By studying and comparing the workhorse cellulose-degrading enzymes (cellobiohydrolases) of two fungi, researchers from the Energy Department’s National Renewable Energy Laboratory (NREL) have pinpointed regions on these enzymes that can be targeted via genetic engineering to help break down cellulose faster.

A newly published open-access paper in Nature Communications describes NREL’s long-running study of the fungal CBHs—enzymes that use hydrolysis as their main chemistry to degrade cellulose—Trichoderma reesei (TrCel7A) and Penicillium funiculosum (PfCel7A).

In both nature and industrial processes, enzymes from this family are among the most significant enzymes for breaking down cellulose. A projected 2,000-ton-per-day cellulosic ethanol plant could potentially use up to 5,000 tons of enzyme per year, and half of that enzyme cocktail could be from this enzyme family.

There’s been a drive over the last several decades of trying to understand and improve biocatalysts from this key enzyme family. The more efficient the enzyme, the less enzyme used, and thus the process is cheaper. However, we still have a long way to go to be able to make enhancements in a predictive capacity.

—Gregg Beckham, group leader at NREL and senior author

In 2005, NREL researchers Mike Himmel, Steve Decker, and Bill Adney discovered a CBH from a different fungus, PfCel7A, and found that it performs 60% better than TrCel7A.

If we could understand the structural differences, then we could potentially use that information to engineer better enzymes, which in turn could help reduce the cost of cellulosic biofuel and biochemical production. Given the challenge working with these enzymes, it took NREL’s team seven years of thorough experimental work to develop the tools needed to ascertain that there are a couple of hot spots on these two CBHs that can be modified to make them perform better.

—Gregg Beckham

At the time, tools for genetic engineering in Trichoderma were very limited, but we knew from previous work that other hosts had issues expressing these proteins. We basically started from scratch and built our own in-house T. reesei system of host strains, vectors, and transformation and screening protocols. Compared to well-developed systems like E. coli, T. reesei’s poor transformation efficiency, tedious selection processes, slow growth, and low protein yield made this a challenging operation. Every strain we built took months from design to final testing.

—Steve Decker

The discovery unfolded as NREL took a close look at the similarities between TrCel7A and PfCel7A and then worked to isolate the differences. Both enzymes have a three-domain architecture: the carbohydrate binding molecule that attaches it to cellulose; the catalytic domain that breaks down cellulose; and the link that connects these two domains together. The research team then conducted domain swapping experiments by creating a chimera library—a collection of mutant enzymes created from the two parent enzymes.

With three domains between two parents, that makes eight combinations in total. We tested the various combinations to find out which area is providing the enzyme with better performance, and perhaps not surprisingly, in hindsight, it’s the catalytic domain.

—Gregg Beckham

With those findings, the researchers then compared the catalytic domains of TrCel7A and PfCel7A and found eight areas that were different. Continuing to narrow down the possibilities, the team took the TrCel7A parent and made modifications, one at a time, in those eight areas and uncovered two important modifications that resulted in TrCel7A performing almost to the level of the PfCel7A parent.

Those two, very small changes on this huge protein basically doubled the performance of TrCel7A. What this teaches researchers doing protein engineering on these incredibly challenging enzymes is that there are very minor changes to this catalytic domain that can be modified to dramatically affect the performance of the enzyme, making it capable of breaking down cellulose faster and thus allowing industrial processes to use less enzyme.

—Gregg Beckham

The NREL team’s ultimate aim is to help other researchers sift through the mountain of genomics data to find better enzymes, based on their genetic sequence alone.

The work reported in Nature Communications was funded by the Energy Department’s Bioenergy Technologies Office. A patent application has been filed on this research, and the NREL Technology Transfer Office will be working with researchers to identify potential licensees of the technology.

Resources

• Larry E. Taylor II, Brandon C. Knott, John O. Baker, P. Markus Alahuhta, Sarah E. Hobdey, Jeffrey G. Linger, Vladimir V. Lunin, Antonella Amore, Venkataramanan Subramanian, Kara Podkaminer, Qi Xu, Todd A. VanderWall, Logan A. Schuster, Yogesh B. Chaudhari, William S. Adney, Michael F. Crowley, Michael E. Himmel, Stephen R. Decker & Gregg T. Beckham (2018) “Engineering enhanced cellobiohydrolase activity” Nature Communications volume 9, Article number: 1186 doi: 10.1038/s41467-018-03501-8