Gene array-based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (parent) to LY01 (resistant mutant).
ABSTRACT Escherichia coli KO11 (parent) and LY01 (mutant) have been engineered for the production of ethanol. Gene arrays were used to identify expression changes that occurred in the mutant, LY01, during directed evolution to improve ethanol tolerance (defined as extent of growth in the presence of added ethanol). Expression levels for 205 (5%) of the ORFs were found to differ significantly (p < 0.10) between KO11 and LY01 under each of six different growth conditions (p < 0.000001). Statistical evaluation of differentially expressed genes according to various classification schemes identified physiological areas of importance. A large fraction of differentially expressed ORFs were globally regulated, leading to the discovery of a nonfunctional fnr gene in strain LY01. In agreement with a putative role for FNR in alcohol tolerance, increasing the copy number of fnr(+) in KO11(pGS196) decreased ethanol tolerance but had no effect on growth in the absence of ethanol. Other differences in gene expression provided additional clues that permitted experimentation. Tolerance appears to involve increased metabolism of glycine (higher expression of gcv genes) and increased production of betaine (higher expression of betIBA and betT encoding betaine synthesis from choline and choline uptake, respectively). Addition of glycine (10 mM) increased ethanol tolerance in KO11 but had no effect in the absence of ethanol. Addition of betaine (10 mM) increased ethanol tolerance by over 2-fold in both LY01 and KO11 but had no effect on growth in the absence of ethanol. Both glycine and betaine can serve as protective osmolytes, and this may be the basis of their beneficial action. In addition, the marAB genes encoding multiple antibiotic resistance proteins were expressed at higher levels in LY01 as compared to KO11. Interestingly, overexpression of marAB in KO11 made this strain more ethanol-sensitive. Overexpression of marAB in LY01 had no effect on ethanol tolerance. Increased expression of genes encoding serine uptake (sdaC) and serine deamination (sdaB) also appear beneficial for LY01. Addition of serine increased the growth of LY01 in the presence and absence of ethanol but had no effect on KO11. Changes in the expression of several genes concerned with the synthesis of the cell envelope components were also noted, which may contribute to increased ethanol tolerance.
- [Show abstract] [Hide abstract]
ABSTRACT: DNA microarrays are currently used to study the transcriptional response of many organisms to genetic and environmental perturbations. Although there is much room for improvement of this technology, its potential has been clearly demonstrated in the past 5 years. The general consensus is that the bottleneck is now located in the processing and analysis of transcriptome data and its use for purposes other than the quantification of changes in gene expression levels. In this article we discuss technological aspects of DNA microarrays, statistical and biological issues pertinent to the design of microarray experiments, and statistical tools for microarray data analysis. A review on applications of DNA microarrays in the study of bacterial systems is presented. Special attention is given to studies in the following areas: (1) bacterial response to environmental changes; (2) gene identification, genome organization, and transcriptional regulation; and (3) genetic and metabolic engineering. Soon, the use of DNA microarray technologies in conjunction with other genome/system-wide analyses (e.g., proteomics, metabolomics, fluxomics, phenomics, etc.) will provide a better assessment of genotype-phenotype relationships in bacteria, which serve as a basis for understanding similar processes in more complex organisms.Biotechnology Progress 01/2004; 20(5):1309-24. · 1.85 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Ubiquitous noxious hydrophobic substances, such as hydrocarbons, pesticides and diverse industrial chemicals, stress biological systems and thereby affect their ability to mediate biosphere functions like element and energy cycling vital to biosphere health. Such chemically diverse compounds may have distinct toxic activities for cellular systems; they may also share a common mechanism of stress induction mediated by their hydrophobicity. We hypothesized that the stressful effects of, and cellular adaptations to, hydrophobic stressors operate at the level of water : macromolecule interactions. Here, we present evidence that: (i) hydrocarbons reduce structural interactions within and between cellular macromolecules, (ii) organic compatible solutes - metabolites that protect against osmotic and chaotrope-induced stresses - ameliorate this effect, (iii) toxic hydrophobic substances induce a potent form of water stress in macromolecular and cellular systems, and (iv) the stress mechanism of, and cellular responses to, hydrophobic substances are remarkably similar to those associated with chaotrope-induced water stress. These findings suggest that it may be possible to devise new interventions for microbial processes in both natural environments and industrial reactors to expand microbial tolerance of hydrophobic substances, and hence the biotic windows for such processes.Microbial Biotechnology 11/2010; 3(6):701-16. · 3.21 Impact Factor
- [Show abstract] [Hide abstract]
ABSTRACT: Ethanol fuel can be produced renewably from numerous plant and waste materials, but harnessing the energy of lignocellulosic feedstocks has been particularly challenging in the development of this alternative fuel as a substitute for petroleum-based fuels. Consolidated bioprocessing has the potential to make the conversion of biomass to fuel an economical process by combining enzyme production, polysaccharide hydrolysis, and sugar fermentation into a single unit operation. This consolidation of steps takes advantage of the synergistic nature of enzyme systems but requires the use of one or a few organisms capable of producing highly efficient cellulolytic enzymes and fermenting most of the resulting sugars to ethanol with minimal byproduct formation while tolerating high levels of ethanol. In this review, conventional ethanol production, consolidated bioprocessing, and simultaneous saccharification and fermentation are described and compared. Several wild-type and genetically engineered microorganisms, including strains of Clostridium thermocellum, Saccharomyces cerevisiae, Klebsiella oxytoca, Escherichia coli, Flammulina velutipes, and Zymomonas mobilis, among others, are highlighted for their potential in consolidated bioprocessing. This review examines the favorable and undesirable qualities of these microorganisms and their enzyme systems, process engineering considerations for particular organisms, characteristics of cellulosomes, enzyme engineering strategies, progress in commercial development, and the impact of these topics on current and future research.BioEnergy Research 06/2013; 6(2):416-435. · 4.25 Impact Factor