Published on 07/24/2023

By André Julião  |  Agência FAPESP – Scientists affiliated with the Brazilian Biorenewables National Laboratory at the Brazilian Center for Research in Energy and Materials (LNBR-CNPEM) have obtained new knowledge of how the fungi Aspergillus niger and Trichoderma reesei produce the enzymes used to break down sugarcane bagasse and other biomasses that serve as feedstocks for second-generation (2G) ethanol.

An article on the study is published in Frontiers in Fungal Biology.

“We studied two of the fungal species most widely used to produce various industrial enzymes. T. reesei is the most important for production of cellulase, and A. niger is used to produce citric acid and other enzymes for several applications in the chemical industry,” said Gustavo Pagotto Borin, first author of the article. He conducted the study for his PhD research at LNBR-CNPEM with a scholarship from FAPESP. 

Imported enzyme cocktails currently account for some 50% of the cost of producing second-generation ethanol from sugarcane bagasse in Brazil. The process depends on these feedstocks and is more complex than making first-generation ethanol from sugarcane juice.

Understanding how microorganisms produce the enzymes can help make the process more efficient and pave the way to developing products that do not require imports, are specific to Brazilian biomass and cost less. 

“This kind of research has advanced a great deal in recent years. Companies are investing heavily in biorefineries that produce first- and second-generation ethanol, as well as other products made from sugarcane bagasse, as substitutes for petroleum and contributions to the low-carbon economy,” said Juliana Velasco de Castro Oliveira, co-author of the article and a researcher at LNBR-CNPEM supported by FAPESP. Oliveira was principal investigator for the study.

Energy source

The researchers who conducted the study used metabolomics to investigate how the fungal metabolism works when its source of carbon is sugarcane bagasse. For these microorganisms, carbon is food – a source of energy for growth and development of its life cycle.

They also analyzed the functioning of the fungal metabolism in connection with other carbon sources, such as lactose, glucose and carboxymethyl cellulose (CMC), three types of sugar frequently used by the industry. These sugars can also be used as “food” for enzyme-producing fungi of interest. The researchers set out to discover how and to what extent carbon sources with different levels of complexity alter the fungal metabolism.

Mannitol and trehalose (sugars), and glutamate, glutamine and alanine (amino acids), were the most abundant metabolites in both fungi regardless of the carbon source. “These five molecules seem to be extremely important to both fungi. Their presence in both could mean they’ve been conserved throughout the evolution of these species,” Borin said.

Among other functions, glutamine contributes to nitrogen recycling in cells. It is also important for its participation in the TOR (target of rapamycin) cell signaling pathway, essential for cell growth and other basic cellular functions.

The researchers were surprised to find metabolites that suggested cellular stress, such as gamma-aminobutyric acid (GABA) and glycerol. “This was interesting. Although they’re highly efficient at breaking down biomass, these fungi may not yet have reached their full potential. If this is confirmed in future, it would be worth thinking about genetic modifications to favor these processes and make them more efficient so as to increase enzyme output,” Oliveira said.

In previous years, since Borin’s research for his master’s degree, the researchers had analyzed the same species of fungus with sugarcane bagasse as the carbon source using other omics techniques, such as secretomics, analyzing proteins secreted by cells, and transcriptomics, studying gene transcription. They also analyzed gene co-expression networks, identifying hitherto unknown genes that could be associated with bagasse degradation in T. reesei.

The study reported in Frontiers in Fungal Biology completes a cycle that extends scientists’ understanding of how these fungi produce important enzymes in the conversion of sugarcane bagasse into other products.

The article “Assessing the intracellular primary metabolic profile of Trichoderma reesei and Aspergillus niger grown on different carbon sources” is at: www.frontiersin.org/articles/10.3389/ffunb.2022.998361/full. 

Source: https://agencia.fapesp.br/41959