Dataset Information

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1.

ABSTRACT:

Background

Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Methylomonas sp. DH-1 must be identified to improve the genetic engineering prospects of this microorganism.

Results

We identified a DNA methylation site, TGGCCA, and its corresponding cytosine methyltransferase for the first time in Methylomonas sp. DH-1 through whole-genome bisulfite sequencing. The methyltransferase was confirmed to methylate the fourth nucleotide of TGGCCA. In general, methylated plasmids exhibited better transformation efficiency under the protection of the RM system than non-methylated plasmids did. As expected, when we transformed Methylomonas sp. DH-1 with plasmid DNA harboring the psy gene, the metabolic flux towards carotenoid increased. The methyltransferase-treated plasmid exhibited an increase in transformation efficiency of 2.5?×?10³ CFU/?g (124%). The introduced gene increased the production of carotenoid by 26%. In addition, the methyltransferase-treated plasmid harboring anti-psy sRNA gene exhibited an increase in transformation efficiency by 70% as well. The production of carotenoid was decreased by 40% when the psy gene was translationally repressed by anti-psy sRNA.

Conclusions

Plasmid DNA methylated by the discovered cytosine methyltransferase from Methylomonas sp. DH-1 had a higher transformation efficiency than non-treated plasmid DNA. The RM system identified in this study may facilitate the plasmid-based genetic manipulation of methanotrophs.

SUBMITTER: Ren J

PROVIDER: S-EPMC7720504 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1.

Ren Jun J Lee Hyang-Mi HM Thai Thi Duc TD Na Dokyun D

Biotechnology for biofuels 20201207 1

<h4>Background</h4>Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Methylomonas sp. DH-1 is a candidate platform for bioconversion that uses methane as a carbon source. Although several genetic engineering techniques have been developed to work with Methylomonas sp. DH-1, the genetic manipulation of plasmids remains difficult because of the restriction-modification (RM) system present in the bacteria. Therefore, the RM system in Me ...[more]

PMID: 33372613

Similar Datasets

Project description:Background:Methane, a main component of natural gas and biogas, has gained much attention as an abundant and low-cost carbon source. Methanotrophs, which can use methane as a sole carbon and energy source, are promising hosts to produce value-added chemicals from methane, but their metabolic engineering is still challenging. In previous attempts to produce lactic acid (LA) from methane, LA production levels were limited in part due to LA toxicity. We solved this problem by generating an LA-tolerant strain, which also contributes to understanding novel LA tolerance mechanisms. Results:In this study, we engineered a methanotroph strain Methylomonas sp. DH-1 to produce d-lactic acid (d-LA) from methane. LA toxicity is one of the limiting factors for high-level production of LA. Therefore, we first performed adaptive laboratory evolution of Methylomonas sp. DH-1, generating an LA-tolerant strain JHM80. Genome sequencing of JHM80 revealed the causal gene watR, encoding a LysR-type transcription factor, whose overexpression due to a 2-bp (TT) deletion in the promoter region is partly responsible for the LA tolerance of JHM80. Overexpression of the watR gene in wild-type strain also led to an increase in LA tolerance. When d form-specific lactate dehydrogenase gene from Leuconostoc mesenteroides subsp. mesenteroides ATCC 8293 was introduced into the genome while deleting the glgA gene encoding glycogen synthase, JHM80 produced about 7.5-fold higher level of d-LA from methane than wild type, suggesting that LA tolerance is a critical limiting factor for LA production in this host. d-LA production was further enhanced by optimization of the medium, resulting in a titer of 1.19 g/L and a yield of 0.245 g/g CH4. Conclusions:JHM80, an LA-tolerant strain of Methylomonas sp. DH-1, generated by adaptive laboratory evolution was effective in LA production from methane. Characterization of the mutated genes in JHM80 revealed that overexpression of the watR gene, encoding a LysR-type transcription factor, is responsible for LA tolerance. By introducing a heterologous lactate dehydrogenase gene into the genome of JHM80 strain while deleting the glgA gene, high d-LA production titer and yield were achieved from methane.

Project description:BackgroundMethylacidiphilum sp. IT6 has been validated its C3 substrate assimilation pathway via acetol as a key intermediate using the PmoCAB3, a homolog of the particulate methane monooxygenase (pMMO). From the transcriptomic data, the contribution of PmoD of strain IT6 in acetone oxidation was questioned. Methylomonas sp. DH-1, a type I methanotroph containing pmo operon without the existence of its pmoD, has been deployed as a biocatalyst for the gas-to-liquid bioconversion of methane and propane to methanol and acetone. Thus, Methylomonas sp. DH-1 is a suitable host for investigation. The PmoD-expressed Methylomonas sp. DH-1 can also be deployed for acetol production, a well-known intermediate for various industrial applications. Microbial production of acetol is a sustainable approach attracted attention so far.ResultsIn this study, bioinformatics analyses elucidated that novel protein PmoD is a C-terminal transmembrane-helix membrane with the proposed function as a transport protein. Furthermore, the whole-cell biocatalyst was constructed in Methylomonas sp. DH-1 by co-expression the PmoD of Methylacidiphilum sp. IT6 with the endogenous pMMO to enable acetone oxidation. Under optimal conditions, the maximum accumulation, and specific productivity of acetol were 18.291 mM (1.35 g/L) and 0.317 mmol/g cell/h, respectively. The results showed the first coupling activity of pMMO with a heterologous protein PmoD, validated the involvement of PmoD in acetone oxidation, and demonstrated an unprecedented production of acetol from acetone in type I methanotrophic biocatalyst. From the data achieved in batch cultivation conditions, an assimilation pathway of acetone via acetol as the key intermediate was also proposed.ConclusionUsing bioinformatics tools, the protein PmoD has been elucidated as the membrane protein with the proposed function as a transport protein. Furthermore, results from the assays of PmoD-heteroexpressed Methylomonas sp. DH-1 as a whole-cell biocatalyst validated the coupling activity of PmoD with pMMO to convert acetone to acetol, which also unlocks the potential of this recombinant biocatalyst for acetol production. The proposed acetone-assimilated pathway in the recombinant Methylomonas sp. DH-1, once validated, can extend the metabolic flexibility of Methylomonas sp. DH-1.

Dataset Information

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1.

Background

Results

Conclusions

Publications

Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets