The National Institute for Computational Sciences

Another Stride in Biofuels Research

Recent Finding is Part of Ongoing Research Supported by the Synergistic Suite of XSEDE Resources

By Scott Gibson

Cellulase enzymes found in nature—from sources such as wood-degrading fungi, cow rumen (stomach compartment, or paunch), and compost piles—form one of the key catalysts for breaking down plant biomass in industrial processes to make biofuels, but they remain quite expensive because they can’t be easily recovered after use and they are costly to make. “At the potential scale of biofuels production, even small gains in enzyme performance potentially could have huge cost implications for biofuels production and help make biofuels more cost-competitive with transportation fuels derived from fossil fuels,” says Gregg Beckham of the U.S. Department of Energy’s National Renewable Energy Laboratory (NREL).

So, in that context, serendipitous findings that point researchers toward a new angle for the investigation of cellulases become all the more significant. Compute allocations from the Extreme Science and Engineering Discovery Environment (XSEDE) have made just such a breakthrough possible, and details are provided in a recently published paper titled “Glycosylated linkers in multimodular lignocellulose-degrading enzymes dynamically bind to cellulose” in the journal Proceedings of the National Academy of Sciences. The work was authored by researchers from NREL in collaboration with colleagues from the University of Colorado (CU-Boulder), the Swedish University of Agricultural Sciences, the University of Kentucky, and University College Ghent.

The content of the paper reflects one of the many aspects of understanding the key Family 6 and Family 7 cellulase enzymes, which are two of the primary enzyme families used industrially to breakdown cellulose, explains Beckham, the paper’s lead author.

A Surprising Discovery

Both in nature and in industrial conditions, a cocktail of cellulases is required to breakdown biomass. Many of the enzymes in cellulase cocktails employ a similar chemical reaction to break bonds between carbohydrate polymers with water in a process known as hydrolysis. The end product of hydrolysis of cellulose is glucose, which can then be fermented into ethanol or converted to transportation fuels such as gasoline, diesel, or jet fuel through various biological or catalytic upgrading processes.

Many of these lignocellulose-degrading enzymes have two ‘ordered domains,’ one that enables very selective stickiness to cellulose, called a carbohydrate-binding module, and one that binds single cellulose chains in a long tunnel where the chain is clipped to soluble sugars, Beckham explains. The surprising finding pertains to the ‘disordered’ linker that connects the two ordered domains.

“For a very long time, it [the linker] was just there to connect two domains with a very specific and synergistic function; however, several years ago, we ran a large-scale simulation of an entire enzyme on cellulose and kept it going for a very long time,” Beckham says. “Surprisingly to us, the linker started to bind to cellulose. We did this for another enzyme and many other cases to make sure we weren’t seeing something anomalous, and in every simulation we ran, the linkers from different enzymes would bind. We eventually verified this experimentally and showed that the simulation predictions were indeed real.”

The discovery reveals that linkers have likely evolved not only for connecting a binding module and catalytic domain but also to serve a binding function themselves, Beckham explains. “This suggests that linkers might be considered as a target of enzyme engineering in biofuels studies, where researchers are intensely focused on enhancing these enzymes. Improving cellulases is one of the biggest research goals of many researchers in biofuels today.”

Molecular Dynamics (MD) Simulations

The work leading to the most recent finding was done on the Athena supercomputer (a now-retired system, managed by NICS) and Kraken, which is housed at Oak Ridge National Laboratory and managed by NICS for the National Science Foundation.

To look at linker function, the researchers performed MD simulations of the Trichoderma reesei Family 6 and Family 7 enzymes (TrCel6A and TrCel7A, respectively) bound to cellulose.

The Trichoderma reesei Family 7 cellobiohydrolase (enzyme) on the surface of cellulose (the main component of plant cell walls). The cellulose microfibril is shown in green, and the enzyme in gray. A microfibril is a fiberlike strand consisting of long chains of glucose linked together to form cellulose, which then pack into long bundles. The cellulase enzymes themselves are decorated with sugar molecules on the catalytic domain. The N-linked glycosylation is shown in blue, and the O-linked glycosylation in yellow. N-linked glycosylation is the attachment of a sugar molecule to a nitrogen atom in an amino acid residue in a protein; O-linked glycosylation is the attachment of a sugar molecule to an oxygen atom in an amino acid residue in a protein. The catalytic domain is the part of the enzyme that threads a cellulose chain where the enzyme clips the cellulose chain to soluble sugars. Image: Peter Ciesielski and Gregg Beckham, National Renewable Energy Laboratory.

The Trichoderma reesei Family 7 cellobiohydrolase (enzyme) on the surface of cellulose. Color coding is the same as in the previous image. The linker binds to the surface of cellulose, which was predicted with molecular dynamics simulation and validated with experimental binding affinity measurements. Image: Peter Ciesielski and Gregg Beckham, National Renewable Energy Laboratory.

Answering Complicated Questions

Beckham says that the overall effort to understand cellulases is a multifaceted challenge that has required the research team of which he is a part to employ resources from across the XSEDE system. During the course of its investigations of cellulases so far, the research team has used supercomputers at the National Institute for Computational Sciences (NICS), the Texas Advanced Computing Center (TACC), the Pittsburgh Supercomputing Center (PSC), and the San Diego Supercomputing Center (SDSC).

“XSEDE has allowed us not only to understand what the linker is doing but it has also allowed us to see how the hydrolysis reactions occur,” Beckham says. “It’s given us the insight we need to know how to engineer the binding module, study the linker in isolation, mutate amino acids in silico, and study how a chain is threaded up into the tunnel, long thought to be the rate-limiting step in the binding process.”

In the broad perspective, Beckham says supercomputers at PSC and SDSC enable the research team to apply quantum chemistry methods, while the NICS-managed Athena (a now-retired system) and Kraken and Ranger (a now-retired system) and Stampede at TACC have enabled them to perform large-scale MD simulations.

“We’re now starting to design and test enzymes experimentally with different linker sequences and lengths,” Beckham says. “We’ll be able to produce these enzymes in fungi and then test their activity to start to understand better the effect of linker sequence and length on enzyme function.”

Associated Paper

C.M. Payne, M.G. Resch, L. Chen, M.F. Crowley, M.E. Himmel, L.E. Taylor, M. Sandgren, J. Ståhlberg, I. Stals, Z. Tan, G.T. Beckham, “Glycosylated linkers in multi-modular lignocellulose degrading enzymes dynamically bind to cellulose,” Proceedings of the National Academy of Sciences 2013, 110(36), pp. 14646-14651.

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About NICS: The National Institute for Computational Sciences (NICS) operates the University of Tennessee supercomputing center, funded in part by the National Science Foundation. NICS is a major partner in NSF’s Extreme Science and Engineering Discovery Environment, known as XSEDE.