Matching epigenetic signatures with epigenetic enzymes is a little more difficult than matching John Hancock’s signature with, well, John Hancock. But the epigenetic equivalent of signature analysis has been accomplished by scientists based at the Technical University of Denmark.

According to the scientists, matching DNA methylation patterns, a kind of epigenetic motif, to DNA methyltransferases may allow scientists to understand how expression hosts accept methylomic signatures on nonnative DNA sequences—or reject them as forgeries. More important, a systematic application of the scientists’ findings could streamline the development of production hosts, or cell factories that incorporate recombinant genomes. If production hosts could be engineered to carry curated methylomes, they would more readily accept nonnative DNA, specifically, methylomically compatible nonnative DNA.

“Working in other bacteria than Escherichia coli, you often have to do a lot of trial and error when it comes to DNA transformation, but that’s just not good enough,” said Torbjørn Ølshøj Jensen, a researcher at the Technical University of Denmark. “You need knowledge and tools. With this, you have a systematic and rational way of fixing the problems.”

Jensen is the first author of an article (“Genome-wide systematic identification of methyltransferase recognition and modification patterns”) that appeared August 19 in the journal Nature Communications. This article describes MetMap, a high-throughput method to experimentally demonstrate the DNA modification specificity of methyltransferases. The method allowed the scientists to couple enzymes with specific methylation patterns in two bacteria.

“[We use] automated cloning and [analyze] methyltransferases in vectors carrying a strain-specific cassette containing all potential target sites,” wrote the article’s authors. “To validate the method, we analyze the genomes of the thermophile Moorella thermoacetica and the mesophile Acetobacterium woodii, two acetogenic bacteria having substantially modified genomes with 12 methylation motifs and a total of 23 methyltransferase genes.

Essentially, the scientists constructed plasmids containing one of the methyltransferases and cassettes holding multiple copies of certain DNA patterns. These DNA patterns, called motifs, are the targets for methyltransferases. By coupling the two, the methyltransferase expressed by the plasmid would mark the DNA in a specific way, thus, revealing the enzyme’s methylation pattern.

This was done for all methyltransferases. Afterwards, all the plasmids (in a pool) were read using a sequencing method designed to reveal methyl groups. This gave the researchers a library of enzyme-to-motif couplings.

To validate the method, the scientists analyzed the genomes of the host bacteria. Both bacteria, incidentally, are hosts with great potential for industrial applications and substantially modified genomes.

In total, the two bacterial organisms hold 23 methyltranstransferase genes, but only show modification on 12 different DNA motifs on their genomes, meaning that not all methyltransferases are active.

“Using our method,” the Nature Communications article indicated, “we characterize the 23 methyltransferases, assign motifs to the respective enzymes, and verify activity for 11 of the 12 motifs.” That is, for 11 of the 12 motifs, the scientists were able to couple activity to specific methyltransferases gene.

The method could allow scientists to design hosts with an unambiguous methylome—meaning that the organism harbors only wanted methyltransferases, which will ease introduction of foreign DNA into non-model organisms. This can be useful both when building cell factories based upon new or lesser-known hosts, and when trying to understand the regulation of gene expression and cell differentiation.

“Knowing which enzyme does what opens up to a lot of applications,” Jensen asserted. “With this knowledge, you can construct model organisms with artificial methylomes, mimicking the methylation pattern of the strain you want to introduce DNA to. In this way you can ensure ‘survival’ of introduced DNA.”

All species mark their DNA with methyl groups. This is done to regulate gene expression, distinguish indigenous DNA from foreign DNA, or to mark old DNA strands during replication. Methylation is carried out by methyltransferases, which decorate DNA with methyl groups in certain patterns to create an epigenetic layer on top of DNA.

Scientists often run into problems with methylation while attempting to introduce foreign DNA to a host organism, for instance bacteria or yeast. But introducing foreign DNA is essential when building production hosts capable of producing medicines, sustainable biochemicals, and food ingredients. Often, the host needs genes from other organisms to produce the sought-for compounds.

But just as often as not, the host will reject the foreign DNA and chop it into pieces, simply because the methylation patterns reveal that the DNA is alien. Scientists working with E. coli as their host, usually don’t have that many problems—or fewer than others—when introducing new DNA, since E. coli is well-known and rather “well behaved.” But moving into lesser known hosts can become a great problem.

In the current study, the two bacterial strains that were used are distant from the expression host E. coli, both with respect to growth temperature and codon usage. These strains, which were chosen specifically to demonstrate the robustness of the developed method, are gas-consuming bacteria with a scarce product spectrum, making them highly interesting hosts for production of biochemicals.

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