Methylorubrum extorquens
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ABSTRACT:
PROVIDER: PRJNA448359 | ENA |
REPOSITORIES: ENA
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Project description:Genome Sequencing of Engineered and Evolved Methylobacterium Isolates
| PRJNA273781 | ENA
Project description:Methylobacterium chloromethanicum overview
| PRJNA36191 | ENA
Project description:Ethylamine versus succinate cells of Methylobacterium extorquens
| PRJNA125461 | ENA
Project description:The genome of three strains of soil-isolated Methylobacterium extorquens
| PRJNA884983 | ENA
Project description:Genetic tools are a prerequisite to engineer cellular factories for synthetic biology and biotechnology. Methylorubrum extorquens AM1 is an important platform organism of a future C1-bioeconomy. However, its application is currently limited by the availability of genetic tools. Here we systematically tested repABC regions to maintain extrachromosomal DNA in M. extorquens. We used three elements to construct mini-chromosomes that are stably inherited at single copy number and can be shuttled between Escherichia coli and M. extorquens. These mini-chromosomes are compatible among each other and with high-copy number plasmids of M. extorquens. We also developed a set of inducible promoters of wide expression range, reaching levels exceeding those currently available, notably the PmxaF-promoter. In summary, we provide a set of tools to control the dynamic expression and copy number of genetic elements in M. extorquens, which opens new ways to unleash the metabolic and biotechnological potential of this organism for future applications.
| S-EPMC6862569 | biostudies-literature
Project description:Methylorubrum extorquens (formerly Methylobacterium extorquens) AM1 is a methylotrophic bacterium with a versatile lifestyle. Various carbon sources including acetate, succinate and methanol are utilized by M. extorquens AM1 with the latter being a promising inexpensive substrate for use in the biotechnology industry. Itaconic acid (ITA) is a high-value building block widely used in various industries. Given that no wildtype methylotrophic bacteria are able to utilize methanol to produce ITA, we tested the potential of M. extorquens AM1 as an engineered host for this purpose. In this study, we successfully engineered M. extorquens AM1 to express a heterologous codon-optimized gene encoding cis-aconitic acid decarboxylase. The engineered strain produced ITA using acetate, succinate and methanol as the carbon feedstock. The highest ITA titer in batch culture with methanol as the carbon source was 31.6 ± 5.5 mg/L, while the titer and productivity were 5.4 ± 0.2 mg/L and 0.056 ± 0.002 mg/L/h, respectively, in a scaled-up fed-batch bioreactor under 60% dissolved oxygen saturation. We attempted to enhance the carbon flux toward ITA production by impeding poly-β-hydroxybutyrate accumulation, which is used as carbon and energy storage, via mutation of the regulator gene phaR. Unexpectedly, ITA production by the phaR mutant strain was not higher even though poly-β-hydroxybutyrate concentration was lower. Genome-wide transcriptomic analysis revealed that phaR mutation in the ITA-producing strain led to complex rewiring of gene transcription, which might result in a reduced carbon flux toward ITA production. Besides poly-β-hydroxybutyrate metabolism, we found evidence that PhaR might regulate the transcription of many other genes including those encoding other regulatory proteins, methanol dehydrogenases, formate dehydrogenases, malate:quinone oxidoreductase, and those synthesizing pyrroloquinoline quinone and thiamine co-factors. Overall, M. extorquens AM1 was successfully engineered to produce ITA using acetate, succinate and methanol as feedstock, further supporting this bacterium as a feasible host for use in the biotechnology industry. This study showed that PhaR could have a broader regulatory role than previously anticipated, and increased our knowledge of this regulator and its influence on the physiology of M. extorquens AM1.
| S-EPMC6520949 | biostudies-literature
Project description:Here, we report the complete genome sequences of Methylorubrum extorquens NBC_00036 and Methylorubrum extorquens NBC_00404. The genomes were sequenced using the Oxford Nanopore Technologies MinION and Illumina NovaSeq systems. Both genomes are circular, with sizes of 5,661,342 bp and 5,869,086 bp, respectively.
| S-EPMC10353431 | biostudies-literature
Project description:Plasmids are one of the most important genetic tools for basic research and biotechnology, as they enable rapid genetic manipulation. Here we present a novel pBBR1-based plasmid for Methylorubrum extorquens, a model methylotroph that is used for the development of C1-based microbial cell factories. To develop a vector with compatibility to the so far mainly used pCM plasmid system, we transferred the pBBR1-based plasmid pMiS1, which showed an extremely low transformation rate and caused a strong growth defect. Isolation of a suppressor mutant with improved growth led to the isolation of the variant pMis1_1B. Its higher transformation rate and less pronounced growth defect phenotype could be shown to be the result of a mutation in the promotor region of the rep gene. Moreover, cotransformation of pMis1_1B and pCM160 was possible, but the resulting transformants showed stronger growth defects in comparison with a single pMis1_1B transformant. Surprisingly, cotransformants carrying pCM160 and a pMis1_1B derivative containing a mCherry reporter construct showed higher fluorescence levels than strains containing only the pMis1_1B-based reporter plasmids or a corresponding pCM160 derivative. Relative plasmid copy number determination experiments confirmed our hypothesis of an increased copy number of pMis1_1B in the strain carrying both plasmids. Despite the slight metabolic burden caused by pMis1_1B, the plasmid strongly expands the genetic toolbox for M. extorquens.
| S-EPMC9531332 | biostudies-literature
Project description:Methylorubrum extorquens AM1 is an efficient platform strain possessing biotechnological potential in formate- and methanol-based single carbon (C1) bioeconomy. Constitutive expression or costly chemical-inducible expression systems are not always desirable. Here, several glucose-, xylose-, and levulinic acid (LA)-inducible promoter systems were assessed for the induction of green fluorescent protein (GFP) as a reporter protein. Among them, the LA-inducible gene expression system (HpdR/P hpdH ) showed a strong expression of GFP (51-fold) compared to the control. The system was induced even at a low concentration of LA (0.1 mM). The fluorescence intensity increased with increasing concentrations of LA up to 20 mM. The system was tunable and tightly controlled with meager basal expression. The maximum GFP yield obtained using the system was 42 mg/g biomass, representing 10% of the total protein content. The efficiency of the proposed system was nearly equivalent (90%-100%) to that of the widely used strong promoters such as P mxaF and P L/O4 . The HpdR/P hpdH system worked equally efficiently in five different strains of M. extorquens. LA is a low-cost, renewable, and sustainable platform chemical that can be used to generate a wide range of products. Hence, the reported system in potent strains of M. extorquens is highly beneficial in the C1-biorefinery industry to produce value-added products and bulk chemicals.
| S-EPMC8714952 | biostudies-literature