Escherichia Coli by Global Transcription Machinery Engineering Fa Zhang, Xiaohong Qian, Haiming Si, Guochao Xu, Ruizhi Han and Ye Ni*
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Zhang et al. Microb Cell Fact (2015) 14:175 DOI 10.1186/s12934-015-0368-4 RESEARCH Open Access Significantly improved solvent tolerance of Escherichia coli by global transcription machinery engineering Fa Zhang, Xiaohong Qian, Haiming Si, Guochao Xu, Ruizhi Han and Ye Ni* Abstract Background: Escherichia coli has emerged as a promising platform microorganism to produce biofuels and fine chemicals of industrial interests. Certain obstacles however remain to be overcome, among which organic-solvent tolerance is a crucial one. Results: We used global transcription machinery engineering (gTME) to improve the organic-solvent tolerance (OST) of E. coli JM109. A mutant library of σ70 encoded by rpoD was screened under cyclohexane pressure. E. coli JM109 strain harboring σ70 mutant C9 was identified with capability of tolerating 69 % cyclohexane. The rpoD mutant con- tains three amino-acid substitutes and a stop-codon mutation, resulting a truncated sequence containing regions σ1.1 and σ1.2. Total protein difference produced by E. coli JM109 strain harboring C9 was examined with 2D-PAGE, and 204 high-abundant proteins showed over twofold variation under different solvent stress. Conclusions: Our results show that several genes (gapA, sdhB, pepB and dppA) play critical roles in enhanced solvent tolerance of E. coli, mainly involving in maintaining higher intracellular energy level under solvent stress. Global tran- scription machinery engineering is therefore a feasible and efficient approach for engineering strain with enhanced OST-phenotype. Keywords: Global transcription machinery engineering, Sigma factor 70, Organic solvent tolerance, Escherichia coli, gapA, sdhB, pepB Background 3.4–3.8 [4]. For example, toluene is toxic to E. coli cells The increasing attention to green chemistry has even at concentrations as low as 0.1 % [5]. Hence, it is prompted the production of non-renewable fuels, mate- of great importance to develop organic-solvent tolerant rials, pharmaceuticals, and fine chemicals by microbial (OST) E. coli strains for industrial applications. factories [1]. Escherichia coli as one of the most impor- Since the first toluene tolerant strain Pseudomonas tant platform microorganisms, could be applied as a putida IH-2000 identified in 1989, extensively work had whole-cell biocatalyst, which provides safe intracellular been focused on P. putida and Clostridium species etc. environment for enzymes [2]. In whole-cell biocatalysis, [6, 7]. Various OST mechanisms have been proposed, nonaqueous system (such as organic solvents) is often including cell membrane adaptations [8], cell morphol- adopted to facilitate the solubility of hydrophobic sub- ogy [9], and efflux pumps etc. [10, 11]. Traditional strain strates and (or) products [3]. Organic solvents are toxic engineering methods, such as adaptation [12], enrich- to most microorganisms. E. coli was reported to barely ment cultivation [13], chemical and physical mutagenesis tolerate organic solvents with LogP values lower than [14], have been widely used for developing OST strains. Global transcription machinery engineering (gTME) is *Correspondence: [email protected] a novel directed evolution strategy to assist in unlock- The Key Laboratory of Industrial Biotechnology, Ministry of Education, ing complex phenotypes by disturbing the transcrip- School of Biotechnology, Jiangnan University, 214122 Wuxi, Jiangsu, tion at genome level. Alper and co-workers obtained China © 2015 Zhang et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Microb Cell Fact (2015) 14:175 Page 2 of 11 yeast strains that tolerated ethanol up to 20 % (v/v) [15]. mutagenesis would not affect the normal growth of the In the past few years, a number of gTME-aided studies E. coli strain. have outperformed those of traditional methods, result- Solvent tolerance towards other solvents was also ing desired phenotypes more effectively. Several tran- investigated. E. coli carrying C9 showed increasing in scription factors, such as sigma factor in bacteria [16], cell density when cultivated in presence of 1.0 % butanol, zinc finger-containing artificial transcription factor [17], 13 % hexane, 0.4 % toluene, and 0.5 % butyl acetate, Spt15 in yeast [18] were widely used as a potential tool whereas WT σ70 could merely survive under 0.1 % to improve strain tolerance and increase biofilm forma- butanol, 5 % hexane, 0.1 % toluene and 0.2 % butyl acetate tion. Sigma factor 70 (σ70) is the most common tran- (Fig. 2). scription factor in E. coli. In addition to binding to RNA Among various organic solvents, C9 mutant strain polymerase and recognizing DNA template strand, it can showed higher tolerance to cyclohexane. Since cyclohex- also alter the affinity of RNA polymerase to the promoter. ane pressure was used in the library screening, isolated Most importantly, the transcriptional efficiency can be mutants often show higher preference to cyclohexane. regulated by mutation of σ factor [19, 20]. Alper and Additionally, it has been proved that cyclohexane could Stephanopoulos successfully constructed an E. coli strain be oxidized into cyclohexanol with less toxicity by micro- that could tolerate as high as 60 g/L ethanol by mutating organisms in our previous study [23]. rpoD [21]. By random mutagenesis of rpoD and rpoS, Yu The sequence alignment of σ70 WT and C9 revealed and coworkers obtained an E. coli mutant that could pro- that rpoD mutant gene C9 contains two amino-acid duce 561.4 mg/L hyaluronic acid [22]. mutations in region 1.1 (D39E, A72V) and two other In our previous study, an OST P. putida JUCT1 that mutations in region 1.2 (T94M, and a stop codon muta- tolerated 60 % cyclohexane was obtained by gradient tion at residue 123). adaptation. Based on two-dimensional electrophoresis (2-DE), two 3-hydroxyacid dehydrogenase family genes, 2‑DE analysis and protein identification by MALDI‑TOF/TOF mmsB (from P. putida) and zwf (from E. coli), were iden- Two-DE, a powerful protein separation technique to tified and proved to be critical for the enhanced solvent illustrate proteins associated with certain phenotype, was tolerance in both P. putida and E. coli [23, 24]. used to investigate the proteomics of E. coli strains har- In this study, we evaluated the efficacy of gTME in E. boring C9 mutant when grown with or without cyclohex- coli by screening rpoD mutant library under cyclohexane ane. 2-DE analysis of WT strain (without cyclohexane) pressure. We aimed at isolating σ70 mutants to improve was also conducted as control. Our results show that the solvent tolerance of E. coli, which could potentially there was no obvious difference between WT and C9 be applied in non-aqueous biocatalysis and biofuel strain in the absence of cyclohexane (Additional file 1: production. Figure S1). Compared with control (C9 without solvent), 204 high-abundant proteins in C9 strain showed over Results twofold difference in the presence of 38 % cyclohexane 70 Screening of solvent tolerance σ mutants (Fig. 3). To improve the solvent tolerance of E. coli, a rpoD Among 204 high-abundant proteins, 43 independent mutant library was constructed and screened under protein spots were cut off and analyzed by MALDI-TOF/ cyclohexane pressure. After preliminary screen- TOF. Finally, 22 proteins including 19 up-regulated and ing, 9 strains resulted in OD660 of over 1.1 were sub- 3 down-regulated proteins were successfully identified jected to secondary screening, where mutants were (Table 1). These up-regulated proteins are involved in enriched through repeated subcultures supplemented nucleotide synthesis, amino acid and glucose metabo- with escalating cyclohexane concentration. Finally, E. lism, transporter and porin proteins synthesis, etc. coli strain carrying σ70 mutant C9 showed the high- Among them, up-regulated genes gapA (glyceralde- est cyclohexane tolerance was selected. When grew in hyde-3-phosphate dehydrogenase A) and sdhB (FeS 38 % cyclohexane, its OD660 could reach 0.83, while the subunit of succinate dehydrogenase) are involved in the parent strain JM109/pHACM-rpoDWT could not even glycolysis process and TCA cycle, respectively [25, 26]. grow under this condition (Fig. 1a). Then solvent tol- Both of them could produce intracellular ATP and pro- erance of σ70 mutant C9 was determined under higher vide high energy storage to overcome solvent stress. The cyclohexane concentrations. The result showed that expression levels of pepB (aminopeptidase B) and yfgM (a E. coli harboring C9 could tolerate 69 % cyclohexane hypothetical protein) were remarkably enhanced in C9 (Fig. 1b). In the absence of cyclohexane, there was mutant under solvent treatment, whereas their functions no significant difference between the cell growth of have barely been reported. Both bcp (thiol peroxidase) WT and C9 mutant strains (Fig. 1c), suggesting rpoD and dppA (dipeptide transporter) genes were significantly Zhang et al. Microb Cell Fact (2015) 14:175 Page 3 of 11 Fig. 1 a Screening of solvent tolerance σ70 mutants C1–C9 at