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. 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/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. 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.

中文翻译：

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鉴定出胞嘧啶甲基转移酶，该酶提高了甲基单孢菌属sp。中的转化效率。DH-1

工业生物燃料和其他增值产品可以由代谢工程微生物生产。甲基单胞菌 DH-1是使用甲烷作为碳源的生物转化候选平台。尽管已经开发了几种基因工程技术来与甲基单孢菌属一起工作。DH-1，由于存在于细菌中的限制性修饰（RM）系统，质粒的遗传操作仍然很困难。因此，Methylomonas sp。中的RM系统。必须鉴定DH-1以改善该微生物的基因工程前景。我们首次在Methylomonas sp中鉴定了一个DNA甲基化位点TGGCCA及其对应的胞嘧啶甲基转移酶。DH-1通过全基因组亚硫酸氢盐测序。确认了甲基转移酶使TGGCCA的第四个核苷酸甲基化。通常，在RM系统的保护下，甲基化的质粒比非甲基化的质粒表现出更好的转化效率。如预期的那样，当我们转化了甲基单孢菌属时。带有带有psy基因的质粒DNA的DH-1，向类胡萝卜素的代谢通量增加。经甲基转移酶处理的质粒的转化效率提高了2.5×103 CFU /μg（124％）。引入的基因使类胡萝卜素的产量增加了26％。另外，带有抗-psy sRNA基因的甲基转移酶处理的质粒也显示出转化效率提高了70％。当psy基因被抗psy sRNA翻译抑制时，类胡萝卜素的产生减少了40％。被发现的来自甲基单胞菌属的胞嘧啶甲基转移酶甲基化的质粒DNA。DH-1的转化效率高于未处理的质粒DNA。在这项研究中确定的RM系统可能有助于甲烷营养生物的基于质粒的遗传操作。