including small molecule biosynthesis, macromolecular synthesis, and aerobic/anaerobic respiration and fermentation. In addition, 119 of these 175 genes were also identified in our previous study of the fnr allele. A MEME/weight matrix method was used to identify a new putative ArcA-binding site for all genes of the E. coli genome. 16 new sites were identified upstream of genes in our gold standard set. The strict statistical analyses that we have performed on our data allow us to predict that 1139 genes in the E. coli genome are regulated either directly or indirectly by the ArcA protein with a 99% confidence level.