Comparative transcriptome analysis of a novel obligate Methylomonas sp. DH-1 reveals key differences in the transcriptional responses in C1 and secondary metabolite pathways during growth on methane and methanol
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ABSTRACT:
ORGANISM(S): Methylomonas sp. DH-1
PROVIDER: GSE101494 | GEO | 2019/02/26
REPOSITORIES: GEO
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Project description:Methylomicrobium buryatense 5GB1 is an obligate methylotroph, which grows on methane or methanol with similar growth rates. Core metabolic pathways are similar on both substrates, but recent studies of methane metabolism suggest that growth on methanol might have significant differences from growth on methane. In this study, both a targeted metabolomics approach as well as a 13C tracer approach have been taken to understand core carbon metabolism in M. buryatense 5GB1 during methanol growth, to determine whether such differences occur. Targeted metabolomics analyses were performed on both methane and methanol cultures to identify metabolic nodes with altered fluxes. Several key metabolites showed significant differences in pool size. Noticeably, 2-keto-3-deoxy-6-phosphogluconate (KDPG) showed much larger pools under methanol culture, suggesting the Entner-Doudoroff (ED) pathway was more active. Intermediates in other parts of metabolism also showed differences in pool sizes under methanol growth. A systematic shift of active core metabolism is proposed to explain the changes. In order to distinguish flux partition differences at the C3-C4 node, 13C tracer analysis was also applied to methanol-grown cultures. Using the experimental results as constraints, we applied flux balance analysis to determine the metabolic flux phenotype of M. buryatense 5GB1 growing on methanol. The resulting new insights into core metabolism of this methanotroph provide an improved basis for future strain design.
2019-04-17 | GSE110541 | GEO
Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane. Differential gene analysis of two growth conditions (three biological replicates each) was performed: (i) M. acetivorans/pES1-MATmcr3 grown on methane and (ii) M. acetivorans/pES1-MATmcr3 grown on methanol. All starter cultures (200 mL) were grown on methanol for 5 days, and harvested by centrifugation. Cell pellets were washed three times with HS medium, and resuspended using 5 mL HS medium, 2 µg/mL puromycin, and 0.1 mM FeCl3. For condition (i), methane was filled into the headspace of the cultures. For condition (ii), 150 mM methanol was added. All cultures were incubated at 37C for 5 days, followed by rapid centrifugation in the presence of 50 µL RNAlater solution (Ambion, Austin, TX) per mL of culture. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) were then digested with terminator 5â-phosphate-dependent exonuclease (Epicentre, Madison, WI) to partially remove ribosomal RNA. Digested RNA were cleaned up using AgenCourt RNAClean XP beads (AgenCourt Bioscience, Beverly, MA) and used for cDNA library construction using the TruSeq Stranded mRNA Library kit (Illumina). Pooled and barcoded cDNA library was then sequenced on a HiSeq sequencing platform (Illumina). Obtained reads were mapped to the reference genome of M. acetivorans (Genbank accession NC_003552.1) using STAR. The mapped reads were assembled using Cufflink v2.2.1 to identify potential novel transcripts. Assembled, unannotated novel transcripts for all the strains were combined with the list of known genes. Differential expression of genes and potential novel transcripts were determined using Cuffdiff at a significance cutoff at q < 0.07 with a false discovery rate of 0.05. Expression levels of gene transcripts are expressed as fragments per kilobase of transcript per million mapped fragments (FPKM), and expression changes are determined by the ratio of FPKM of culture replicates grown on methane to FPKM of culture replicates grown on methanol.
2015-12-20 | E-GEOD-66445 | biostudies-arrayexpress
Project description:Tris(4-chlorophenyl)methane (TCPM) and tris(4-chlorophenyl)methanol (TCPMOH) are two marine pollutants of emerging concern, found in marine mammals and coastal bird populations. Here, we assess the response to these compounds due to acute exposures in zebrafish embryos (from 96-100 hours post fertilization).
2021-09-09 | GSE165920 | GEO
Project description:Tris(4-chlorophenyl)methane (TCPM) and tris(4-chlorophenyl)methanol (TCPMOH) are two marine pollutants of emerging concern, found in marine mammals and coastal bird populations. Here, we assess the response to these compounds due to developmental (24-100 hourrs post fertilization) exposures in zebrafish embryos
2022-12-31 | GSE198106 | GEO
Project description:BackgroundMethanotrophs play an important role in biotechnological applications, with their ability to utilize single carbon (C1) feedstock such as methane and methanol to produce a range of high-value compounds. A newly isolated obligate methanotroph strain, Methylomonas sp. DH-1, became a platform strain for biotechnological applications because it has proven capable of producing chemicals, fuels, and secondary metabolites from methane and methanol. In this study, transcriptome analysis with RNA-seq was used to investigate the transcriptional change of Methylomonas sp. DH-1 on methane and methanol. This was done to improve knowledge about C1 assimilation and secondary metabolite pathways in this promising, but under-characterized, methane-bioconversion strain.ResultsWe integrated genomic and transcriptomic analysis of the newly isolated Methylomonas sp. DH-1 grown on methane and methanol. Detailed transcriptomic analysis indicated that (i) Methylomonas sp. DH-1 possesses the ribulose monophosphate (RuMP) cycle and the Embden-Meyerhof-Parnas (EMP) pathway, which can serve as main pathways for C1 assimilation, (ii) the existence and the expression of a complete serine cycle and a complete tricarboxylic acid (TCA) cycle might contribute to methane conversion and energy production, and (iii) the highly active endogenous plasmid pDH1 may code for essential metabolic processes. Comparative transcriptomic analysis on methane and methanol as a sole carbon source revealed different transcriptional responses of Methylomonas sp. DH-1, especially in C1 assimilation, secondary metabolite pathways, and oxidative stress. Especially, these results suggest a shift of central metabolism when substrate changed from methane to methanol in which formaldehyde oxidation pathway and serine cycle carried more flux to produce acetyl-coA and NADH. Meanwhile, downregulation of TCA cycle when grown on methanol may suggest a shift of its main function is to provide de novo biosynthesis, but not produce NADH.ConclusionsThis study provides insights into the transcriptomic profile of Methylomonas sp. DH-1 grown on major carbon sources for C1 assimilation, providing in-depth knowledge on the metabolic pathways of this strain. These observations and analyses can contribute to future metabolic engineering with the newly isolated, yet under-characterized, Methylomonas sp. DH-1 to enhance its biochemical application in relevant industries.
| S-EPMC6373157 | biostudies-literature
Project description:Our goal is to convert methane efficiently into liquid fuels that may be more readily transported. Since aerobic oxidation of methane is less efficient, we focused on anaerobic processes to capture methane, which are accomplished by anaerobic methanotrophic archaea (ANME) in consortia. However, no pure culture capable of oxidizing and growing on methane anaerobically has been isolated. In this study, Methanosarcina acetivorans, an archaeal methanogen, was metabolically engineered to take up methane, rather than to generate it. To capture methane, we cloned the DNA coding for the enzyme methyl-coenzyme M reductase (Mcr) from an unculturable archaeal organism from a Black Sea mat into M. acetivorans to effectively run methanogenesis in reverse. The engineered strain produces primarily acetate, and our results demonstrate that pure cultures can grow anaerobically on methane.
2015-12-20 | GSE66445 | GEO
Project description:Methanol is considered as an interesting carbon source in biobased microbial production processes. As Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies on the response of this organism to methanol. C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be up-regulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The deletion mutants aldadhE and aldmshC were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF recently identified by us. Similar to ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogeneous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway.
2013-09-27 | GSE49936 | GEO
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.
2020-09-01 | GSE153477 | GEO
Project description:Methanol is considered as an interesting carbon source in biobased microbial production processes. As Corynebacterium glutamicum is an important host in industrial biotechnology, in particular for amino acid production, we performed studies on the response of this organism to methanol. C. glutamicum wild type was able to convert 13C-labeled methanol to 13CO2. Analysis of global gene expression in the presence of methanol revealed several genes of ethanol catabolism to be up-regulated, indicating that some of the corresponding enzymes are involved in methanol oxidation. Indeed, a mutant lacking the alcohol dehydrogenase gene adhA showed a 62% reduced methanol consumption rate, indicating that AdhA is mainly responsible for methanol oxidation to formaldehyde. Further studies revealed that oxidation of formaldehyde to formate is catalyzed predominantly by two enzymes, the acetaldehyde dehydrogenase Ald and the mycothiol-dependent formaldehyde dehydrogenase AdhE. The deletion mutants M-oM-^AM-^DaldM-oM-^AM-^DadhE and M-oM-^AM-^DaldM-oM-^AM-^DmshC were severely impaired in their ability to oxidize formaldehyde, but residual methanol oxidation to CO2 was still possible. The oxidation of formate to CO2 is catalyzed by the formate dehydrogenase FdhF recently identified by us. Similar to ethanol, methanol catabolism is subject to carbon catabolite repression in the presence of glucose and is dependent on the transcriptional regulator RamA, which was previously shown to be essential for expression of adhA and ald. In conclusion, we were able to show that C. glutamicum possesses an endogeneous pathway for methanol oxidation to CO2 and to identify the enzymes and a transcriptional regulator involved in this pathway. Whole-genome DNA microarray analyses were performed to monitor changes in the global gene expression of C. glutamicum wild type in response to the presence of methanol.
2013-09-27 | E-GEOD-49936 | biostudies-arrayexpress
Project description:JHM80 strain was obtained by adaptive evolution of Methylomonas sp. DH-1 strain against 8.0 g/L of lactate. The tolerance against lactate of JHM80 was obtained by the overexpression of watR (weak acid tolerance regulator). In order to discover the target genes of WatR, FLAG epitope tag was added to WatR of JHM80 strain and ChIP-seq was performed.
2023-08-08 | GSE206216 | GEO