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/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.

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: Cytosine methyltransferase; DNA methylation; Methylomonas sp. DH-1; Transformation efficiency.

Fig. 1

Identification of a methylation site and its corresponding methyltransferase in Methylomonas sp. DH-1. a The TGGCCA methylation site was the only site discovered by WGBS. b The potential methyltransferases in Methylomonas sp. DH-1. The REBASE-predicted methyltransferases in its genome and native plasmid are shown

Fig. 2

Agarose gel electrophoresis of the plasmid DNA cleaved by restriction enzymes. a Restriction patterns of non-methylated and methylated plasmid DNA. The plasmid map and restriction sites are also shown. Methylation was induced by 0.1 mM IPTG, which initiates the expression of the cytosine methyltransferase gene. M denotes a DNA marker. The treated restriction enzymes are shown in each lane. b The conversion of non-methylated and methylated TGGCCA sequences during bisulfite sequencing. Only non-methylated cytosines are converted to thymines during bisulfite sequencing and PCR. The left-hand figure shows that the two cytosines were converted to uracils, which means there were no methylated cytosines. Conversely, the right-hand figure shows that the fourth cytosine was not changed to uracil, indicating that it was methylated by the cytosine methyltransferase

Fig. 3

Plasmid maps of the constructed plasmids. a The cytosine methyltransferase-containing plasmid and psy-containing plasmid are shown. The psy-containing plasmid was constructed in a proof-of-concept metabolic engineering process to increase the metabolic flux towards carotenoid, and the cytosine methyltransferase-containing plasmid was used to methylate the psy-containing plasmid. The psy gene was under the control of the mxaF promoter, encoding subunit of methanol dehydrogenase [32], which was predicted using Promoter Hunter [37]. b The overall strategy of plasmid methylation in E. coli (JM110) and transformation into Methylomonas sp. DH-1. The first step was to methylate the target plasmid using cytosine methyltransferase, and the second step was to transform it into Methylomonas sp. DH-1 after separation from the methylase-containing plasmid

Fig. 4

The transformation efficiency of methylated plasmid DNAs. a The overall biosynthetic pathway towards carotenoid. The psy gene is indicated. b Transformation efficiencies of non-methylated plasmids (light gray bar) and methylated plasmids (dark gray bar) in Methylomonas sp. DH-1. The maps of the two plasmids are shown in Fig. 3a. Standard deviations were calculated from triplicates. The asterisk (*) denotes p values < 0.05. c Carotenoid intensity in Methylomonas sp. DH-1 cells after transformation with the methylated psy plasmid. The intensity was measured using multi-detection microplate reader, and the carotenoid intensity was obtained 8 h after cultivation. Standard deviations were calculated from triplicates

Fig. 5

Transformation efficiency of a methylated plasmid harboring anti-psy synthetic sRNA gene. a An anti-psy synthetic sRNA gene was designed to reduce the expression of the psy gene. The synthetic sRNA binds to the coding region of the psy gene and represses the translation of the psy mRNA. The binding energy was calculated by mfold [38]. b Transformation efficiencies of the non-methylated plasmid (light gray bar) and methylated plasmid (dark gray bar) in Methylomonas sp. DH-1. Standard deviations were calculated from triplicates. The asterisk (*) denotes p values < 0.05. c Carotenoid intensity (arbitrary unit) in Methylomonas sp. DH-1 cells. The intensity was measured using multi-detection microplate reader, and the carotenoid intensity was obtained 8 h after cultivation. Standard deviations were calculated from triplicates. The asterisk (*) denotes p values < 0.05