Pesquisadores brasileiros descobriram, caracterizaram e validaram as funções de duas novas famílias de enzimas com potencial biotecnológico.
Um método para reduzir a dependência de petróleo e outros combustíveis fósseis é converter resíduos agroindustriais em moléculas de relevância social, como biocombustíveis e bioquímicos. O Brasil está bem posicionado para liderar essa mudança como um dos maiores produtores mundiais de biomassa vegetal, mas as matérias-primas lignocelulósicas (contendo lignina, hemicelulose e celulose) são difíceis de desconstruir ou (mais tecnicamente) recalcitrantes à degradação microbiana e enzimática .
Cientistas brasileiros buscam na natureza pistas de como melhorar a despolimerização desses materiais aumentando a disponibilidade dos açúcares que eles contêm. Uma equipe de pesquisa do Laboratório Nacional de Biorrenováveis (LNBR), braço do Centro Brasileiro de Pesquisa em Energia e Materiais (CNPEM), realizou um estudo interdisciplinar envolvendo ômica (genômica, proteômica, metabolômica etc.) e luz síncrotron em Campinas (SP), e descobriu duas novas famílias de enzimas com potencial biotecnológico produzidas por microrganismos no intestino de capivaras. O CNPEM é uma organização privada sem fins lucrativos que é supervisionada pelo Ministério da Ciência, Tecnologia e Inovação (MCTI).
Ambas as famílias de enzimas atuam em componentes das paredes celulares das plantas e, portanto, podem ser usadas para produzir biocombustíveis, produtos bioquímicos e biomateriais. Um deles também tem aplicações potenciais na indústria de laticínios, pois promove a degradação da lactose.
Microrganismos presentes no trato digestivo do animal podem ter estratégias moleculares únicas para despolimerizar essa biomassa. Crédito: Gabriela Félix Persinoti
“Uma de nossas linhas de pesquisa explora a diversidade brasileira em busca de novos mecanismos microbianos que reduzam a recalcitrância de resíduos lignocelulósicos. Constatamos que a capivara é um herbívoro altamente adaptado, capaz de obter energia a partir de resíduos vegetais recalcitrantes e pouco estudado”, disse Mário Tyago Murakami, diretor científico do LNBR e último autor do artigo que relata o estudo publicado na revista científica Comunicações da Natureza.
A capivara (Hydrochoerus hydrochaeris) é o maior roedor vivo do mundo e converte com muita eficiência os açúcares contidos nas plantas em energia, embora seja detestada em alguns lugares porque pode abrigar o carrapato que transmite a febre maculosa brasileira, uma doença infecciosa rara, mas altamente letal causada pela bactéria Rickettsia rickettsii.
“Há muitos estudos com ruminantes, principalmente bovinos, mas as informações sobre herbívoros monogástricos são relativamente escassas. Ao contrário dos ruminantes, as capivaras digerem grama e outras matérias vegetais no ceco, a primeira parte do intestino grosso. Em função de sua conversão de açúcar altamente eficiente, e porque as capivaras na região de Piracicaba [of São Paulo state] se alimentam de cana-de-açúcar, entre outras plantas, partimos da hipótese de que microrganismos presentes no trato digestivo do animal podem ter estratégias moleculares únicas para despolimerizar essa biomassa, o que é muito importante para a indústria brasileira”, disse Gabriela Felix Persinoti, pesquisadora de bioinformática do LNBR e autor correspondente do artigo.
O estudo foi apoiado pela FAPESP por meio de Projeto Temático e bolsa de pós-doutorado concedida a Mariana Abrahão Bueno de Morais.
Metodologia inovadora
A abordagem interdisciplinar utilizada no estudo incluiu multi-ômica (genômica, transcriptômica e metabolômica para caracterizar aspectos moleculares da microbiota intestinal da capivara) e bioinformática, além de aceleradores de partículas do CNPEM para analisar as enzimas descobertas em nível atômico. “Não me lembro de nenhum estudo que tenha combinado todas essas técnicas, incluindo o uso de luz síncrotron [a source of extremely bright electromagnetic radiation that helps scientists observe the inner structures of materials]”, disse Murakami. “Nesta pesquisa, nossa análise perfurou todo o caminho desde a comunidade microbiana até a estrutura atômica de certas proteínas”.
Os cientistas analisaram amostras coletadas do ceco e do reto de três capivaras fêmeas sacrificadas em Tatuí (SP) em 2017 sob a política local de controle da população de capivaras. Os animais não estavam prenhes nem infectados por R. rickettsii.
“As amostras cecal e retal foram coletadas por cirurgia abdominal. O material foi congelado em nitrogênio líquido.[{” attribute=””>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 explained. “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.”
The researchers also wanted to know which microorganisms were most active at the time the samples were collected – in other words, which genes the microorganisms were actually expressing. To this end, they used metatranscriptomics, for which the raw material is RNA. “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 analyzed all this data to identify genes that could play a key role in reducing plant fiber recalcitrance, focusing mainly on hitherto unknown 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.”
Having identified these candidates, they moved on to a biochemical demonstration of their functions. “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.”
According to Murakami, dual validation ensured that novel families were indeed involved. “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
According to Persinoti, one of the newly found families, GH173, has potential uses in the food sector, while another, CBM89, is related with carbohydrate recognition and might help with the manufacturing of second-generation ethanol from sugarcane bagasse and straw.
The researchers are also developing enzyme cocktails with enzyme-hyperproducing fungi, and the newly discovered enzymes could naturally be included in 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.”
Reference: “Gut microbiome of the largest living rodent harbors unprecedented enzymatic systems to degrade plant polysaccharides” by Lucelia Cabral, Gabriela F. Persinoti, Douglas A. A. Paixão, Marcele P. Martins, Mariana A. B. Morais, Mariana Chinaglia, Mariane N. Domingues, Mauricio L. Sforca, Renan A. S. Pirolla, Wesley C. Generoso, Clelton A. Santos, Lucas F. Maciel, Nicolas Terrapon, Vincent Lombard, Bernard Henrissat and Mario T. Murakami, 2 February 2022, Nature Communications.
DOI: 10.1038/s41467-022-28310-y
Discussion about this post