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Caltech Researchers Create Group of Synthetic, Thermostable Enzymes for Cellulosic Biofuel Production

Portions of three natural fungal cellulase enzymes that have been recombined to produce a synthetic, thermostable cellulase are denoted by blue, green and red coloring. The recombined cellulase enzyme modeled here functions at higher temperatures than any of the three parents. Source: Caltech. Click to enlarge.

Researchers at the California Institute of Technology (Caltech) led by Frances H. Arnold, the Dick and Barbara Dickinson Professor of Chemical Engineering and Biochemistry at Caltech, and gene-synthesis company DNA2.0 have a new group of 15 highly stable fungal enzyme catalysts that efficiently break down cellulose into sugars at high temperatures for conversion into a variety of renewable fuels and chemicals.

Previously, fewer than 10 such fungal cellobiohydrolase II (CBH II) enzymes were known. In addition to their remarkable stabilities, Arnold’s enzymes degrade cellulose over a wide range of conditions. A paper on the work was published 23 March in the early edition of the Proceedings of the National Academy of Sciences.

This is a really nice demonstration of the power of synthetic biology. You can rapidly generate novel, interesting biological materials in the laboratory, and you don’t have to rely on what you find in nature. We just emailed DNA2.0 sequences based on what we pulled out of a database and our recombination design, and they synthesized the DNA. We never had to go to any organism to get them. We never touched a fungus.

—Dr. Frances Arnold

Generating this group of diverse, thermostable fungal CBH II chimeras is the first step in building an inventory of stable cellulases from which optimized enzyme mixtures for biomass conversion can be formulated.

Breaking down cellulose to make the sugars available for fermentation is far tougher than breaking down starch. An additional complication is that while the fermentation reaction that breaks down corn starch needs just one enzyme, the degradation of cellulose requires a suite of enzymes, or cellulases, working in concert.

The cellulases currently used industrially, all of which were isolated from various species of plant-decaying filamentous fungi, are both slow and unstable, and, as a result, the process remains prohibitively expensive. “Even a two-fold reduction in their cost could make a big difference to the economics of renewable fuels and chemicals,” says Arnold.

Arnold and Caltech postdoctoral scholar Pete Heinzelman created the 15 new enzymes using a process called structure-guided recombination. Using a computer program to design where the genes recombine, the Caltech researchers mated the sequences of three known fungal cellulases to make more than 6,000 progeny sequences that were different from any of the parents, yet encoded proteins with the same structure and cellulose-degradation ability.

By analyzing the enzymes encoded by a small subset of those sequences, the Caltech and DNA2.0 researchers were able to predict which of the more than 6,000 possible new enzymes would be the most stable, especially under higher temperatures (a characteristic called thermostability).

Thermostability is a requirement of efficient cellulases, because at higher temperatures (e.g., 70-80 °C) chemical reactions are more rapid. In addition, cellulose swells at higher temperatures, which makes it easier to break down. The known cellulases from nature typically won”t function at temperatures higher than about 50 °C.

Enzymes that are highly thermostable also tend to last for a long time, even at lower temperatures. And, longer-lasting enzymes break down more cellulose, leading to lower cost.

—Frances Arnold

Using the computer-generated sequences, coauthor Jeremy Minshull and colleagues from DNA2.0 of Menlo Park, California, synthesized actual DNA sequences, which were transferred into yeast in Arnold’s laboratory. The yeast produced the enzymes, which were then tested for their cellulose-degrading ability and efficiency. Each of the 15 new cellulases reported in the PNAS paper was more stable, worked at significantly higher temperatures (70-75 °C), and degraded more cellulose than the parent enzymes at those temperatures.

The total of 15 validated thermostable CBH II enzymes have high sequence diversity, differing from their closest natural homologs at up to 63 amino acid positions. Selected purified thermostable chimeras hydrolyzed phosphoric acid swollen cellulose at temperatures 7 to 15 °C higher than the parent enzymes. These chimeras also hydrolyzed as much or more cellulose than the parent CBH II enzymes in long-time cellulose hydrolysis assays and had pH/activity profiles as broad, or broader than, the parent enzymes.

—Heinzelman et al. (2009)

Next, the researchers plan to use the structure-guided recombination process to perfect each of the half-dozen or so cellulases that make up the cocktail of enzymes required for the industrial degradation of cellulose.

We’ve demonstrated the process on one of the components. Now we have to create families of all of the other components, and then look for the ideal mixtures for each individual application,” Arnold says, with the ultimate goal of creating a cost-efficient recipe for cellulosic biofuel.

The work was supported by the Army-Industry Institute for Collaborative Biotechnologies and the Caltech Innovation Institute.

Resources

  • Pete Heinzelman, Christopher D. Snow, Indira Wu, Catherine Nguyen, Alan Villalobos, Sridhar Govindarajan, Jeremy Minshull, and Frances H. Arnold (2009) A family of thermostable fungal cellulases created by structure-guided recombination. PNAS published online before print doi:

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