Abstract
BackgroundIndustrial 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.ResultsWe 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/μg (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.ConclusionsPlasmid 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.
Highlights
Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms
According to REBASE, it was predicted that the cytosine methyltransferase AYM39_01025 (Additional file 1) would recognize the GGCC sequence for methylation, which is similar to the identified methylation site TGGCCA, in which the fourth nucleotide (C) was methylated in our results
This cytosine methyltransferase was selected as a potential methylase for TGGCCA
Summary
Industrial biofuels and other value-added products can be produced from metabolically engineered microorganisms. Several genetic engineering techniques have been developed to work with Methylomonas sp. Genetic manipulation tools have been developed to work with methanotrophs [14,15,16,17]. DH-1 was isolated from brewery waste sludge, and several engineering tools have been developed [18]. This bacterium has been favored in diverse examples of metabolic engineering: the conversion of methane to methanol [18] and the production of value-added. Foreign DNA is not methylated and is destroyed by host restriction enzymes. The process of DNA methylation is utilized for epigenetic regulation [27] and nanopore sequencing [28]
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