Gene Array-Based identification of changes that contribute to ethanol tolerance in ethanologenic Escherichia coli: comparison of KO11 (Parent) to LY01 (resistant mutant). Biotechnol Prog

Department of Chemical and Biological Engineering, Iowa State University, Ames, Iowa, United States
Biotechnology Progress (Impact Factor: 2.15). 04/2003; 19(2):612-23. DOI: 10.1021/bp025658q
Source: PubMed


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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    • "This approach has resulted in increase in microbial biocatalysts efficiency in production of ethanol with increasing tolerance to LCM inhibitory compounds. For example, the Escherichia coli LY01 strain was found to show high tolerance to toxic aldehydes than its wild type KO11, by expressing high levels of genes involved in safeguarding osmolytic balance, stress response proteins and cell envelope components 76, 175. Furthermore, Escherichia coli strain LY168 engineered from wild type K011 from was shown to produce higher level of ethanol in a minimal nutritional supplement from various lignocellulose biomass containing LCM inhibitory compounds 176. "
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    • "Genome-level studies on biofuel tolerant strains have consistently found that it is necessary to alter the expression of multiple genes to provide the greatest benefit [9,27,33,53]. For example, simultaneous disruption of five unrelated genes provided significant improvements to isobutanol tolerance in a study by Atsumi et al. [9]. "
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    • "The response to ethanol is more like salt or desiccation stress in that ethanol appears to have a water exclusion effect. Consequently, studies implicate the role of osmoprotectants in stress mitigation (Gonzalez et al. 2003; Underwood et al. 2004). E. coli shows a similar response in cell wall fatty acid composition in response to salt and ethanol stresses and pretreatment with salt resulted in greater resistance to ethanol (Ingram and Vreeland 1980); specifically, an increase in unsaturated fatty acids was found in response to ethanol stress (Ingram et al. 1980). "
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    ABSTRACT: The need for renewable alternative sources of liquid biofuels has lead to tremendous interest in the conversion of lignocellulosic biomass to fuel compounds via microbial routes. A key aspect of the research involves the engineering of robust and stable microbial host platforms that can produce these compounds at high titer. Impact on growth caused by inhibitory compounds in the deconstructed biomass and accumulation of toxic metabolic intermediates and final product are bottlenecks that severely limit product titers. This chapter reviews known sources of toxicity arising from various aspects of this process and discusses native and heterologous mechanisms of microbial stress response and defense that can be used to engineer better production hosts.
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