Converting agro-industrial waste into molecules of interest to society, such as biofuels and biochemicals, is one of the ways to mitigate dependence on oil and its derivatives. Brazil, one of the world’s largest producers of plant biomass, is privileged in this regard. However, this type of lignocellulosic raw material (composed of lignin, hemicellulose and cellulose) is difficult to deconstruct — or, as they say in scientific jargon, is recalcitrant.
In an attempt to improve this depolymerization process, researchers from the National Biorenewables Laboratory (LNBR), an agency linked to the National Center for Research in Energy and Materials (CNPEM), in Campinas, have been studying and learning from nature strategies to facilitate access to sugars contained in these materials. Through an interdisciplinary project, they discovered two new families of enzymes with biotechnological potential produced by microorganisms present in the intestine of capybaras.
Both families act on plant cell wall components and, therefore, can be used in biofuel, biochemical and biomaterial manufacturing processes. One of them, in particular, also has potential for the dairy industry, as it promotes the degradation of lactose.
“One of our lines of research is to explore the Brazilian biodiversity in search of new microbial mechanisms that reduce the recalcitrance of lignocellulosic residues. In our studies, we identified the capybara as a herbivore highly adapted to obtain energy from recalcitrant plant residues and still little studied”, reveals Mário Murakami, scientific director of the LNBR and responsible for the work, recently published in the journal Nature Communications.
The largest rodent on the planet, the capybara is very efficient at converting sugars contained in lignocellulosic materials into energy — although it is known by most of the population for its sins (as it can host the tick that transmits Rocky Mountain spotted fever) than for its virtues.
“There are several studies with ruminants, mainly cattle, but in relation to monogastric herbivores [com estômago simples] information is scarcer. In capybaras, unlike ruminants, digestion of ingested food, mainly grasses, takes place in the cecum, the initial part of the large intestine. As the capybara has high efficiency in the conversion of sugars, and capybaras from the Piracicaba region have incorporated sugarcane in their diet, our hypothesis was that the microorganisms present in their digestive tract could present novel molecular strategies for the depolymerization of this biomass of great industrial relevance”, summarizes the researcher Gabriela Felix Persinoti, co-author of the article.
The investigation was supported by Fapesp through a Thematic Project and a Post-Doctoral Scholarship granted to Mariana Abrahão Bueno de Morais.
unpublished methodology
The work used an interdisciplinary approach that includes multiomic analyzes (such as genomics, transcriptomics and metabolomics), employed in the large-scale characterization of different molecular aspects of the mammalian intestinal microbial community, as well as bioinformatics tools and particle accelerators to characterize the discovered enzymes. at the atomic level.
“I don’t recall any work that has integrated all these approaches, including the use of synchrotron light. [uma fonte de radiação eletromagnética de alto brilho usada para a observação das estruturas internas dos materiais]”, says Murakami. “In this study, we went from understanding the microbial community to the level of the atomic structure of proteins.”
The scientists worked with samples taken directly from the cecum and rectum of capybaras. The material was obtained from three young females euthanized in Tatuà (SP), in 2017, in compliance with local population control policies for these animals. They were neither pregnant nor infected with Rickettsia rickettsii, the bacterium that causes Rocky Mountain spotted fever.
“By means of abdominal surgery, samples were collected from the cecum and rectum of three animals. The material was frozen in liquid nitrogen and, in the laboratory, we extracted DNA and RNA, used to perform large-scale sequencing using omics approaches. integrative”, details Persinoti.
Initially, the researchers performed the sequencing of marker genes. In this case, the 16S gene, present in all bacteria and archaea.
“With this first sequencing, we were able to identify differences between the cecum and rectum samples and verify the main microorganisms present in each one. The 16S gene gives us a superficial answer, that is, which microorganisms are there, to a greater or lesser extent. abundance; but it does not provide information about which enzymes they produce or which enzyme-coding genes are present in their genome. For this, we used another omic technique, metagenomics. With the DNA extracted from the entire microbial community of the gastrointestinal tract of the capybara, we made a large-scale sequencing. Then, with the help of several bioinformatics tools, we identified the genomes that were present in each of the samples, which genes each of the genomes contained, which were new and which microorganisms had never been described. we were able to make predictions of the functions of these genes with the potential to act in the depolymerization of biomass, in the conversion that of sugars into energy and so on.”
The team also wanted to know which microorganisms were most active at the time of sample collection, that is, which genes were actually being expressed. For this, they used metatranscriptomics, a technique that uses RNA as its raw material.
“Another omic tool used was metabolomics, to confirm which metabolites the microorganisms were producing. We combined all this omic, bioinformatics, expression and gene potential information to decipher the role of microorganisms present in the capybara intestine in the efficient conversion of plant fibers into energy.”
Armed with this information, the scientists sought to find out which genes could play a key role in reducing the recalcitrance of plant fibers, focusing mainly on previously unknown targets.
“The selection strategy was focused on novel genomes that presented a rich repertoire of genes involved in the depolymerization of plant biomass. We verified how these genes were organized in the genome of microorganisms to identify if there were nearby genes whose function was unknown, but which, possibly , could be involved in the same processes of deconstruction of recalcitrant plant fibers. This is important information, which helps us to direct the search. However, it is only when we are able to demonstrate these results experimentally, in a later stage, that we establish the creation of these new enzyme families.”
Once the new candidates were identified, the team set out to demonstrate their biochemical functions. “We synthesized the genes in vitro and expressed them using a bacterium to produce the corresponding proteins. We performed several enzymatic and biochemical assays to find the function of these proteins and find out where they would act. We determined the atomic structure of the proteins using synchrotron light beams and other techniques. Armed with this functional and structural information, further experiments were carried out to determine which region of the protein is critical for its activity and the molecular mechanism by which it performs its function.”
Murakami emphasizes that to make sure they are describing a new family, the group did a double validation. “We selected another member of the set of gene sequences that would theoretically form the universe of the new family discovered and that had low similarity to what we had initially studied. We synthesized the gene, purified it, biochemically characterized it and showed that this sequence has the same functional properties as In other words, we characterized a second member of the new family to be absolutely sure that these proteins actually constituted a new family.”
New enzymes and cocktails
Persinoti reveals that one of the new families discovered, called GH173, has potential application in the food industry, while the CBM89 family, related to carbohydrate recognition, could contribute to facilitating the production of fuels such as second-generation ethanol, for example, obtained from from sugarcane bagasse and straw.
The LNBR group develops enzymatic cocktails with enzyme-producing fungi and the natural continuity of this work would be to include the enzymes discovered in the capybara microbiota in these fungal platforms.
“There is an integration from the discovery of new families of enzymes to the transfer of technology to support innovation. In our group, we are very interested in exploring this great treasure of Brazilian biodiversity, in particular what we call dark genomic matter, that is, , these complex microbial communities that contain as yet unknown potentials. CNPEM’s unique infrastructure and partnerships with public universities allowed 99% of the work, from conceptual design to execution, analysis and writing, to be done here in Brazil. Looking at the immense richness of Brazilian biodiversity, it was more than expected that we were in a position to make such impactful discoveries”, emphasizes Murakami.
The article “Gut microbiome of the largest living rodent harbors unprecedented enzymatic systems to degrade plant polysaccharides” can be accessed here.
