Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance

Zhang, Hongfang; Chong, Huiqing; Ching, Chi Bun; Song, Hao; Jiang, Rongrong

doi:10.1007/s00253-012-4012-5

Cited by 64 publications

(47 citation statements)

References 66 publications

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“…The amino acid substitution at position D138 of A2 (D138Y) is of particular interest as some other CRP mutants with either improved osmotolerance or 1-butanol tolerance also had this D138 modification [32,35], but unlike A2, those mutants also carried modifications at other locations. D138 is known to play essential roles during the hinge reorientation and helical adjustment upon allosteric activation by cAMP [43] through the formation of hydrogen bonds with G141 [44].…”

Section: Resultsmentioning

confidence: 99%

Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

Chong

Yeow²,

Wang³

et al. 2013

PLoS ONE

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The presence of acetate exceeding 5 g/L is a major concern during E. coli fermentation due to its inhibitory effect on cell growth, thereby limiting high-density cell culture and recombinant protein production. Hence, engineered E. coli strains with enhanced acetate tolerance would be valuable for these bioprocesses. In this work, the acetate tolerance of E. coli was much improved by rewiring its global regulator cAMP receptor protein (CRP), which is reported to regulate 444 genes. Error-prone PCR method was employed to modify crp and the mutagenesis libraries (~3×106) were subjected to M9 minimal medium supplemented with 5–10 g/L sodium acetate for selection. Mutant A2 (D138Y) was isolated and its growth rate in 15 g/L sodium acetate was found to be 0.083 h-1, much higher than that of the control (0.016 h-1). Real-time PCR analysis via OpenArray® system revealed that over 400 CRP-regulated genes were differentially expressed in A2 with or without acetate stress, including those involved in the TCA cycle, phosphotransferase system, etc. Eight genes were chosen for overexpression and the overexpression of uxaB was found to lead to E. coli acetate sensitivity.

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Section: Resultsmentioning

confidence: 99%

Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

Chong

Yeow²,

Wang³

et al. 2013

PLoS ONE

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“…Random mutation and expression of an exogenous global regulator irrE from radiation-resistant Deinococcus radiodurans in E. coli resulted in improved biofuel tolerances of E. coli cells from several to a hundred fold [20]. Random mutagenesis of a global transcription factor cyclic AMP receptor protein (CRP) of E. coli resulted in a twofold growth rate increase when grown under 1.2% 1-butanol [21]. These studies demonstrated that direct manipulation of regulatory systems could provide a useful tool in tolerance engineering.…”

Section: Introductionmentioning

confidence: 99%

Butanol tolerance regulated by a two-component response regulator Slr1037 in photosynthetic Synechocystis sp. PCC 6803

Chen

Wang

et al. 2014

Biotechnol Biofuels

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BackgroundButanol production directly from CO2 in photosynthetic cyanobacteria is restricted by the high toxicity of butanol to the hosts. In previous studies, we have found that a few two-component signal transduction systems (TCSTSs) were differentially regulated in Synechocystis sp. PCC 6803 after butanol treatment.ResultsTo explore regulatory mechanisms of butanol tolerance, in this work, by constructing gene knockout mutants of the butanol-responsive TCSTS genes and conducting tolerance analysis, we uncovered that an orphan slr1037 gene encoding a novel response regulator was involved in butanol tolerance in Synechocystis. Interestingly, the ∆slr1037 mutant grew similarly to the wild type under several other stress conditions tested, which suggests that its regulation on butanol tolerance is specific. Using a quantitative iTRAQ LC-MS/MS proteomics approach coupled with real-time reverse transcription PCR, we further determined the possible butanol-tolerance regulon regulated by Slr1037. The results showed that, after slr1037 deletion, proteins involved in photosynthesis and glycolysis/gluconeogenesis of central metabolic processes, and glutaredoxin, peptide methionine sulfoxide reductase and glucosylglycerol-phosphate synthase with stress-responsive functions were down-regulated, suggesting that Slr1037 may exhibit regulation to a wide range of cellular functions in combating butanol stress.ConclusionsThe study provided a proteomic description of the putative butanol-tolerance regulon regulated by the slr1037 gene. As the first signal transduction protein identified directly related to butanol tolerance, response regulator Slr1037 could be a natural candidate for transcriptional engineering to improve butanol tolerance in Synechocystis.

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“…In addition, many genes were related to organic solvent tolerance in the E. coli strain, for example, mutant cyclic AMP receptor protein (CRP) [32], overexpression of groESL [33–35], mutant Δ lon (cell envelope-related gene) [36] and control membrane-related functions (overexpression of ATF, fabD , feoA and srpABC ) [37]. Moreover, Rutherford et al [17] reported that n-butanol stress-response genes are also involved in many stress responses, such as oxidative stress ( sodA , sodC and yqhD ), heat shock and cell envelope stress ( rpoE , clpB , htpG , cpxR and cpxP ).…”

Section: Resultsmentioning

confidence: 99%

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

et al. 2017

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BackgroundN-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell.ResultsThe host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis.ConclusionsThese results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD+ and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were downregulated, which further assisted in regulating NADH/NAD+ redox and preventing additional ATP depletion. These results indicated that more NADH and ATP were required in the n-butanol synthetic pathway. Our study demonstrates a potential approach to increase the robustness of microorganisms and the production of toxic chemicals through the ability to reduce oxidative stress.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0356-3) contains supplementary material, which is available to authorized users.

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Engineering global transcription factor cyclic AMP receptor protein of Escherichia coli for improved 1-butanol tolerance

Cited by 64 publications

References 66 publications

Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

Improving Acetate Tolerance of Escherichia coli by Rewiring Its Global Regulator cAMP Receptor Protein (CRP)

Butanol tolerance regulated by a two-component response regulator Slr1037 in photosynthetic Synechocystis sp. PCC 6803

Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli

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