## WUSTL team receives $3.9M to engineer bacteria to produce drop-in fuels from lignin ##### 29 December 2017 Researchers at Washington University in St. Louis have received a$3.9-million grant from the Department of Energy (DOE) to develop bacteria that manufacture renewable biofuels.

Building on prior funding from the DOE Office of Biological and Environmental Research (BER) for systems biology work on Rhodococcus opacus, this project will apply innovative combinations of -omics and mass spectrometry data analysis, flux analysis, and machine learning to develop improved genome-scale models that will guide multiplex genome engineering of R. opacus strains with branched chain fatty acid esters (BCFAE) biosynthetic capacity and improved tolerance to lignin degradation products.

The project will take advantage of this organism’s capability to use both polysaccharides and lignin as carbon sources as well as its natural tolerance to phenolic compounds such as those produced by lignin breakdown.

The grant supports research in five Washington University labs, including those led by co-principal investigators Gautam Dantas, an associate professor of pathology and immunology; Tae Seok Moon, an assistant professor of energy, environment and chemical engineering; Marcus B. Foston, an assistant professor of energy, environment and chemical engineering; Yinjie Tang, an associate professor of energy, environment and chemical engineering; and Fuzhong Zhang, an associate professor of energy, environment and chemical engineering. Hector Garcia Martin of Lawrence Berkeley National Laboratory is another collaborator.

Rhodococcus opacus was originally discovered growing on toxic compounds outside a chemical plant. These bacteria thrive on these toxic compounds, using them as a source of food for the production of biofuels.

We are taking advantage of the fact that the compounds that R. opacus grows on are not just random toxins. These compounds are related closely to lignin, complex polymers that make up roughly 30 percent of plant matter. Our team is using a combination of chemistry, systems biology and synthetic biology to try to process lignin plant matter into biofuels that can be added directly to current petroleum-based engines.

—Gautam Dantas

Millions of tons of lignin are generated yearly from papermaking and lignocellulose-based biofuel industries. Currently, the value of lignin is restricted to its application as a fuel for on-site boiler operations. This project seeks to expand its uses.

The Foston lab is focused on developing chemical transformation processes that extract lignin from biomass, breaking apart that lignin into a form that can be “fed” to the bacteria. Lignin is a large and complex molecule designed to give plants mechanical stability and protect them from attack. Therefore, special conditions such as the presence of a catalyst and the application of heat and pressure are required to break apart lignin. The goal is to create a flexible process that generates compounds R. opacus essentially likes to eat.

The Moon lab is focused on optimizing the different strains of R. opacus through adaptive evolution and the use of synthetic biology tools. The researchers are growing the bacteria in these lignin-based compounds, making the compounds progressively more toxic with each generation, selecting for microbes that are becoming increasingly efficient at processing the compounds. More importantly, according to Moon, his team is developing the synthetic biology tools needed to engineer and improve R. opacus strains for fuel production.

The Dantas lab is focused on sequencing the DNA and RNA of R. opacus as it adapts to more efficiently process the raw (and toxic) biomass. As the microbes evolve, the researchers will identify patterns in genes and metabolic pathways that are the most important to the processing of the compounds and that are the easiest to genetically engineer so that in the future these pathways could be “dialed up” without waiting for generations of the bacteria to adapt and evolve.

The Tang and Garcia Martin labs will use computer models and machine learning to map the interconnecting networks of genes and metabolism and predict what the microbe will do under a given set of circumstances. One little change could propagate through the network and result in multiple changes to the resulting compounds that the microbe produces. Understanding these networks could allow for fine-tuning of the bacteria to make use of changing sources of biomass.

The Zhang lab is taking the compounds made by the bacteria and processing them into refined forms of fuel that so closely resemble petroleum or diesel, they could be pumped into vehicles that are on the road today.

While the process to manufacture this biofuel likely would be energy-intensive, at least initially, the researchers said that it would not require drilling and that the burning of the fuel itself would be carbon-neutral.