Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH-1
- PDF / 2,088,174 Bytes
- 8 Pages / 595.276 x 790.866 pts Page_size
- 45 Downloads / 146 Views
Biotechnology for Biofuels Open Access
RESEARCH
Identification of a cytosine methyltransferase that improves transformation efficiency in Methylomonas sp. DH‑1 Jun Ren, Hyang‑Mi Lee, Thi Duc Thai and Dokyun Na*
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 engineer‑ ing 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 effi‑ ciency 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 plas‑ mid 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. Keywords: Methylomonas sp. DH-1, Transformation efficiency, DNA methylation, Cytosine methyltransferase Background Although methane contributes to the greenhouse effect much more than carbon dioxide does, it is a useful feedstock for methanotrophs, which are bacteria that utilize methane as a carbon source [1–3]. Methane can be converted into methanol [4], and methanol can be metabolized to many other value-added chemicals such *Correspondence: [email protected] Department of Biomedical Engineering, Chung-Ang University, Seoul 06974, Republic of Korea
as l-glutamate [5, 6], l-lysine [7, 8], cadaverine [9, 10], α-humulene [11], mesaconate, and (2S)-methyl-succinate [12] in metabolically engineered methanotrophs [13]. For efficient genetic engineering, genetic manipulation tools have been developed to work with methanotrophs [14–17]. Recently, the type l Methylomonas sp. DH-1 was isolated from brewery waste sludge, and s
Data Loading...
