Adaptive laboratory evolution reveals general and specific chemical tolerance mechanisms and enhances biochemical production
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ABSTRACT:
PROVIDER: PRJEB32954 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| ERR3380180_1.fastq.gz | Fastqsanger.gz | |||
| ERR3380180_2.fastq.gz | Fastqsanger.gz | |||
| ERR3380190_1.fastq.gz | Fastqsanger.gz | |||
| ERR3380190_2.fastq.gz | Fastqsanger.gz | |||
| ERR3382870_1.fastq.gz | Fastqsanger.gz |
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Project description:Synthetic methylotrophy has recently been intensively studied to achieve methanol-based biomanufacturing of fuels and chemicals. However, attempts to engineer platform microorganisms to utilize methanol mainly focus on enzyme and pathway engineering. Herein, we enhanced methanol bioconversion of synthetic methylotrophs by improving cellular tolerance to methanol. A previously engineered methanol-dependent Corynebacterium glutamicum is subjected to adaptive laboratory evolution with elevated methanol content. Unexpectedly, the evolved strain not only tolerates higher concentrations of methanol but also shows improved growth and methanol utilization. Transcriptome analysis suggests increased methanol concentrations rebalance methylotrophic metabolism by down-regulating glycolysis and up-regulating amino acid biosynthesis, oxidative phosphorylation, ribosome biosynthesis, and parts of TCA cycle. Mutations in the O-acetyl-L-homoserine sulfhydrylase Cgl0653 catalyzing formation of L-methionine analog from methanol and methanol-induced membrane-bound transporter Cgl0833 are proven crucial for methanol tolerance. This study demonstrates the importance of tolerance engineering in developing superior synthetic methylotrophs.
| S-EPMC7205612 | biostudies-literature
Project description:Acetate is a promising economical and sustainable carbon source for bioproduction, but it is also a known cell-growth inhibitor. In this study, adaptive laboratory evolution (ALE) with acetate as selective pressure was applied to Halomonas bluephagenesis TD1.0, a fast-growing and contamination-resistant halophilic bacterium that naturally accumulates poly(3-hydroxybutyrate) (PHB). After 71 transfers, the evolved strain, B71, was isolated, which not only showed better fitness (in terms of tolerance and utilization rate) to high concentrations of acetate but also produced a higher PHB titer compared with the parental strain TD1.0. Subsequently, overexpression of acetyl-CoA synthetase (ACS) in B71 resulted in a further increase in acetate utilization but a decrease in PHB production. Through whole-genome resequencing, it was speculated that genetic mutations (single-nucleotide variation (SNV) in phaB, mdh, and the upstream of OmpA, and insertion of TolA) in B71 might contribute to its improved acetate adaptability and PHB production. Finally, in a 5 L bioreactor with intermittent feeding of acetic acid, B71 was able to produce 49.79 g/L PHB and 70.01 g/L dry cell mass, which were 147.2% and 82.32% higher than those of TD1.0, respectively. These results highlight that ALE provides a reliable method to harness H. bluephagenesis to metabolize acetate for the production of PHB or other high-value chemicals more efficiently.
| S-EPMC9103988 | biostudies-literature
Project description:There is great interest in developing synthetic methylotrophs that harbor methane and methanol utilization pathways in heterologous hosts such as Escherichia coli for industrial bioconversion of one-carbon compounds. While there are recent reports that describe the successful engineering of synthetic methylotrophs, additional efforts are required to achieve the robust methylotrophic phenotypes required for industrial realization. Here, we address an important issue of synthetic methylotrophy in E. coli: methanol toxicity. Both methanol, and its oxidation product, formaldehyde, are cytotoxic to cells. Methanol alters the fluidity and biological properties of cellular membranes while formaldehyde reacts readily with proteins and nucleic acids. Thus, efforts to enhance the methanol tolerance of synthetic methylotrophs are important. Here, adaptive laboratory evolution was performed to improve the methanol tolerance of several E. coli strains, both methylotrophic and non-methylotrophic. Serial batch passaging in rich medium containing toxic methanol concentrations yielded clones exhibiting improved methanol tolerance. In several cases, these evolved clones exhibited a > 50% improvement in growth rate and biomass yield in the presence of high methanol concentrations compared to the respective parental strains. Importantly, one evolved clone exhibited a two to threefold improvement in the methanol utilization phenotype, as determined via 13C-labeling, at non-toxic, industrially relevant methanol concentrations compared to the respective parental strain. Whole genome sequencing was performed to identify causative mutations contributing to methanol tolerance. Common mutations were identified in 30S ribosomal subunit proteins, which increased translational accuracy and provided insight into a novel methanol tolerance mechanism. This study addresses an important issue of synthetic methylotrophy in E. coli and provides insight as to how methanol toxicity can be alleviated via enhancing methanol tolerance. Coupled improvement of methanol tolerance and synthetic methanol utilization is an important advancement for the field of synthetic methylotrophy.
| S-EPMC7904680 | biostudies-literature
Project description:BackgroundCadmium has been a significant threat to environment and human health due to its high toxicity and wide application in fossil-fuel burning and battery industry. Cyanobacteria are one of the most dominant prokaryotes, and the previous studies suggested that they could be valuable in removing Cd2+ from waste water. However, currently, the tolerance to cadmium is very low in cyanobacteria. To further engineer cyanobacteria for the environmental application, it is thus necessary to determine the mechanism that they respond to high concentration of cadmium.ResultsIn this study, a robust strain of Synechocystis PCC 6803 (named ALE-9.0) tolerant to CdSO4 with a concentration up to 9.0 µM was successfully isolated via adaptive laboratory evolution over 802-day continuous passages under cadmium stress. Whole-genome re-sequencing was then performed and nine mutations were identified for the evolved strain compared to the wild-type strain. Among these mutations, a large fragment deletion in slr0454 encoding a cation or drug efflux system protein was found to contribute directly to the resistance to Cd2+ stress. In addition, five other mutations were also demonstrated related to the improved Cd2+ tolerance in ALE-9.0. Moreover, the evolved ALE-9.0 strain was found to obtain cross tolerance to some other heavy metals like zinc and cobalt as well as higher resistance to high light.ConclusionsThe work here identified six genes and their mutations related to Cd2+ tolerance in Synechocystis PCC 6803, and demonstrated the feasibility of adaptive laboratory evolution in tolerance modifications. This work also provided valuable information regarding the cadmium tolerance mechanism in Synechocystis PCC 6803, and useful insights for cyanobacterial robustness and tolerance engineering.
| S-EPMC6058365 | biostudies-literature
Project description:BackgroundThere is a need to replace petroleum-derived with sustainable feedstocks for chemical production. Certain biomass feedstocks can meet this need as abundant, diverse, and renewable resources. Specific ionic liquids (ILs) can play a role in this process as promising candidates for chemical pretreatment and deconstruction of plant-based biomass feedstocks as they efficiently release carbohydrates which can be fermented. However, the most efficient pretreatment ILs are highly toxic to biological systems, such as microbial fermentations, and hinder subsequent bioprocessing of fermentative sugars obtained from IL-treated biomass.MethodsTo generate strains capable of tolerating residual ILs present in treated feedstocks, a tolerance adaptive laboratory evolution (TALE) approach was developed and utilized to improve growth of two different Escherichia coli strains, DH1 and K-12 MG1655, in the presence of two different ionic liquids, 1-ethyl-3-methylimidazolium acetate ([C2C1Im][OAc]) and 1-butyl-3-methylimidazolium chloride ([C4C1Im]Cl). For multiple parallel replicate populations of E. coli, cells were repeatedly passed to select for improved fitness over the course of approximately 40 days. Clonal isolates were screened and the best performing isolates were subjected to whole genome sequencing.ResultsThe most prevalent mutations in tolerant clones occurred in transport processes related to the functions of mdtJI, a multidrug efflux pump, and yhdP, an uncharacterized transporter. Additional mutations were enriched in processes such as transcriptional regulation and nucleotide biosynthesis. Finally, the best-performing strains were compared to previously characterized tolerant strains and showed superior performance in tolerance of different IL and media combinations (i.e., cross tolerance) with robust growth at 8.5% (w/v) and detectable growth up to 11.9% (w/v) [C2C1Im][OAc].ConclusionThe generated strains thus represent the best performing platform strains available for bioproduction utilizing IL-treated renewable substrates, and the TALE method was highly successful in overcoming the general issue of substrate toxicity and has great promise for use in tolerance engineering.
| S-EPMC5691611 | biostudies-literature
Project description:To understand the mechanism of isopropanol tolerance of Escherichia coli for improvement of isopropanol production, we performed genome re-sequencing and transcriptome analysis of isopropanol tolerant E. coli strains obtained from parallel adaptive laboratory evolution under IPA stress.
2017-06-21 | GSE89685 | GEO
Project description:Background: The toxic gas carbon monoxide (CO) is abundantly present in synthesis gas (syngas) and certain industrial waste gases that can serve as feedstocks for the biological production of industrially significant chemicals and fuels. For efficient bacterial growth to occur, and to increase productivity and titres, a high resistance to the gas is required. The aerobic bacterium Cupriavidus necator H16 can grow on CO2 + H2, although it cannot utilise CO as a source of carbon and energy. This study aimed to increase its CO resistance through adaptive laboratory evolution. Results: To increase the tolerance of C. necator to CO, the organism was continually subcultured in the presence of CO both heterotrophically and autotrophically. Ten individual cultures were evolved heterotrophically with fructose in this manner and eventually displayed a clear growth advantage over the wild type strain. Next-generation sequencing revealed several mutations, including a single point mutation upstream of a cytochrome bd ubiquinol oxidase operon (cydA2B2), which was present in all evolved isolates. When a subset of these mutations was engineered into the parental H16 strain, only the cydA2B2 upstream mutation enabled faster growth in the presence of CO. Expression analysis, mutation, overexpression and complementation suggested that cydA2B2 transcription is upregulated in the evolved isolates, resulting in increased CO tolerance under heterotrophic but not autotrophic conditions. However, through subculturing on a syngas-like mixture with increasing CO concentrations, C. necator could also be evolved to tolerate high CO concentrations under autotrophic conditions. A mutation in the gene for the soluble [NiFe]-hydrogenase subunit hoxH was identified in the evolved isolates. When the resulting amino acid change was engineered into the parental strain, autotrophic CO resistance was conferred. A strain constitutively expressing cydA2B2 and the mutated hoxH gene exhibited high CO tolerance under both heterotrophic and autotrophic conditions. Conclusion: C. necator was evolved to tolerate high concentrations of CO, a phenomenon which was dependent on the terminal respiratory cytochrome bd ubiquinol oxidase when grown heterotrophically and the soluble [NiFe]-hydrogenase when grown autotrophically. A strain exhibiting high tolerance under both conditions was created and presents a promising chassis for syngas-based bioproduction processes.
| S-EPMC10164946 | biostudies-literature
Project description:Reinforcing microbial thermotolerance is a strategy to enable fermentation with flexible temperature settings and thereby to save cooling costs. Here, we report on adaptive laboratory evolution (ALE) of the amino acid-producing bacterium Corynebacterium glutamicum under thermal stress. After 65 days of serial passage of the transgenic strain GLY3, in which the glycolytic pathway is optimized for alanine production under oxygen deprivation, three strains adapted to supraoptimal temperatures were isolated, and all the mutations they acquired were identified by whole-genome resequencing. Of the 21 mutations common to the three strains, one large deletion and two missense mutations were found to promote growth of the parental strain under thermal stress. Additive effects on thermotolerance were observed among these mutations, and the combination of the deletion with the missense mutation on otsA, encoding a trehalose-6-phosphate synthase, allowed the parental strain to overcome the upper limit of growth temperature. Surprisingly, the three evolved strains acquired cross-tolerance for isobutanol, which turned out to be partly attributable to the genomic deletion associated with the enhanced thermotolerance. The deletion involved loss of two transgenes, pfk and pyk, encoding the glycolytic enzymes, in addition to six native genes, and elimination of the transgenes, but not the native genes, was shown to account for the positive effects on thermal and solvent stress tolerance, implying a link between energy-producing metabolism and bacterial stress tolerance. Overall, the present study provides evidence that ALE can be a powerful tool to refine the phenotype of C. glutamicum and to investigate the molecular bases of stress tolerance.
| S-EPMC4357955 | biostudies-literature
Project description:Base editing of general transcription factor Spt15 to enhance yeast stress tolerance
| PRJNA672711 | ENA