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.

A vector set for systematic metabolic engineering in Saccharomyces cerevisiae.

A set of shuttle vectors was constructed to facilitate expression of genes for metabolic engineering in Saccharomyces cerevisiae. Selectable markers include the URA3, TRP1, MET15, LEU2-d8, HIS3 and CAN1 genes. Differential expression of genes can be achieved as each marker is available on both CEN/ARS- and 2 µ-containing plasmids. Unique restriction sites downstream of TEF1, PGK1 or HXT7-391 promoters and upstream of the CYC1 terminator allow insertion of open-reading frame cassettes for expression. Furthermore, a fragment appropriate for integration into the genome via homologous recombination can be readily generated in a polymerase chain reaction. Vector marker genes are flanked by loxP recognition sites for the CreA recombinase to allow efficient site-specific marker deletion and recycling. Expression and copy number were characterized for representative high- and low-copy vectors carrying the different marker and promoter sequences. Metabolic engineering typically requires the stable introduction of multiple genes and genomic integration is often preferred. This requires an expanded number of stable expression sites relative to standard gene expression studies. This study demonstrated the practicality of polymerase chain reaction amplification of an expression cassette and genetic marker, and subsequent replacement of endogenous retrotransposons by homologous recombination with flanking sequences. Such reporters were expressed comparably to those inserted at standard integration loci. This expands the number of available characterized integration sites and demonstrates that such sites provide a virtually inexhaustible pool of integration targets for stable expression of multiple genes. Together these vectors and expression loci will facilitate combinatorial gene expression for metabolic engineering.

All-codon scanning identifies p53 cancer rescue mutations.

In vitro scanning mutagenesis strategies are valuable tools to identify critical residues in proteins and to generate proteins with modified properties. We describe the fast and simple All-Codon Scanning (ACS) strategy that creates a defined gene library wherein each individual codon within a specific target region is changed into all possible codons with only a single codon change per mutagenesis product. ACS is based on a multiplexed overlapping mutagenesis primer design that saturates only the targeted gene region with single codon changes. We have used ACS to produce single amino-acid changes in small and large regions of the human tumor suppressor protein p53 to identify single amino-acid substitutions that can restore activity to inactive p53 found in human cancers. Single-tube reactions were used to saturate defined 30-nt regions with all possible codon changes. The same technique was used in 20 parallel reactions to scan the 600-bp fragment encoding the entire p53 core domain. Identification of several novel p53 cancer rescue mutations demonstrated the utility of the ACS approach. ACS is a fast, simple and versatile method, which is useful for protein structure-function analyses and protein design or evolution problems.

Predicting positive p53 cancer rescue regions using Most Informative Positive (MIP) active learning.

Many protein engineering problems involve finding mutations that produce proteins with a particular function. Computational active learning is an attractive approach to discover desired biological activities. Traditional active learning techniques have been optimized to iteratively improve classifier accuracy, not to quickly discover biologically significant results. We report here a novel active learning technique, Most Informative Positive (MIP), which is tailored to biological problems because it seeks novel and informative positive results. MIP active learning differs from traditional active learning methods in two ways: (1) it preferentially seeks Positive (functionally active) examples; and (2) it may be effectively extended to select gene regions suitable for high throughput combinatorial mutagenesis. We applied MIP to discover mutations in the tumor suppressor protein p53 that reactivate mutated p53 found in human cancers. This is an important biomedical goal because p53 mutants have been implicated in half of all human cancers, and restoring active p53 in tumors leads to tumor regression. MIP found Positive (cancer rescue) p53 mutants in silico using 33% fewer experiments than traditional non-MIP active learning, with only a minor decrease in classifier accuracy. Applying MIP to in vivo experimentation yielded immediate Positive results. Ten different p53 mutations found in human cancers were paired in silico with all possible single amino acid rescue mutations, from which MIP was used to select a Positive Region predicted to be enriched for p53 cancer rescue mutants. In vivo assays showed that the predicted Positive Region: (1) had significantly more (p<0.01) new strong cancer rescue mutants than control regions (Negative, and non-MIP active learning); (2) had slightly more new strong cancer rescue mutants than an Expert region selected for purely biological considerations; and (3) rescued for the first time the previously unrescuable p53 cancer mutant P152L.

Identification of genes involved in salt adaptation in the archaeon Methanosarcina mazei Gö1 using genome-wide gene expression profiling.

Methanosarcina mazei is a nonhalophilic methanogen that can adapt to 800 mM NaCl. Microarray studies have been used to examine the effect of elevated salinities on the regulation of gene expression in M. mazei. Eighty-four genes of different functional categories, such as solute transport and biosynthesis, Na(+) export, stress response, ion, protein and phosphate transport, metabolic enzymes, regulatory proteins, DNA-modification systems, and cell-surface modulators, were found to be stronger expressed at high salinities. Moreover, 10 genes encoding different metabolic functions including potassium uptake and ATP synthesis were reduced in expression under high salt. The overall expression profiles suggest that M. mazei is able to adapt to high salinities by multiple upregulation of many different cellular functions including protective pathways such as solute transport and biosynthesis, import of phosphate, export of Na(+), and upregulation of pathways for modification of DNA and cell surface architecture.

Quantitative proteomic and microarray analysis of the archaeon Methanosarcina acetivorans grown with acetate versus methanol.

Methanosarcina acetivorans strain C2A is an acetate- and methanol-utilizing methane-producing organism for which the genome, the largest yet sequenced among the Archaea, reveals extensive physiological diversity. LC linear ion trap-FTICR mass spectrometry was employed to analyze acetate- vs methanol-grown cells metabolically labeled with 14N vs 15N, respectively, to obtain quantitative protein abundance ratios. DNA microarray analyses of acetate- vs methanol-grown cells was also performed to determine gene expression ratios. The combined approaches were highly complementary, extending the physiological understanding of growth and methanogenesis. Of the 1081 proteins detected, 255 were > or =3-fold differentially abundant. DNA microarray analysis revealed 410 genes that were > or =2.5-fold differentially expressed of 1972 genes with detected expression. The ratios of differentially abundant proteins were in good agreement with expression ratios of the encoding genes. Taken together, the results suggest several novel roles for electron transport components specific to acetate-grown cells, including two flavodoxins each specific for growth on acetate or methanol. Protein abundance ratios indicated that duplicate CO dehydrogenase/acetyl-CoA complexes function in the conversion of acetate to methane. Surprisingly, the protein abundance and gene expression ratios indicated a general stress response in acetate- vs methanol-grown cells that included enzymes specific for polyphosphate accumulation and oxidative stress. The microarray analysis identified transcripts of several genes encoding regulatory proteins with identity to the PhoU, MarR, GlnK, and TetR families commonly found in the Bacteria domain. An analysis of neighboring genes suggested roles in controlling phosphate metabolism (PhoU), ammonia assimilation (GlnK), and molybdopterin cofactor biosynthesis (TetR). Finally, the proteomic and microarray results suggested roles for two-component regulatory systems specific for each growth substrate.

Global transcriptional analysis of Methanosarcina mazei strain Gö1 under different nitrogen availabilities.

Certain archaeal species can fix molecular nitrogen under nitrogen limiting conditions although little is known about this process at either the genetic or molecular level. To address this on a genome-wide scale, transcriptional analysis was performed on the model methanogen Methanosarcina mazei strain Gö1 using DNA-microarrays. The genomic expression patterns for cells grown under nitrogen fixing conditions versus nitrogen sufficiency (10 mM ammonium) revealed that approximately 5% of all genes are differentially expressed. Besides a small set of genes previously known to be up-regulated under nitrogen limitation, 14 additional genes involved in nitrogen metabolism were identified plus 10 genes encoding potential transcriptional regulators, 13 genes involved in carbon metabolism, 3 genes in general stress response, 8 putative transporter genes, and an additional 21 genes with unknown function. Quantitative reverse transcriptase PCR experiments confirmed the differential expression of a subset of these genes. Promoter analysis revealed a palindromic DNA motif centered nearby the transcriptional start point for several genes up-regulated under nitrogen limitation. A bioinformatics study demonstrated the presence of this motif in the up-stream region of 52 genes genome-wide, the majority of which showed nitrogen dependent differential transcription. We therefore hypothesize that this DNA element is involved in nitrogen control in M. mazei where it may act as a binding site for a regulatory protein.

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.

Heat shock response of Archaeoglobus fulgidus.

The heat shock response of the hyperthermophilic archaeon Archaeoglobus fulgidus strain VC-16 was studied using whole-genome microarrays. On the basis of the resulting expression profiles, approximately 350 of the 2,410 open reading frames (ORFs) (ca. 14%) exhibited increased or decreased transcript abundance. These span a range of cell functions, including energy production, amino acid metabolism, and signal transduction, where the majority are uncharacterized. One ORF called AF1298 was identified that contains a putative helix-turn-helix DNA binding motif. The gene product, HSR1, was expressed and purified from Escherichia coli and was used to characterize specific DNA recognition regions upstream of two A. fulgidus genes, AF1298 and AF1971. The results indicate that AF1298 is autoregulated and is part of an operon with two downstream genes that encode a small heat shock protein, Hsp20, and cdc48, an AAA+ ATPase. The DNase I footprints using HSR1 suggest the presence of a cis-binding motif upstream of AF1298 consisting of CTAAC-N5-GTTAG. Since AF1298 is negatively regulated in response to heat shock and encodes a protein only distantly related to the N-terminal DNA binding domain of Phr of Pyrococcus furiosus, these results suggest that HSR1 and Phr may belong to an evolutionarily diverse protein family involved in heat shock regulation in hyperthermophilic and mesophilic Archaea organisms.

DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates.

Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.

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.

Global gene expression profiling in Escherichia coli K12. The effects of oxygen availability and FNR.

The work presented here is a first step toward a long term goal of systems biology, the complete elucidation of the gene regulatory networks of a living organism. To this end, we have employed DNA microarray technology to identify genes involved in the regulatory networks that facilitate the transition of Escherichia coli cells from an aerobic to an anaerobic growth state. We also report the identification of a subset of these genes that are regulated by a global regulatory protein for anaerobic metabolism, FNR. Analysis of these data demonstrated that the expression of over one-third of the genes expressed during growth under aerobic conditions are altered when E. coli cells transition to an anaerobic growth state, and that the expression of 712 (49%) of these genes are either directly or indirectly modulated by FNR. The results presented here also suggest interactions between the FNR and the leucine-responsive regulatory protein (Lrp) regulatory networks. Because computational methods to analyze and interpret high dimensional DNA microarray data are still at an early stage, and because basic issues of data analysis are still being sorted out, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence. In particular, we describe an approach for identifying gene expression patterns (clusters) obtained from multiple perturbation experiments based on a subset of genes that exhibit high probability for differential expression values.

A chromosomal ars operon homologue of Pseudomonas aeruginosa confers increased resistance to arsenic and antimony in Escherichia coli.

Operons encoding homologous arsenic-resistance determinants (ars) have been discovered in bacterial plasmids from Gram-positive and Gram-negative organisms, as well as in the Escherichia coli chromosome. However, evidence for this arsenic-resistance determinant in the medically and environmentally important bacterial species Pseudomonas aeruginosa is conflicting. Here the identification of a P. aeruginosa chromosomal ars operon homologue via cloning and complementation of an E. coli ars mutant is reported. The P. aeruginosa chromosomal ars operon contains three potential ORFs encoding proteins with significant sequence similarity to those encoded by the arsR, arsB and arsC genes of the plasmid-based and E. coli chromosomal ars operons. The cloned P. aeruginosa chromosomal ars operon confers augmented resistance to arsenic and antimony oxyanions in an E. coli arsB mutant and in wild-type P. aeruginosa. Expression of the operon was induced by arsenite at the mRNA level. DNA sequences homologous with this operon were detected in some, but not all, species of the genus Pseudomonas, suggesting that its conservation follows their taxonomic-based evolution.