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Newly identified enzymes from herbivore gut fungi may lead to cheaper cellulosic biofuels

A team of researchers led by Dr. Michelle O’Malley at UC Santa Barbara has identified several promising new enzyme candidates for breaking down lignocellulsoic biomass for biofuel production from relatively unexplored gut fungi in herbivores. To do so, they developed a systems-level approach that integrates transcriptomic sequencing (RNA-Seq); proteomics; phenotype; and biochemical studies.

The biomass-degrading enzymes from the anaerobic gut fungi are competitive with optimized commercial enzyme preparations from Aspergillus and Trichoderma. Further, compared to the model platforms, the gut fungal enzymes are unbiased in substrate preference due to a wealth of xylan-degrading enzymes. The findings suggest that industry could modify the gut fungi so that they produce improved enzymes that will outperform the best available ones, potentially leading to cheaper biofuels and bio-based products. A paper on their work is published in the journal Science.

Lignocellulosic biomass from plant matter is an abundant, renewable starting material for biofuel and industrial chemical production. Industrial-scale processes require fungal enzymes to convert biomass into fermentable sugars. However, lignin must be removed from crude biomass with costly pretreatment processes to permit enzymatic degradation and sugar release. The need for multiple enzyme production processes increases this cost further, as genetically modified fungal platforms such as Trichoderma reesei and Aspergillus nidulans over produce limited subsets of enzymes that are unable to independently digest even pretreated substrates completely to sugars. Economical chemical production will require a versatile, unbiased platform to produce all enzymes needed to hydrolyze diverse lignocellulose feedstocks into fermentable sugars without pretreatment.

Microbes found in the digestive tract of large herbivores are attractive enzyme platforms for lignocellulose processing. … However, their strict anaerobic lifestyle, complex nutritional requirements, and culture recalcitrance have severely hindered early attempts at isolation, exploitation, and molecular characterization. We isolated three previously uncharacterized cultures from the feces of different herbivorous mammals with varied diets.

—Solomon et al.

To make the finding, O’Malley drew upon two US Department of Energy Office of Science User Facilities: the Environmental Molecular Science Laboratory at Pacific Northwest National Laboratory and the DOE Joint Genome Institute. O’Malley’s study is the first to result from a partnership between the two facilities called Facilities Integrating Collaborations for User Science or FICUS. The partnership allows scientists around the world to draw on capabilities at both Office of Science user facilities to get a more complete understanding of fundamental scientific questions. O'Malley’s team also included scientists from PNNL, DOE JGI, the Broad Institute of MIT and Harvard, and Harper Adams University.

By tapping the RNA sequencing and protein characterization capabilities at the respective facilities, we have advanced biofuel research in ways not otherwise possible. This collaborative program was established to encourage and enable researchers to more easily integrate the expertise and capabilities of multiple user facilities into their research. FICUS offers a one-stop shopping approach for access to technology infrastructure that is rapidly becoming a model for collaboration.

—Susannah Tringe, DOE JGI deputy for User Programs

Gut fungi are known to play a significant role in helping herbivores digest plants. One reason has to do with the swarming behavior of some fungi. When the fungi reproduce, they release dozens of spores with tail-like appendages called flagella. These baby fungi swim around like tadpoles and find new food in the gut. They then trade tails for root-like structures called hyphae, which dig into plant material. The fungi’s hyphae excrete enzymes that break down plant material.

Despite their fascinating biology, anaerobic gut fungi can be difficult to isolate and study. By utilizing the cutting-edge scientific capabilities at EMSL and JGI, O’Malley showed how the huge catalog of anaerobic gut fungi enzymes could advance biofuel production.

—Scott Baker, EMSL’s science theme lead for Biosystem Dynamics and Design

The DOE JGI sequenced the messenger RNA (mRNA) of several gut fungi to come up with their transcriptome, which represents all the possible proteins they could make.

O’Malley compared this effort to re-assembling a map from its pieces, only without seeing the complete picture. Since not all proteins are enzymes, the researchers needed to cross check their map with another one. EMSL researchers created a second map that identified enzymes the fungi actually produced. This so-called proteome acted like landmarks that matched up to JGI’s map, highlighting the biomass-degrading enzymes in the transcriptome.

Together, the maps from JGI and EMSL pointed to the enzymes gut fungi can produce. Compared to the industrial varieties, which top out around 100 enzymes, gut fungi can produce hundreds more. Of note, the fungi produce enzymes better at breaking down a hemicellulose found in wood, called xylan. And when the scientists changed the fungi’s diet from canary grass to sugar, the fungi responded by changing the enzymes it produced. In other words, the fungi can update their enzyme arsenal on the fly.

Because gut fungi have more tools to convert biomass to fuel, they could work faster and on a larger variety of plant material. That would open up many opportunities for the biofuel industry.

—Michelle O’Malley

The study was funded by the US Department of Energy Office of Science, the US Department of Agriculture and the Institute for Collaborative Biotechnologies. Additionally, O’Malley was the recipient of a DOE Office of Science Early Career Award within the Biological and Environmental Research Program.

O’Malley will present her findings at the DOE JGI’s 11th Annual Genomics of Energy & Environment Meeting in Walnut Creek, California, on 24 March.

Resources

  • Kevin V. Solomon, Charles H. Haitjema, John K. Henske, Sean P. Gilmore, Diego Borges-Rivera, Anna Lipzen, Heather M. Brewer, Samuel O. Purvine, Aaron T. Wright, Michael K. Theodorou, Igor V. Grigoriev, Aviv Regev, Dawn A. Thompson, Michelle A. O’Malley (2016) “Early-branching gut fungi possess a large, comprehensive array of biomass degrading enzymes,” Science doi: 10.1126/science.aad1431

Comments

SJC

There have been advances in making cellulose renewable fuels, there will be more to come.

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