Dataset Information

A Novel Butanol Tolerance-Promoting Function of the Transcription Factor Rob in Escherichia coli.

ABSTRACT: Producing high concentrations of biobutanol is challenging, primarily because of the toxicity of butanol toward cells. In our previous study, several butanol tolerance-promoting genes were identified from butanol-tolerant Escherichia coli mutants and inactivation of the transcriptional regulator factor Rob was shown to improve butanol tolerance. Here, the butanol tolerance characteristics and mechanism regulated by inactivated Rob are investigated. Comparative transcriptome analysis of strain DTrob, with a truncated rob in the genome, and the control BW25113 revealed 285 differentially expressed genes (DEGs) to be associated with butanol tolerance and categorized as having transport, localization, and oxidoreductase activities. Expression of 25 DEGs representing different functional categories was analyzed by quantitative reverse transcription PCR (qRT-PCR) to assess the reliability of the RNA-Seq data, and 92% of the genes showed the same expression trend. Based on functional complementation experiments of key DEGs, deletions of glgS and yibT increased the butanol tolerance of E. coli, whereas overexpression of fadB resulted in increased cell density and a slight increase in butanol tolerance. A metabolic network analysis of these DEGs revealed that six genes (fadA, fadB, fadD, fadL, poxB, and acs) associated with acetyl-CoA production were significantly upregulated in DTrob, suggesting that Rob inactivation might enhance butanol tolerance by increasing acetyl-CoA. Interestingly, DTrob produced more acetate in response to butanol stress than the wild-type strain, resulting in the upregulation expression of some genes involved in acetate metabolism. Altogether, the results of this study reveal the mechanism underlying increased butanol tolerance in E. coli regulated by Rob inactivation.

SUBMITTER: Wang Z

PROVIDER: S-EPMC7537768 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Novel Butanol Tolerance-Promoting Function of the Transcription Factor Rob in <i>Escherichia coli</i>.

Wang Zhiquan Z Xue Tingli T Hu Dongsheng D Ma Yuanyuan Y

Frontiers in bioengineering and biotechnology 20200922

Producing high concentrations of biobutanol is challenging, primarily because of the toxicity of butanol toward cells. In our previous study, several butanol tolerance-promoting genes were identified from butanol-tolerant <i>Escherichia coli</i> mutants and inactivation of the transcriptional regulator factor Rob was shown to improve butanol tolerance. Here, the butanol tolerance characteristics and mechanism regulated by inactivated Rob are investigated. Comparative transcriptome analysis of st ...[more]

PMID: 33072717

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Project description:Backgroundn-Butanol is a promising emerging biofuel, and recent metabolic engineering efforts have demonstrated the use of several microbial hosts for its production. However, most organisms have very low tolerance to n-butanol (up to 2% (v/v)), limiting the economic viability of this biofuel. The rational engineering of more robust n-butanol production hosts relies upon understanding the mechanisms involved in tolerance. However, the existing knowledge of genes involved in n-butanol tolerance is limited. The goal of this study is therefore to identify E. coli genes that are involved in n-butanol tolerance.Methodology/principal findingsUsing a genomic library enrichment strategy, we identified approximately 270 genes that were enriched or depleted in n-butanol challenge. The effects of these candidate genes on n-butanol tolerance were experimentally determined using overexpression or deletion libraries. Among the 55 enriched genes tested, 11 were experimentally shown to confer enhanced tolerance to n-butanol when overexpressed compared to the wild-type. Among the 84 depleted genes tested, three conferred increased n-butanol resistance when deleted. The overexpressed genes that conferred the largest increase in n-butanol tolerance were related to iron transport and metabolism, entC and feoA, which increased the n-butanol tolerance by 32.8±4.0% and 49.1±3.3%, respectively. The deleted gene that resulted in the largest increase in resistance to n-butanol was astE, which enhanced n-butanol tolerance by 48.7±6.3%.Conclusions/significanceWe identified and experimentally verified 14 genes that decreased the inhibitory effect of n-butanol tolerance on E. coli. From the data, we were able to expand the current knowledge on the genes involved in n-butanol tolerance; the results suggest that an increased iron transport and metabolism and decreased acid resistance may enhance n-butanol tolerance. The genes and mechanisms identified in this study will be helpful in the rational engineering of more robust biofuel producers.

Project description:BackgroundEscherichia coli has been proved to be one promising platform chassis for the production of various natural products, such as biofuels. Product toxicity is one of the main bottlenecks for achieving maximum production of biofuels. Host strain engineering is an effective approach to alleviate solvent toxicity issue in fermentation.ResultsThirty chaperones were overexpressed in E. coli JM109, and SecB recombinant strain was identified with the highest n-butanol tolerance. The tolerance (T) of E. coli overexpressing SecB, calculated by growth difference in the presence and absence of solvents, was determined to be 9.13% at 1.2% (v/v) butanol, which was 3.2-fold of the control strain. Random mutagenesis of SecB was implemented and homologously overexpressed in E. coli, and mutant SecBT10A was identified from 2800 variants rendering E. coli the highest butanol tolerance. Saturation mutagenesis on T10 site revealed that hydrophobic residues were required for high butanol tolerance of E. coli. Compared with wild-type (WT) SecB, the T of SecBT10A strain was further increased from 9.14 to 14.4% at 1.2% butanol, which was 5.3-fold of control strain. Remarkably, E. coli engineered with SecBT10A could tolerate as high as 1.8% butanol (~ 14.58 g/L). The binding affinity of SecBT10A toward model substrate unfolded maltose binding protein (preMBP) was 11.9-fold of that of WT SecB as determined by isothermal titration calorimetry. Residue T10 locates at the entrance of hydrophobic substrate binding groove of SecB, and might play an important role in recognition and binding of cargo proteins.ConclusionsSecB chaperone was identified by chaperone mining to be effective in enhancing butanol tolerance of E. coli. Maximum butanol tolerance of E. coli could reach 1.6% and 1.8% butanol by engineering single gene of SecB or SecBT10A. Hydrophobic interaction is vital for enhanced binding affinity between SecB and cargo proteins, and therefore improved butanol tolerance.

Dataset Information

A Novel Butanol Tolerance-Promoting Function of the Transcription Factor Rob in Escherichia coli.

Publications

A Novel Butanol Tolerance-Promoting Function of the Transcription Factor Rob in <i>Escherichia coli</i>.

Similar Datasets

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