Improvement of isopropanol tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies

Horinouchi, Takaaki; Sakai, Aki; Kotani, Hazuki; Tanabe, Kumi; Furusawa, Chikara

doi:10.1016/j.jbiotec.2017.06.408

Cited by 39 publications

(23 citation statements)

References 70 publications

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“…Based on information about target genes and metabolites obtained via omics analyses, it is possible to rationally engineer alcohol-tolerant bacterial strains. As described previously, several reports noted a relationship between alcohol tolerance and specific amino acids (Gonzalez et al 2003 ; Horinouchi et al 2010 , 2017a ; Wang et al 2013 ; Haft et al 2014 ). Based on these results, several groups investigated the biosynthesis or supplementation of these amino acids for improving alcohol tolerance (Gonzalez et al 2003 ; Horinouchi et al 2010 , 2017a ; Haft et al 2014 ).…”

Section: Engineering Alcohol Tolerancesupporting

confidence: 52%

“…As described previously, several reports noted a relationship between alcohol tolerance and specific amino acids (Gonzalez et al 2003 ; Horinouchi et al 2010 , 2017a ; Wang et al 2013 ; Haft et al 2014 ). Based on these results, several groups investigated the biosynthesis or supplementation of these amino acids for improving alcohol tolerance (Gonzalez et al 2003 ; Horinouchi et al 2010 , 2017a ; Haft et al 2014 ). In addition, it is possible to identify mutations that contribute to alcohol tolerance via genome re-sequencing analyses of tolerant strains obtained using ALE, and it is possible to evaluate the effects of specific mutations on alcohol tolerance using genome-editing technology (Pósfai et al 1999 ; Wang et al 2009 ; Jiang et al 2013 ).…”

Section: Engineering Alcohol Tolerancesupporting

confidence: 52%

“…In E. coli , metabolomic analyses of its responses to ethanol, n -butanol, and isobutanol stresses revealed that several amino acids and osmoprotectants, such as isoleucine, valine, glycine, glutamate, and trehalose, are key metabolites that protect against these stresses (Wang et al 2013 ). Several transcriptomic studies also demonstrated the relationship between alcohol stress and these low-molecular-weight compounds (Gonzalez et al 2003 ; Horinouchi et al 2010 , 2017a ; Swings et al 2017 ). Although the roles of these compounds in the alcohol stress response remain unclear, they may mitigate the growth inhibition caused by alcohol stress.…”

Section: Understanding Alcohol Tolerance Using Omics Technologiesmentioning

confidence: 98%

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Understanding and engineering alcohol-tolerant bacteria using OMICS technology

Horinouchi

Maeda

Furusawa

2018

World J Microbiol Biotechnol

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Microbes are capable of producing alcohols, making them an important source of alternative energy that can replace fossil fuels. However, these alcohols can be toxic to the microbes themselves, retaring or inhibiting cell growth and decreasing the production yield. One solution is improving the alcohol tolerance of such alcohol-producing organisms. Advances in omics technologies, including transcriptomic, proteomic, metabolomic, and genomic technologies, have helped us understand the complex mechanisms underlying alcohol toxicity, and such advances could assist in devising strategies for engineering alcohol-tolerant strains. This review highlights these advances and discusses strategies for improving alcohol tolerance using omics analyses.

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Section: Engineering Alcohol Tolerancesupporting

confidence: 52%

Section: Engineering Alcohol Tolerancesupporting

confidence: 52%

Section: Understanding Alcohol Tolerance Using Omics Technologiesmentioning

confidence: 98%

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Understanding and engineering alcohol-tolerant bacteria using OMICS technology

Horinouchi

Maeda

Furusawa

2018

World J Microbiol Biotechnol

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“…The genomic DNA was extracted from cell pellets of strain P and evolved strains A‐E by DNeasy Blood & Tissue Kit (Qiagen, Germany) in accordance with the manufacturer's instructions. Genome sequence analysis were performed with MiSeq Desktop Sequencer (Illumina, Inc., San Diego, CA) as described previously (Horinouchi, Sakai, Kotani, Tanabe, & Furusawa, ). The reads obtained were aligned to the reference sequence of E. coli BW25113 genomic DNA (GenBank: NZ_CP009273) using breseq pipeline, version 0.28 (Deatherage & Barrick, ).…”

Section: Methodsmentioning

confidence: 99%

Application of adaptive laboratory evolution to overcome a flux limitation in an Escherichia coli production strain

Tokuyama

Toya

Horinouchi³

et al. 2018

Biotech & Bioengineering

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Gene deletion strategies using flux balance analysis (FBA) have improved the growth-coupled production of various compounds. However, the productivities were often below the expectation because the cells failed to adapt to these genetic perturbations. Here, we demonstrate the productivity of the succinate of the designed gene deletion strain was improved by adaptive laboratory evolution (ALE). Although FBA predicted deletions of adhE-pykAF-gldA-pflB lead to produce succinate from glycerol with a yield of 0.45 C-mol/C-mol, the knockout mutant did not produce only 0.08 C-mol/Cmol, experimentally. After the ALE experiments, the highest succinate yield of an evolved strain reached to the expected value. Genome sequencing analysis revealed all evolved strains possessed novel mutations in ppc of I829S or R849S. In vitro enzymatic assay and metabolic profiling analysis revealed that these mutations desensitizing an allosteric inhibition by L-aspartate and improved the flux through Ppc, while the activity of Ppc in the unevolved strain was tightly regulated by L-aspartate. These result demonstrated that the evolved strains achieved the improvement of succinate production by expanding the flux space of Ppc, realizing the predicted metabolic state by FBA.

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“…It has been demonstrated to be a simple and effective strategy in enhancing tolerance and improving productivity of strains to organic solvents, and combination of ALE with next-generation sequencing and modern omics technologies enable researchers to gain insight into the genetic basis and molecular mechanism of dynamic adaptation. For example, ALE was successfully performed in E. coli to improve its tolerance to isopropanol (IPA) and mutations contributed to IPA tolerance were identified through genome resequencing [25]. The evolved strain of S. cerevisiae by ALE showed significantly enhanced tolerance to limonene and monoterpenes-containing biojet fuels, and genes/ proteins involved in phenotypic changes were identified through genome resequencing, transcriptomics, and proteomics analyses [26].…”

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confidence: 99%

Improvement of sabinene tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies

Liu

et al. 2020

Biotechnol Biofuels

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Background: Biosynthesis of sabinene, a bicyclic monoterpene, has been accomplished in engineered microorganisms by introducing heterologous pathways and using renewable sugar as a carbon source. However, the efficiency and titers of this method are limited by the low host tolerance to sabinene (in both eukaryotes and prokaryotes). Results: In this study, Escherichia coli BL21(DE3) was selected as the strain for adaptive laboratory evolution. The strain was evolved by serial passaging in the medium supplemented with gradually increasing concentration of sabinene, and the evolved strain XYF(DE3), which exhibited significant tolerance to sabinene, was obtained. Then, XYF(DE3) was used as the host for sabinene production and an 8.43-fold higher sabinene production was achieved compared with the parental BL21(DE3), reaching 191.76 mg/L. Whole genomes resequencing suggested the XYF(DE3) strain is a hypermutator. A comparative analysis of transcriptomes of XYF(DE3) and BL21(DE3) was carried out to reveal the mechanism underlying the improvement of sabinene tolerance, and 734 up-regulated genes and 857 downregulated genes were identified. We further tested the roles of the identified genes in sabinene tolerance via reverse engineering. The results demonstrated that overexpressions of ybcK gene of the DLP12 family, the inner membrane protein gene ygiZ, and the methylmalonyl-CoA mutase gene scpA could increase sabinene tolerance of BL21(DE3) by 127.7%, 71.1%, and 75.4%, respectively. Furthermore, scanning electron microscopy was applied to monitor cell morphology. Under sabinene stress, the parental BL21(DE3) showed increased cell length, whereas XYF(DE3) showed normal cell morphology. In addition, overexpression of ybcK, ygiZ or scpA could partially rescue cell morphology under sabinene stress and overexpression of ygiZ or scpA could increase sabinene production in BL21(DE3). Conclusions: This study not only obtained a sabinene-tolerant strain for microbial production of sabinene but also revealed potential regulatory mechanisms that are important for sabinene tolerance. In addition, for the first time, ybcK, ygiZ, and scpA were identified to be important for terpene tolerance in E. coli BL21(DE3).

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Improvement of isopropanol tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies

Cited by 39 publications

References 70 publications

Understanding and engineering alcohol-tolerant bacteria using OMICS technology

Understanding and engineering alcohol-tolerant bacteria using OMICS technology

Application of adaptive laboratory evolution to overcome a flux limitation in an Escherichia coli production strain

Improvement of sabinene tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies

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