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.
"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. "
[Show abstract][Hide abstract] ABSTRACT: Current international interest in finding alternative sources of energy to the diminishing supplies of fossil fuels has encouraged research efforts in improving biofuel production technologies. In countries which lack sufficient food, the use of sustainable lignocellulosic feedstocks, for the production of bioethanol, is an attractive option. In the pre-treatment of lignocellulosic feedstocks for ethanol production, various chemicals and/or enzymatic processes are employed. These methods generally result in a range of fermentable sugars, which are subjected to microbial fermentation and distillation to produce bioethanol. However, these methods also produce compounds that are inhibitory to the microbial fermentation process. These compounds include products of sugar dehydration and lignin depolymerisation, such as organic acids, derivatised furaldehydes and phenolic acids. These compounds are known to have a severe negative impact on the ethanologenic microorganisms involved in the fermentation process by compromising the integrity of their cell membranes, inhibiting essential enzymes and negatively interact with their DNA/RNA. It is therefore important to understand the molecular mechanisms of these inhibitions, and the mechanisms by which these microorganisms show increased adaptation to such inhibitors. Presented here is a concise overview of the molecular adaptation mechanisms of ethanologenic bacteria in response to lignocellulose-derived inhibitory compounds. These include general stress response and tolerance mechanisms, which are typically those that maintain intracellular pH homeostasis and cell membrane integrity, activation/regulation of global stress responses and inhibitor substrate-specific degradation pathways. We anticipate that understanding these adaptation responses will be essential in the design of 'intelligent' metabolic engineering strategies for the generation of hyper-tolerant fermentation bacteria strains.
International journal of biological sciences 06/2013; 9(6):598-612. DOI:10.7150/ijbs.6091 · 4.51 Impact Factor
"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. . "
[Show abstract][Hide abstract] ABSTRACT: ABSTRACT: A major challenge when using microorganisms to produce bulk chemicals such as biofuels is that the production targets are often toxic to cells. Many biofuels are known to reduce cell viability through damage to the cell membrane and interference with essential physiological processes. Therefore, cells must trade off biofuel production and survival, reducing potential yields. Recently, there have been several efforts towards engineering strains for biofuel tolerance. Promising methods include engineering biofuel export systems, heat shock proteins, membrane modifications, more general stress responses, and approaches that integrate multiple tolerance strategies. In addition, in situ recovery methods and media supplements can help to ease the burden of end-product toxicity and may be used in combination with genetic approaches. Recent advances in systems and synthetic biology provide a framework for tolerance engineering. This review highlights recent targeted approaches towards improving microbial tolerance to next-generation biofuels with a particular emphasis on strategies that will improve production.
Biotechnology for Biofuels 09/2011; 4(1):32. DOI:10.1186/1754-6834-4-32 · 6.04 Impact Factor
"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). "
[Show abstract][Hide abstract] 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
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