Improvement of isopropanol tolerance of Escherichia coli using adaptive laboratory evolution and omics technologies
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE89685 | GEO | 2017/06/21
SECONDARY ACCESSION(S): PRJNA352950
REPOSITORIES: GEO
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Project description:BackgroundBiosynthesis 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).ResultsIn 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 down-regulated 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).ConclusionsThis 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).
| S-EPMC7181518 | biostudies-literature
Project description:Due to increasing ecological concerns, recombinant microbial production of biochemicals from sustainable carbon sources like acetate is rapidly gaining importance. However, for establishing successful large-scale production scenarios, a solid understanding of metabolic driving forces is required to inform bioprocess design. To generate such knowledge, we constructed isopropanol-producing Escherichia coli W strains. Based on strain screening and metabolic considerations a 2-stage process was designed incorporating a growth phase followed by a nitrogen starvation phase. This process design yielded the highest isopropanol titers on acetate to date (13.3 g L-1). Additionally, we performed shotgun and acetylated proteomics and identified several stress conditions in the bioreactor scenarios, such as acid stress and impaired sulfur uptake. Metabolic modelling allowed for an in-depth characterization of intracellular flux distributions, uncovering ATP availability as a determining factor for routing carbon towards the isopropanol pathway. Moreover, we asserted the importance of a balance between fluxes of the NADPH-providing isocitrate dehydrogenase (ICDH) and the product pathway. Using the newly gained system-level understanding for isopropanol production from acetate, we assessed possible engineering approaches and propose process designs to maximize production. Collectively, our work contributes to the establishment and optimization of acetate-based bioproduction systems.
2024-10-17 | PXD047939 | Pride
Project description:We performed massive screening of the genes in yeast that were involved in the tolerance to isopropanol using the non-essential genes deleted yeast collection, and identified sixty-five disruptants that grew slower than the wild type strain in the presence of isopropanol. The isopropanol sensitive mutants were tested to know their behaviour under other alcohol stresses. Besides, we conducted microarray analysis to reveal the transcriptional response to isopropanol stress in yeast. Our results certainly provide new insights into yeast response to C3 alcohol isopropanol.
2013-05-13 | GSE46384 | GEO
Project description:We carried out adaptive laboratory evolution of an E. coli strain lacking four genes (adhE, pta, ldhA, frdA) involved in acetyl-CoA consumption, allowing the efficient utilization of acetate as its sole carbon and energy source. The transcriptomes according to the medium status (M9 aceate, M9 glucose) of the evolved strain (SBA01) and its parent strain (DSM01) were compared using RNA-seq.
2020-09-15 | GSE157942 | GEO
Project description:We used RNA-seq to profile E. coli K-12 MG1655 strains subjected to adaptive laboratory evolution after knockout of endogenous glucose-6-phosphate isomerase (pgi) and subsequent expression of heterologous version of the pgi gene from Pseudomonas aeruginosa and Bacillus megaterium.
2023-02-09 | GSE224989 | GEO
Project description:We have performed adaptive laboratory evolution of E. coli ndh gene deletion strain to examine the adaptive strategies of E. coli.
2023-09-12 | GSE242875 | GEO
Project description:We have performed adaptive laboratory evolution of E. coli pdhR gene deletion strain to examine the adaptive strategies of E. coli.
2021-03-08 | GSE167389 | GEO
Project description:We used RNA-seq to profile E. coli K-12 MG1655 strains subjected to adaptive laboratory evolution after chorismate synthase knockouts. Either isochorismate synthase (menF) or isochorismate synthase AND chorismate lyase (ubiC) was deleted from a strain of E. coli K-12 MG1655 that had already been previously adapted for growth on glucose minimal media. RNA-seq profiles of the original glucose-adapted strain, the 2 deletion strains, and 4 laboratory-evolved strains from each deletion are included in duplicate. ubiC catalyzes the first committed step of ubiquinone synthesis, an important molecule for the electron transport chain. Thus, these experiments allowed assessment of cellular adaptations to restore energy metabolism capability.
2023-02-08 | GSE224889 | GEO
Project description:We performed massive screening of the genes in yeast that were involved in the tolerance to isopropanol using the non-essential genes deleted yeast collection, and identified sixty-five disruptants that grew slower than the wild type strain in the presence of isopropanol. The isopropanol sensitive mutants were tested to know their behaviour under other alcohol stresses. Besides, we conducted microarray analysis to reveal the transcriptional response to isopropanol stress in yeast. Our results certainly provide new insights into yeast response to C3 alcohol isopropanol. Strain BY4743 was used in this test. The prepared cells were inoculated into 120 ml of fresh media (YPD without isopropanol, and YPD with 20 g/l or 40 g/l of isopropanol) in 500-ml Sakaguchi-flasks. They were cultivated for 3 h at 30M-BM-0C with 130-rpm shaking. At least three independent experiments were performed at each concentration of isopropanol.
2013-05-13 | E-GEOD-46384 | biostudies-arrayexpress
Project description:Various environmental bacteria were adapted to the presence of toxic and recalcitrant organic solvents. Cupriavidus metallidurans CH34, a β-proteobacterium that was found in industrial environments is known as a bacterial model to study heavy metal resistance. Interestingly, genome screening of CH34 also reveals the existence of genes involved in the degradation of recalcitrant organic solvents, such acetone and aromatic compounds. Here, we showed that this bacterium could resist a large variety of organic solvents, and was also able to metabolize some of them. In particular, investigations were focused on acetone and isopropanol catabolism. Integrative studies based on transcriptomic (DNA microarrays), proteomic (2D-DIGE and Isotope-Coded Protein Label technology) and biochemical analyses (enzyme purification and characterization) showed a similar catabolic pathway for both molecules which involved the AcxABC acetone carboxylase. First, isopropanol is oxidized into acetone by the Adh alcohol dehydrogenase. Acetoacetate production from acetone is then catalyzed by the acetone carboxylase. The generated acetoacetate molecules are then transformed by the PacIJ 3-oxoacid CoA-transferase, to acetoacetyl-CoA and succinate. Finally, an acetyl-CoA acetyltransferase catalyses the hydrolysis of acetoacetyl-CoA into 2 acetyl-CoA that are introduced into the glyoxylate cycle. As demonstrated, key enzymes of the acetone/isopropanol catabolism (encoded by acx and ald genes) are under the control of a σ54-dependent RNA polymerase. Moreover, the catabolic pathway involved in acetone and isopropanol consumption was repressed when gluconate was given as alternative carbon substrate, displaying a new example of diauxie. The results presented here provide a comprehensive picture of acetone and isopropanol biodegradation in Cupriavidus metallidurans CH34 and strongly support the fact that CH34 can be considered as a solvent tolerant bacterium.
2015-08-31 | GSE33578 | GEO