ORNL’s tSAGE fast-tracks microbial design for high-temp manufacturing

Scientists at the Department of Energy's Oak Ridge National Laboratory have developed a platform that engineers heat-loving microbes for industrial-scale manufacturing in a matter of weeks compared with previous approaches that can take months to years. This biotechnology tool strengthens American manufacturing capacity, supporting long-term energy security and global competitiveness.

The platform, thermophilic Serine recombinase Assisted Genome Engineering, or SAGE, quickly inserts DNA into the chromosomes of heat-tolerant microorganisms called thermophiles, "allowing much faster, much higher throughput strain engineering," said Adam Guss, principal investigator and lead for ORNL's Microbial Engineering Group. With tSAGE, "we can build and test hundreds of strains in a matter of a few weeks, compared with prior approaches that would have taken multiple months for just a handful of strains."

The tSAGE tool was created to accelerate synthetic biology work in Clostridium thermocellum, a heat-tolerant microbe that's good at breaking down plant biomass and producing valuable chemicals, as reported in the Journal of Industrial Microbiology and Biotechnology. The work supports the mission of the Center for Bioenergy Innovation (CBI) at ORNL, developing low-cost, highly efficient solutions for the manufacturing of advanced fuels and products from abundant, domestic biomass.

"Using biology to break down lignocellulose or other low-cost feedstocks happens much more efficiently at higher temperatures," Guss said. But a major obstacle to developing thermophiles has been that they are notoriously difficult to engineer. "tSAGE makes thermophilic organisms more accessible to the biotech industry for companies that want to take advantage of the faster reactions that happen at higher temperatures."

tSAGE builds on ORNL's earlier success deploying the SAGE platform across a wide range of microbes as a high-throughput, reliable method to insert DNA into chromosomes. tSAGE "extends this proven approach to heat-loving organisms, enabling the same high-throughput workflow but under thermophilic growth conditions," Guss said.

"The good thing about SAGE is that it just works," Guss said. In contrast to other genetic methods that require extensive optimization to reproduce, tSAGE is designed to do one job extremely well: "If your goal is to have your DNA efficiently put into the chromosome, that's what tSAGE does. By using tSAGE, our group at ORNL has built more strains of C. therm in the last two years than all researchers around the world have over the last 20 years."

tSAGE complements other genome-editing approaches such as the widely adopted CRISPR gene-editing tool, Guss noted. "CRISPR excels at targeted DNA cutting and gene knockouts, while tSAGE is really good at inserting DNA. The combination of these two tools can solve a lot of engineering challenges within microorganisms."

The team also used tSAGE to go beyond inserting genes to build and characterize a large genetic parts library for reliable, predictable downstream microbial engineering and biotechnology development.

The engineered microbes could also be used to enable efficient microbial breakdown of end-of-life plastics, turning what is now a waste stream into high-value commercial products.

tSAGE is available for licensing at ORNL.

The tSAGE development team also included Nandhini Ashok, Yasemin Kaygusuz, Heidi Schindel, Sarah Thurmon and Carrie Eckert. The research was funded by CBI, a Bioenergy Research Center supported by the DOE Office of Science Biological and Environmental Research program.

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