New tool cracks microbial defense codes for faster, precise bioengineering

Scientists at the Department of Energy's Oak Ridge National Laboratory have developed software that reduces the time needed for a key task in the development of custom microbes from a week to just hours. The new tool cracks a key defense mechanism of microorganisms, expediting the creation of microbes with desired traits for the production of new biofuels and other valuable products for the bioeconomy.

The software, named MIJAMP, detects patterns of DNA sequences centered around methylation chemical markers, which function as gene on/off switches. These chemical modifications influence how microbes grow, defend themselves against viruses, and prevent new genetic material from entering a cell.

Microbes add these methyl groups to their DNA to signal enzymes to bypass the DNA segments while attacking any foreign DNA that doesn't have the markers. This defense mechanism, called a restriction-modification system, can create challenges when scientists try to introduce new genetic material into microbes to give them enhanced properties for biomanufacturing.

MIJAMP uses outputs from synthetic biology tools, including nanopore sequencing data that identifies DNA letter order, to identify which parts of the microbial genome are marked with methyl groups. The process works by reading long stretches of DNA and then using pattern recognition algorithms to detect where the methyl groups are located.

The software, described in the Journal of Industrial Microbiology and Biotechnology, predicts which regions of the DNA are likely to contain the methyl markers that protect the cell, so that scientists can use those same markers to bypass the cell's defense system. MIJAMP is open-source software, available on ORNL's Gitlab.

A key feature of MIJAMP is its inclusion of a "human-in-the-loop" validation and refinement process to review predictions, recognizing that biological data doesn't always behave in perfect, predictable ways.

MIJAMP discovered methylated motifs in dozens of naturally isolated microbial strains and modified bacteria. By identifying the exact positions and patterns of DNA methylation, scientists can use the tool to mimic these natural patterns and modify microbial DNA in a way that will not trigger defense mechanisms.

"The greatest impact is expediting the domestication of non-model microbes for specific, efficient functions," said project lead Bill Alexander in ORNL's Synthetic Biology Group. "A task that might have taken a synthetic biologist a week to work on, starting with the genome and requiring high-performance computing resources, can now take just an hour on a laptop."

The new tool builds on a method developed by ORNL and other scientists as part of the DOE Center for Bioenergy Innovation to trick non-model microbes into accepting foreign DNA to enhance desired traits.

MIJAMP is part of a growing body of computational and other tools developed at ORNL for bioeconomy research. The tools automate synthetic biology workflows and accelerate biotechnology innovations, enhancing the nation's energy security, innovation culture and global competitiveness.

These tools include:

• Adaptation of the SAGE gene editing tool to give scientists the ability to insert and test new DNA designs in a variety of microbes faster.
• Integration of the latest in mass spectrometry capabilities and expertise to analyze the plant root environment and help engineer plant-microbe interactions;
• A new CRISPR interference technique to customize microbial traits faster; and
• AI and molecular dynamics simulation tools to predict and facilitate beneficial plant-microbe relationships, supporting the DOE Genesis Mission for AI-driven scientific discovery.

Other ORNL contributors to MIJAMP include Alyssa Tidwell, Evelyn Faust, Carrie Eckert and Adam Guss. The project was supported by the DOE Office of Science Biological and Environmental Research program and the DOE Bioenergy Technologies Office's Agile BioFoundry consortium that facilitates partnerships between the national labs and industry to accelerate biomanufacturing.

UT-Battelle manages ORNL for the Department of Energy's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. The Office of Science is working to address some of the most pressing challenges of our time. For more information, please visit energy.gov/science. - Stephanie Seay