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.
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ABSTRACT: Pretreatment of grasses is required to maximize ethanol yield during fermentation. T85 bermudagrass and Merkeron napiergrass (Pennisetum purpureum Schumach.) were either left untreated or were pressurized batch hot water (PBHW) pretreated for 2 min at 230 °C at 5% w/v whole grass solids loading. Following a 24 h enzymatic digestion, untreated and PBHW pretreated grasses were evaluated for ethanol production and co-product generation including potential fermentation inhibitors. Fermentations of PBHW pretreated grasses with E. coli LY01 produced twice the ethanol of their untreated counterparts. PBHW pretreated Merkeron napiergrass produced 224.5 mg/g grass ethanol (73% maximum theoretical yield) and PBHW pretreated T85 bermudagrass reached 213.0 mg/g grass (70% maximum theoretical ethanol yield). Pretreatment by PBHW resulted in increased solubilization of hemicelluloses. PBHW pretreatment also produced potential fermentation inhibitors such as acetic, formic, cinnamic acids, and aldehydes. Despite some of these inhibitors remaining with the solids after PBHW pretreatment, there was more efficient hydrolysis of the cellulose and remaining hemicellulose during the enzymatic digestion of the grasses prior to fermentation when compared to the untreated grasses. This increase in digestibility observed with enzymes prior to fermentation resulted in increased ethanol yields during bioconversion using E. coli LY01 as the biocatalyst.Biomass and Bioenergy 08/2011; 35(8):3667-3673. DOI:10.1016/j.biombioe.2011.05.021 · 3.41 Impact Factor
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ABSTRACT: Escherichia coli has been engineered to produce isobutanol, with titers reaching greater than the toxicity level. However, the specific effects of isobutanol on the cell have never been fully understood. Here, we aim to identify genotype-phenotype relationships in isobutanol response. An isobutanol-tolerant mutant was isolated with serial transfers. Using whole-genome sequencing followed by gene repair and knockout, we identified five mutations (acrA, gatY, tnaA, yhbJ, and marCRAB) that were primarily responsible for the increased isobutanol tolerance. We successfully reconstructed the tolerance phenotype by combining deletions of these five loci, and identified glucosamine-6-phosphate as an important metabolite for isobutanol tolerance, which presumably enhanced membrane synthesis. The isobutanol-tolerant mutants also show increased tolerance to n-butanol and 2-methyl-1-butanol, but showed no improvement in ethanol tolerance and higher sensitivity to hexane and chloramphenicol than the parental strain. These results suggest that C4, C5 alcohol stress impacts the cell differently compared with the general solvent or antibiotic stresses. Interestingly, improved isobutanol tolerance did not increase the final titer of isobutanol production.Molecular Systems Biology 12/2010; 6:449. DOI:10.1038/msb.2010.98 · 14.10 Impact Factor
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ABSTRACT: Microbes represent a valuable source of commercially significant natural products that have improved our quality of life. Precision engineering can be used to precisely identify and specifically modify genes responsible for production of natural products and improve this production or modify the genes creating products that would not otherwise be produced. There have been several success stories concerning the manipulation of regulatory genes, pathways, and genomes to increase the productivity of industrial microbes. This review will focus on the strategies and integrated approaches for precisely deciphering regulatory genes by various modern techniques. The applications of precision engineering in rational strain improvement also shed light on the biology of natural microbial systems.Antonie van Leeuwenhoek 08/2010; 98(2):151-63. DOI:10.1007/s10482-010-9442-4 · 2.14 Impact Factor