Project description:Methanol, being electron-rich and derivable from methane or CO2, is a potentially renewable one-carbon (C1) feedstock for microorganisms. Although the ribulose monophosphate (RuMP) cycle used by methylotrophs to assimilate methanol differs from the typical sugar metabolism by only three enzymes, turning a non-methylotrophic organism to a synthetic methylotroph that grows to a high cell density has been challenging. Here, we reprogrammed E. coli using metabolic robustness criteria followed by laboratory evolution to establish a strain that can utilize methanol as the sole carbon source efficiently. This synthetic methylotroph alleviated a heretofore uncharacterized hurdle, DNA-protein crosslinking (DPC), by insertion sequence (IS) mediated copy number variations (CNV) and balanced the metabolic flux by mutations. Being capable of growing at a rate comparable to natural methylotrophs in a wide-range of methanol concentrations, this synthetic methylotrophic strain illustrates genome editing and evolution for microbial tropism changes, and expands the scope of biological C1 conversion.
Project description:Reprogramming a non-methylotrophic industrial host, such as Saccharomyces cerevisiae, to a synthetic methylotroph reprents a huge challenge due to the complex regulation in yeast. Through TMC strategy together with ALE strategy, we completed a strict synthetic methylotrophic yeast that could use methanol as the sole carbon source. However, how cells respond to methanol and remodel cellular metabolic network on methanol were not clear. Therefore, genome-scale transcriptional analysis was performed to unravel the cellular reprograming mechanisms underlying the improved growth phenotype.
Project description:Proteome characterization of three E. coli strains: a methanol-dependent strain (MEvo1-0) and two synthetically methylotrophic strains (MEcoli_ref_1, MEcoli_ref_2)
Project description:A total gene expression approach was applied to study the methylotrophic nature of B. methanolicus by comparing the gene expression in bacteria grown methylotropic compared to non-methylotrophic. Genes of interest with different gene expression were quantified in the same RNA samples by real-time PCR, confirming the results found in the microarray experiment. Genes of special interest that are expressed higher when grown methylotrophic, were the RuMP pathway genes located on the pBM19.
Project description:A total gene expression approach was applied to study the methylotrophic nature of B. methanolicus by comparing the gene expression in bacteria grown methylotropic compared to non-methylotrophic. Genes of interest with different gene expression were quantified in the same RNA samples by real-time PCR, confirming the results found in the microarray experiment. Genes of special interest that are expressed higher when grown methylotrophic, were the RuMP pathway genes located on the pBM19. Bacillus methanolicus was grown in minimal media with either methanol or mannitol as carbon source. The experiment was preformed in triplicate, with bacterial cultures grown on 3 different days.
Project description:The increasing demand for non-food competitive carbon sources such as methanol for biotechnology has brought methanol-utilizing bacteria, so-called methylotrophs, to focus. The product spectrum of natural methylotrophs and their genetic accessibility is limited and as an alternative approach, the introduction of methylotrophic metabolism into a biotechnologically well-established organism, such as Escherichia coli, represents a promising concept. By performing long-term evolution over 600 days, we obtained an E. coli strain that is able to grow on methanol as its sole carbon source at rates comparable to natural methylotrophic organisms. We confirmed that the strain forms its entire biomass from methanol. Furthermore, we sequenced the genome of the evolved strain and compared it to the genome of its ancestor. Intriguingly, we found several hundreds of mutations targeting genes of various functions, such as catalysis and regulation. Like the comparison of the genome before and after evolution, the investigation of the proteome would be of high interest. Proteomics would reveal the consequences of the regulatory mutations found in the genome and provide an overall picture of the adaptations by the cell enabling it to grow on methanol. The increasing demand for non-food competitive carbon sources such as methanol for biotechnology has brought methanol-utilizing bacteria, so-called methylotrophs, to focus. The product spectrum of natural methylotrophs and their genetic accessibility is limited and as an alternative approach, the introduction of methylotrophic metabolism into a biotechnologically well-established organism, such as Escherichia coli, represents a promising concept. By performing long-term evolution over 600 days, we obtained an E. coli strain that is able to grow on methanol as its sole carbon source at rates comparable to natural methylotrophic organisms. We confirmed that the strain forms its entire biomass from methanol. Furthermore, we sequenced the genome of the evolved strain and compared it to the genome of its ancestor. Intriguingly, we found several hundreds of mutations targeting genes of various functions, such as catalysis and regulation. Like the comparison of the genome before and after evolution, the investigation of the proteome would be of high interest. Proteomics would reveal the consequences of the regulatory mutations found in the genome and provide an overall picture of the adaptations by the cell enabling it to grow on methanol.
Project description:The synthetic flexicate compound Λ-5b shows potent antimicrobial activity against E. coli. In this study we investigated the transcriptomic response of a clinically relevant E. coli, EHEC O157:H7, to a sublethal concentration of the compound.