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ORNL scientists engineer multifunctional microbe for “one-pot” biomass processing

In the United States (US), scientists at the Oak Ridge National Laboratory (ORNL) have announced that they have successfully modified a single microbe to simultaneously digest five of the most abundant components of lignocellulosic biomass. This is a big step forward in the development of a cost-effective biochemical conversion process to turn plants into renewable fuels and chemicals.

ORNL’s Adam Guss is part of a research team that optimized the Pseudomonas putida bacterium to simultaneously digest five of the most abundant components of lignocellulosic biomass (photo courtesy Jason Richards/ORNL, DOE).

With support from the Department of Energy (DOE) Office of Energy Efficiency and Renewable Energy (EERE) Bioenergy Technologies Office (BETO), the Agile BioFoundry team at ORNL engineered the Pseudomonas putida (P. Putida) bacterium to consume glucose, xylose, arabinose, coumaric acid, and acetic acid simultaneously in a single bioreactor.

This achievement eliminates the need for multiple tanks and microbes for each of those components. The “one-pot” process also breaks down lignin—traditionally a waste product of biomass conversion—so that every part of the plant can be used to create valuable products.

A research paper “Engineered Pseudomonas putida simultaneously catabolizes five major components of lignocellulosic biomass: glucose, xylose, arabinose, coumaric acid, and acetic acid” detailing the findings has been published in Metabolic Engineering in August 2020.

We were pleasantly surprised at how quickly and well the microbe consumed these components, as they are structurally different and utilized via very different pathways. You had all of this carbon converging in the central metabolism and being co-utilized. It was pretty exciting, said Adam Guss, who led ORNL’s research as detailed in Metabolic Engineering.

Upgrading lignin and cellulosic sugars with one organism key

Upgrading lignin, as well as sugars from biomass, is vital to creating a highly efficient, cost-effective biorefinery. To reach that goal, lignin, which accounts for about 10-30 percent of lignocellulose biomass by weight and represents up to 40 percent of total carbon, must be converted to value-added products to increase yield and reduce the cost of the overall bioconversion process.

ORNL scientists took P. putida, a hardy microorganism efficient at digesting glucose, coumarate, and acetate, and optimized pathways for digesting those compounds, as well as xylose and arabinose.

One of the major challenges in synthetic biology is to get an organism to co-utilize multiple compounds. The researchers used rational metabolic engineering, evolution, and reverse engineering to cultivate these desired traits in the microbe.

ORNL scientists have optimized the Pseudomonas putida bacterium to digest five of the most abundant components of lignocellulosic biomass simultaneously, supporting a highly efficient conversion process to create renewable fuels and chemicals from plants (photo courtesy Alli Werner/NREL, DOE).

The engineered microbe was then tested in a bioreactor on corn stover-derived biomass by project partners at the National Renewable Energy Laboratory (NREL) as part of the Agile BioFoundry, a Department of Energy consortium that brings together the expertise and capabilities of nine national labs to advance state-of-the-art biomanufacturing.

The accomplishment also overcomes the problem of trying to use more than one microorganism in a single process bioreactor.

It’s hard to engineer two organisms that like the exact same conditions and play well with each other. Using a single, optimized organism eliminates a lot of those challenges, Adam Guss said.

Work to continue the process optimization

Researchers will work to further optimize the microbe to valorize plant components to create clean, domestic, sustainably sourced fuels and chemicals to support the bioeconomy.

The next steps in the work include further expansion of the number of substrates that P. putida can digest and gaining a better understanding of how these different pathways interact with each other to make the overall process as efficient as possible, Guss noted.

This study builds on a rich legacy of metabolic engineering at ORNL, part of its research that spans the spectrum from the development of hardy biomass crops, multifunctional microbes, and other processes to valorize plant components to create clean, domestic, sustainably sourced fuels and chemicals that can support rural economies.

We’re still in the early stages of making commercially viable processes that turn biomass into fuels and chemicals, and one of the big goals is reducing the cost of breaking down biomass into individual components. With this technology, you won’t need multiple tanks — one doing fermentation and sugar conversion, another doing aromatics, and another doing acetate, for instance. Instead, it can all be done together, Adam Guss said.

ORNL is managed by UT-Battelle for the DOE’s Office of Science, the single largest supporter of basic research in the physical sciences in the United States.

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