Improving Nature's Top Recyclers

The National Renewable Energy Laboratory teams with the Texas Advanced Computing Center to explore designer enzymes for renewable fuels

If a tree falls in the forest and there are no enzymes to digest it, does it decompose? A scientific, rather than an existential question, to be sure, but one that has important ramifications for our renewable energy economy.

Many scientists and engineers are focused on methods to transform non-food based plant material (lignocellosic biomass, in technical terms), into sugars that can be converted into liquid transportation fuels. Think alfalfa stalks, wood-chips, or the corn plant as opposed to the edible corn grains that currently make ethanol.

"Cellulose in the biosphere can last for years," said Gregg Beckham, a scientist in the National Bioenergy Center at the U.S. Department of Energy's National Renewable Energy Laboratory (NREL). "It's really tough, and we want to know why, at the molecular scale."

Despite the strength of plant cell walls, over billions of years, fungi and bacteria have evolved enzymes to convert abundant cellulosic plant matter into sugars to use as energy sources to sustain life.

It's a great trick, but unfortunately, these enzymes don't work fast enough to break down cellulose in an industrial process at a price competitive with fossil fuels…yet. So computational scientists at NREL, in collaboration with a large experimental enzyme engineering group, set about trying to understand and design enhanced enzymes to speed up bio-fuel production and lower the cost of biomass-derived fuel to serve the global population.

A coarse-grained model of the bacterial cellulosome system during the self-assembly process. The long scaffold (in blue) contains binding sites for the free enzymes (red, yellow, and green) of different sizes.

"It's a Goldilocks problem," Beckham said. "The enzymes have to be ‘just right,' and we're trying to find out what just right is, why, and how to make mutations to the enzymes to make them most efficient."

NREL's mission is to develop renewable energy and energy efficiency technologies and practices, advance related science and engineering, and transfer knowledge and innovations to address the nation's energy and environmental goals.

This includes developing biomass-based fuels. Because of the magnitude of the problem, the field is expanding quickly. However, much about the behavior of these enzymes — and even the plant walls themselves — still is unknown.

Virtual Laboratories

Over the past two decades, several research groups have resolved the 3D models of the central enzymes and cellulosic materials involved in biofuel production. This led to a surge of computational experiments probing the behavior of nature's top recyclers.

In a series of linked research projects, NREL scientists and engineers used the Ranger supercomputer at the Texas Advanced Computing Center (TACC) and NREL's Red Mesa system to probe the world of enzymes – in particular, single enzymes from the prodigiously-digesting fungus, Trichoderma reesei, and enzymes from bacteria such as Clostridium thermocellum, which produce large enzyme complexes, termed cellulosomes. Both of these organisms in nature are quite effective at biomass deconstruction, though they use different strategies.

"TeraGrid and Ranger have been 100 percent instrumental in the work we're doing to understand these individual enzymes and large enzyme complexes," Beckham said. (The NREL group is also a heavy user of the Athena system at the National Institute for Computational Science, another TeraGrid partner, for a separate research project.)

It turns out, these enzymes have a toolbox full of instruments that enable them to extract and cut fibers of cellulose from its knitted, solid matrix, breaking the chains into water-soluble sugars that can be converted to ethanol or other molecules for fuels. Bacteria and fungi accomplish this action in slightly different ways, and each has unanswered questions.

When bacteria in the biosphere detect cellulosic material, they secrete enzymes complexes out of their cell walls that let them come into contact with the plant cells.

"Nature cleverly designed machinery for single-cell organisms to locate cellulose, then secrete large enzyme complexes that hold the cells near biomass while the enzymes degrade it," Beckham said.

The bacteria forms scaffolds for its enzymes, which work together as a team to break apart the plant, whereas the fungal enzymes are not tethered to a large complex, but act independently.

It isn't clear how the enzyme-laden scaffolds form. To understand this molecular self-assembly process, NREL researchers created a coarse-grained computational model of the active molecules and set them into motion in a virtual environment. Contrary to expectation, the larger, slower-moving enzymes lingered near the scaffold longer, allowing them to get tangled and to bind to the frame more frequently; the smaller ones moved more freely through the solution, but bound less often.

These results were recently reported in the Journal of Biological Chemistry in a study lead by Yannick, Bomble, and Mike Crowley. They also provided the outcomes of the simulations to the large experimental group at NREL who are using the information in the creation of designer cellulose enzymes to make biomass conversion faster, more efficient, and less expensive.

Group picture of the biomass group at NREL. Back row (from left to right): Yannick Bomble, Mike Crowley, and Gregg Beckham. Front row: Antti-Pekka Hynninen, Mark Nimlos, Christy Payne, and Deanne Sammond. (Not shown: Lintao Bu, James Matthews)

Unexplored enzyme function

The scientists also studied regions of the enzymes that were relatively unexplored, such as the carbohydrate binding molecule (CBM) — a sticky "foot" that helps the enzymes find and guide the cellulose into their active site — and the linker region, which joins the CBM to the catalytic domain. These parts of the enzyme were long thought to play a minor role in enzyme function; yet without them, the enzyme can't convert cellulose to glucose effectively. The questions was, why?

Using the experimentally determined 3D structures of the enzyme's CBM, computational researchers input new models into Ranger to explore its role.

They made several important discoveries. First, they learned that the cellulose surface has energy wells that are a natural fit for the CBM, set one nanometer apart.

"This is an interesting length scale," Beckham said, "because the carbohydrate binding module alone has evolved to process on the same length scale as the overall enzyme."

Second, they found that the linker region, previously believed to contain both stiff and flexible regions, behaves more like a highly flexible tether. These insights would have been more difficult to determine experimentally, but, now hypothesized and backed up with advanced computing simulations, they can be tested in the laboratory.

"It's a very messy problem for the experimentalists, since there's no way to use the traditional tried and true shotgun approach to protein engineering," said Mike Crowley, a principal scientist at NREL and Beckham's colleague. "We're using rational design, where we try to understand how the enzyme works and then predict the best place to change something. Then, we make those specific changes and test them."

Testing the strength of plant walls

Understanding the enzyme is only half of the equation. The plant walls themselves require more study, too. In part, this is because the real-world varieties of biomass — wood chips or corn stover denatured by solvents such as acid, ammonia, or ionic liquids — can form different polymorphs, or altered crystal structures of the same molecule (cellulose) that behave differently than native plant matter.

Beckham and Crowley thus performed large-scale, thermodynamic free energy simulations on both Ranger and on Department of Energy supercomputers to calculate how much energy was required for the enzymes to pull out four different polymorphs of cellulose. The research determined the least "recalcitrant" chains, those most easily digested by the enzymes. This discovery will eventually aid engineers in designing pre-treatment processes to lower cellulose resistance to enzymes.

Overall, this computational research addresses the practical bottlenecks in enzymatic activity that keep renewable energy from biomass from being cost competitive with fossil fuels. The long-term goal of the NREL scientists is to work with industry to drive down costs to aid in further investments in large-scale biofuel production.

"By increasing the production of renewable transportation fuels from biomass, we'll be able to reduce carbon dioxide emissions and utilize renewable, domestic sources of energy in a cost effective manner," Beckham said. "If we can help industry understand and improve these processes for renewable fuel production, we'll be able to offset a significant fraction of fossil fuel use in the long term."

Published March 9, 2011

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The Texas Advanced Computing Center (TACC) at The University of Texas at Austin is one of the leading centers of computational excellence in the United States. The center's mission is to enable discoveries that advance science and society through the application of advanced computing technologies. To fulfill this mission, TACC identifies, evaluates, deploys, and supports powerful computing, visualization, and storage systems and software. TACC's staff experts help researchers and educators use these technologies effectively, and conduct research and development to make these technologies more powerful, more reliable, and easier to use. TACC staff also help encourage, educate, and train the next generation of researchers, empowering them to make discoveries that change the world.