Enzymes found in capybara gut can promote the application of agroindustrial waste

One option to reduce dependence on oil and other fossil fuels is to transform agroindustrial waste into molecules of interest to society, like biofuels and biochemicals. Brazil is well-positioned to pioneer this shift as one of the world’s major producers of plant biomass, however, lignocellulosic raw materials (including lignin, hemicellulose, and cellulose) are difficult to deconstruct, or (to put it another way) resistant to microbial and enzymatic destruction.

Brazilian researchers are looking to nature for clues on how to optimize the depolymerization of these materials by increasing the supply of the sugars they contain.

A research team at the Brazilian Biorenewables National Laboratory (LNBR), a branch of the Brazilian Center for Research in Energy and Materials (CNPEM), performed interdisciplinary research involving omics (genomics, proteomics, metabolomics, etc.) and synchrotron light in Campinas (São  Paulo state), and found two new families of enzymes with the biotechnological potential generated by microorganisms in capybara guts.

The Ministry of Science, Technology, and Innovation (MCTI) controls CNPEM, which is a private nonprofit.

Both enzyme families function on plant cell wall components, allowing them to be exploited to make biofuels, biochemicals, and biomaterials. Since it stimulates lactose breakdown, one of them could be useful in the dairy business.

One of our research lines explores Brazilian diversity in pursuit of novel microbial mechanisms that reduce the recalcitrance of lignocellulosic waste. We noted that the capybara is a highly adapted herbivore capable of obtaining energy from recalcitrant plant waste and that it hasn’t been studied very much.”

Mário Tyago Murakami, Study Last Author and Scientific Director, Brazilian Biorenewables National Laboratory

The study was published in Nature Communications.

The capybara (Hydrochoerus hydrochaeris) is the world’s biggest living rodent and transforms plant sugars into energy very effectively, even if some hate it as it can harbor the tick that transfers Brazilian spotted fever—a rare but deadly infectious disease caused by the bacterium Rickettsia rickettsii.

There are plenty of studies of ruminants, especially bovines, but information about monogastric herbivores is relatively scarce. Unlike ruminants, capybaras digest grass and other plant matter in the cecum, the first part of the large intestine.”

Gabriela Felix Persinoti, Study Corresponding Author and Bioinformatics Researcher, Brazilian Biorenewables National Laboratory

“In light of their highly efficient sugar conversion, and because capybaras in the Piracicaba region [of São Paulo state] feed on sugarcane, among other plants, we started from the hypothesis that microorganisms present in the animal’s digestive tract might have unique molecular strategies to depolymerize this biomass, which is very important to Brazilian industry,” Persinoti added.

Novel methodology

Multi-omics (genomics, transcriptomics, and metabolomics were employed to define molecular characteristics of the capybara’s gut microbiota) and bioinformatics were used in the research, as well as CNPEM’s particle accelerators to investigate the enzymes identified at the atomic level.

I can’t recall any studies that have combined all these techniques, including the use of synchrotron light [a source of extremely bright electromagnetic radiation that helps scientists observe the inner structures of materials]. In this research, our analysis drilled all the way down from the microbial community to the atomic structure of certain proteins.”

Mário Tyago Murakami, Study Last Author and Scientific Director, Brazilian Biorenewables National Laboratory

The researchers examined samples taken from the cecum and rectum of three female capybaras executed in Tatuí (São Paulo state) in 2017 as part of a local capybara population management effort. Neither the animals were pregnant nor were they infected with R. rickettsii.

“The cecal and rectal samples were collected by abdominal surgery. The material was frozen in liquid nitrogen. DNA and RNA samples were extracted in the laboratory and submitted to large-scale sequencing using integrative omics,” Persinoti said.

They began by sequencing marker genes, in this case, 16S, present in all bacteria and archaea.

“With this first sequencing, we were able to detect differences between the cecal and rectal samples and to identify the main microorganisms in them. The gene 16S gave us a superficial answer as to which microorganisms were present and abundant to a greater or lesser extent, but didn’t tell us which enzymes the microorganisms produced or which enzymes were present in their genomes,” she clarified.

“For this purpose, we used another omics technique, metagenomics. We submitted DNA from the entire microbial community in the capybaras’ gastrointestinal tract to large-scale sequencing, obtaining a larger amount of data.”

“By deploying an array of bioinformatics tools, we were able not only to identify the genomes present in each of the samples, and the genes in each of the genomes, but also to find out which genes were new and which microorganisms had never been described. In this manner, we were able to predict the functions of the genes that had the potential to help depolymerize biomass and convert sugar into energy,” Persinoti added.

The researchers also sought to determine which microbes were most active at the time when the samples were taken, or which genes they were expressing. They did this by using metatranscriptomics, which uses RNA as its basic material.

“Another technique we used was metabolomics, to confirm which metabolites the microorganisms were producing,” Persinoti said. “Combining all this information from omics, bioinformatics, and actual and potential gene expression, we were able to decipher the role of gut microorganisms in achieving such highly efficient conversion of plant fibers and to find out which genes were involved in the process.”

They then used all of this information to find genes that potentially play a crucial role in lowering plant fiber recalcitrance, with an emphasis on previously discovered targets.

“The selection strategy focused on novel genomes with an abundance of genes involved in plant biomass depolymerization,” Persinoti said.

“We saw how these genes were organized in the genomes of the microorganisms, and leveraged this information to find out whether there were nearby genes with unknown functions that might be involved in breaking down recalcitrant plant fiber. This is important because it guides the search for novel genes, but only when we were able to demonstrate these results experimentally at a later stage could we establish the creation of these novel families of enzymes,” she said.

They proceeded on to a molecular demonstration of their roles after identifying these possibilities.

“We synthesized the genes in vitro and expressed them using a bacterium to produce the corresponding proteins,” Persinoti said.

“We performed several enzyme and biochemical assays to discover the functions of these proteins and where they acted. We determined the proteins’ atomic structures using synchrotron light and other techniques. With this functional and structural information, we were able to do other experiments to find out which regions of the proteins were critical to their activity and analyze the molecular mechanisms underlying their functions.”

Murakami claims that dual validation assured that new families were included.

“We selected a gene not very similar to one we had studied previously in the set of sequences that theoretically formed the universe of a newly discovered family. We synthesized the gene, purified it, characterized it biochemically, and showed that the sequence had the same functional properties as the previous one,” he explained.

“In other words, we characterized a second member of the new family in order to be absolutely sure these proteins did indeed constitute a novel family.”

Novel enzymes and cocktails

One of the newly found families, GH173, has possible applications in the food industry, according to Persinoti, while the other, CBM89, is linked to carbohydrate recognition and could help with the manufacturing of second-generation ethanol from sugarcane bagasse and straw.

The researchers are also working with enzyme-hyperproducing fungi to create enzyme cocktails, and the recently found enzymes could easily be incorporated into these fungal platforms. “The discovery of novel enzyme families can be integrated with the transfer of technology to support innovation,” Murakami said.

“In our group, we’re very interested in exploring this great Brazilian biodiversity treasure, particularly to understand what we call dark genomic matter—parts of these complex microbial communities with unknown potential. Our center has excellent infrastructure for this purpose and, together with our partnerships with public universities, this has enabled competitive research of this kind to be done in Brazil.”

“Indeed, 99% of the work, from conceptual design to execution, analysis, and writing up, was done here. Given the immense richness of Brazilian biodiversity, it was only to be expected that we would have the conditions and capabilities to make high-impact discoveries such as these.”