One way to reduce dependence on oil and other fossil fuels is to convert agro industrial waste into socially meaningful molecules, such as biofuels and biochemicals. As one of the world's major producers of plant biomass, Brazil is fully capable of leading this transformation, but lignocellulosic raw materials are difficult to decompose, or more professionally, stubborn to the degradation of microorganisms and enzymes.
Now, Brazilian scientists are looking for clues from nature to understand how to improve the depolymerization of these materials by increasing the availability of the sugars they contain. An interdisciplinary study involving holographic technology (genomics, proteomics, metabolomics, etc.) and synchrotron radiation was carried out in Campinas by a research team of Brazil's National Laboratory for biological renewable resources (LNBR) under the Brazilian center for energy and materials research (cnpem). They found two new enzyme families with biotechnology potential produced by microorganisms in the intestine of capybara. Cnpem is a private non-profit organization supervised by the country's Ministry of science, technology and innovation (MCTI).
It is understood that these two enzyme families act on the components of plant cell wall, so they can be used to produce biofuels, biochemicals and biomaterials. One of them also has potential applications in the dairy industry because it can promote the degradation of lactose.
"One of our research directions is to explore the diversity of Brazil in order to find new microbial mechanisms to reduce the resistance of lignocellulose waste. We note that capybara is a highly adapted herbivore, which can obtain energy from stubborn plant waste, and there is not much research on it," said M á Rio tyago Murakami, scientific director of LNBR and the last author of the article reporting the study.
Capybara is the world's largest living rodent. It can very effectively convert sugar contained in plants into energy, but it is not popular in some ways - because it may harbor ticks that transmit Brazilian spotted fever, a rare but highly deadly infectious disease caused by Rickettsia bacteria.
"There is a lot of research on ruminants, especially cattle, but there is relatively little information on monogastric herbivores. Unlike ruminants, buffalo digest grass and other plant substances in the cecum," said Gabriela Felix persinati, a bioinformatics researcher at LNBR and corresponding author of the article, "Considering their efficient sugar conversion and because buffalo in pirasicaba feed on sugarcane and other plants, we proceed from the hypothesis that microorganisms in the animal digestive tract may have a unique molecular strategy to depolymerize this biomass, which is very important for Brazilian industry."
Novel method
The interdisciplinary approach used in this study includes multiomics and bioinformatics, as well as cnpem's particle accelerator. "I don't remember any research that combined all these technologies, including the use of synchrotron radiation (an extremely bright source of electromagnetic radiation that helps scientists observe the internal structure of materials). In this study, our analysis went from microbial communities to the atomic structure of some proteins," Murakami said
The scientists analyzed samples from the cecum and rectum of three female capybara that were euthanized in tatui in 2017 in accordance with local policies to control the number of capybara. These animals were neither pregnant nor infected with Rickettsia.
"Fecal and rectal samples were collected through abdominal surgery. The materials were frozen in liquid nitrogen. DNA and RNA samples were extracted in the laboratory and sequenced on a large scale using comprehensive omnipotence technology," persinoti said
They first sequenced the marker genes, in this case 16S, which are present in all bacteria and archaea. "Through this first sequencing, we were able to detect the differences between fecal and rectal samples and identify the main microorganisms. Gene 16S gives us a superficial answer to which microorganisms exist and are more or less abundant, but does not tell us which enzymes these microorganisms produce or which enzymes are present in their genomes. For this purpose, we used another holographic technology, metagenomics 。 We submitted the DNA of the whole microbial community in the gastrointestinal tract of capybara to large-scale sequencing and obtained more data. By deploying a series of bioinformatics tools, we can not only determine the genome in each sample and the genes in each genome, but also find out which genes are new and which microorganisms have never been described. In this way, we can predict the function of genes that may help depolymerize biomass and convert sugar into energy, "persinoti said.
The researchers also want to know which microorganisms are most active when collecting samples - in other words, which genes are actually expressed by microorganisms. To this end, they used metatranscriptomics, which is made of RNA. "Another technique we use is metabonomics to identify which metabolites are being produced by microorganisms. Combined with all this information from holography, bioinformatics and actual and potential gene expression, we can decipher the role of intestinal microorganisms in achieving such efficient transformation of plant fibers and find out which genes are involved in this process," persinoti said
They then analyzed all these data to identify genes that may play a key role in reducing plant fiber stubbornness - focusing on hitherto unknown targets. "The selection strategy focuses on novel genomes with a wealth of genes involved in the depolymerization of plant biomass," persinati said, "We see how these genes are organized in the microbial genome and use this information to find out whether nearby genes with unknown functions may be involved in the decomposition of stubborn plant fibers. This is very important because it guides the search for new genes, but we can determine the production of these new enzyme families only when we can prove these results later."
After identifying these candidates, the researchers turned to biochemical demonstrations of their functions. "We synthesized these genes in vitro and used a bacterium to express them to produce the corresponding proteins. We conducted several enzyme and biochemical experiments to discover the functions of these proteins and their action sites. We used synchrotron radiation and other technologies to determine the atomic structure of these proteins. With this functional and structural information, we can do other experiments to find out which regions of these proteins are right Its activity is crucial and the molecular mechanism of its function is analyzed, "persinati explained.
According to Murakami, double verification ensures that the new family is indeed involved.
New enzymes and cocktail parties
According to persinoti, one of the newly discovered families gh173 has potential applications in the field of food, while another family cbm89 is related to carbohydrate recognition, which may help to produce second-generation ethanol from bagasse and straw.
Researchers are also developing enzyme cocktails from enzyme producing fungi, and the newly discovered enzymes can also be naturally included in these fungal platforms. "The discovery of novel enzyme families can be combined with technology transfer to support innovation," Murakami said, "In our group, we are very interested in exploring this great treasure of biodiversity in Brazil, especially to understand what we call dark genomic matter - the potential unknown part of these complex microbial communities. Our center has excellent infrastructure for this, coupled with our partnership with public universities, which makes this competitive research possible in Brazil. In fact, 99% of the work, From conceptual design to implementation, analysis and writing are completed here. In view of the huge biodiversity of Brazil, it is expected that we have the conditions and ability to make such high impact discoveries. "