Project description:The goal of this work was to measure the half-lives of RNA transcripts and identify which genes in Methanosarcina acetivorans C2A were differentially expressed between methylotrophic and acetitrophic growth substrates. We used this data to predict metabolic phenotype under different growth condition and hypothesize whether key metabolic genes were transcriptionally or degradationally regulated.
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: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: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:3-hydroxypropionic acid (3-HP) is a promising platform chemical with various industrial applications. Several metabolic routes to produce 3-HP from organic substrates such as sugars or glycerol have been implemented in yeast, enterobacterial species and other microorganisms. In this work, we investigated 3-HP metabolism of the well-studied ‘Knallgas bacterium’ Cupriavidus necator, a potential C1-chassis for the production of 3-HP and other fatty acid derivatives from CO2 and H2. When testing C. necator for its tolerance towards 3-HP, it was noted that it could utilise the compound as the sole source of carbon and energy.
