An enzymatic cocktail developed in Brazil feeds the production of second generation ethanol

Researchers at the Brazilian Center for Energy and Materials Research (CNPEM) genetically modified a fungus to produce a cocktail of enzymes that break down carbohydrates in biomass, such as sugarcane waste (leaves and leaves) and bagasse, fermentable sugar for industry. efficient conversion to biofuel.

The development of low-cost enzyme cocktails is one of the main challenges in the production of second generation ethanol.

Second generation biofuels are made from various types of non-food biomass, including agricultural residues, wood chips and used cooking oils. The process of the CNPEM research group paves the way for an optimized use of sugar cane residues to produce biofuels.

The fungus Trichoderma reesei is one of the most prolific producers of plant cell wall degrading enzymes and is widely used in the biotechnology industry. To improve its productivity as a biofactory for the enzymatic cocktail in question, the researchers introduced six genetic modifications in RUT-C30, a strain of the fungus available to the public. They patented the process and reported it in an article published in the journal Biotechnology for biofuels.

“The fungus has been rationally modified to maximize the production of these enzymes of biotechnological interest. Using the CRISPR / Cas9 gene editing technique, we have modified the transcription factors to regulate the expression of genes associated with the enzymes, the suppressed proteases which caused problems with the stability of the enzyme cocktail, and added important enzymes that the fungus lacks in nature. As a result, we were able to allow the fungus to produce a large amount of enzymes from waste agro-industries, a cheap and abundant raw material in Brazil, “Mario T. Murakami, scientist director of the CNPEM Biorenewables Laboratory (LNBR), told Agência FAPESP.

Some 633 million tonnes of sugar cane are processed per harvest in Brazil, generating 70 million tonnes of sugar cane waste (dry mass) annually, according to the National Food Supply Company (CONAB). This waste is underutilized for the production of fuel ethanol.

Murakami pointed out that virtually all of the enzymes used in Brazil to break down biomass are imported from a few foreign producers who keep the technology under the protection of trade secrets. In this context, the imported enzymatic cocktail can represent up to 50% of the production cost of a biofuel.

“Within the traditional paradigm, decades of study were required to develop a competitive platform for the production of enzyme cocktails,” he said. “In addition, cocktails could not be obtained only by synthetic biology techniques from publicly available strains because growers used different methods to develop them, such as adaptive evolution, exposure of the fungus to chemical reagents and the induction of genomic mutations in order to select the most interesting phenotype. Now, however, using advanced gene editing tools such as CRISPR / Cas9, we have succeeded in establishing a competitive platform with only a few rational modifications in two and a half years. “

The bioprocess developed by CNPEM researchers produced 80 grams of enzymes per liter, the highest titer experimentally tested so far for T. reesei from a low-cost sugar-based raw material. This is more than double the concentration previously reported in the scientific literature for the fungus (37 grams per liter).

“An interesting aspect of this research is that it was not limited to the lab,” said Murakami. “We tested the bioprocess in a semi-industrial production environment, scaling it up for a pilot plant to assess its economic feasibility.”

Although the platform has been customized for the production of cellulosic ethanol from sugarcane residue, he added, it can break down other types of biomass and the advanced sugars can be used to produce other biorenewables such as plastics and chemical intermediates.

New class of enzymes

The process was the practical (in terms of industrial application) result of extensive research conducted by the LNBR to develop enzymes capable of breaking down carbohydrates. In another study supported by FAPESP and published in Chemical Nature Biology, the researchers revealed seven new classes of enzymes found mainly in fungi and bacteria.

The new enzymes belong to the glycoside hydrolase (GH) family. According to Murakami, these enzymes have great potential for applications not only in the field of biofuels but also in medicine, food and textiles, for example. Enzymes will inspire new industrial processes by taking advantage of the different ways in which nature breaks down polysaccharides (carbohydrates made up of many simple sugars).

These enzymes break down beta-glucans, some of the most abundant polysaccharides found in the cell walls of grains, bacteria and fungi, and much of the biomass available globally, indicating the potential use of the enzymes in preservatives. food and textiles. In the case of biofuels, the key property is their ability to digest materials rich in plant fibers.

“We set out to study the diversity of nature in the degradation of polysaccharides and how this knowledge can be applied to processes in different industries,” said Murakami. “In addition to the discovery of new enzymes, another important aspect of this research is the network of similarity approach that we use to produce a systematic and in-depth knowledge of this family of enzymes. The approach allowed us to start from zero and in a relatively short time, to arrive at the most studied family of enzymes active on beta-1,3-glucans to date, with information available on specificity and mechanisms of action. “

The main criterion for classifying enzymes is usually phylogeny, that is, the evolutionary history of the molecule, while CNPEM researchers focus on functionality.

“Thanks to advances in DNA sequencing technology, we now have many known genetic sequences and a well-established ability to study and characterize molecules and enzymes in terms of functionality. Therefore, we were able to refine the methodology network of similarity and use it for the first time to study enzymes active on polysaccharides, ”said Murakami.

Using the network of similarity approach, the group classified seven subfamilies of enzymes according to their functionality. Characterizing at least one member of each subfamily, the researchers systematically accessed the diversity of molecular strategies for the degradation of beta-glucans contained in thousands of members of the enzyme family.

Biochemical tour de force

Phylogenetic analysis focuses on regions of DNA that have been conserved over time, while classification by functionality is based on non-conserved regions associated with functional differentiation. “It gave us efficiency and allowed us to group over 1,000 sequences into just seven subgroups or classes with the same function,” Murakami said.

Because the approach was new, the researchers performed several other studies to recheck and validate the classification method. From the seven groups of enzymes capable of breaking down polysaccharides, they obtained 24 entirely new structures, including various substrate-enzyme complexes, considered crucial in providing information for understanding the mechanisms of action involved.

The study included functional and structural analyzes to understand how these enzymes act on the carbohydrates involved. “Polysaccharides are available in dozens of configurations and are capable of many types of chemical bonds,” Murakami said. “We wanted to observe exactly which chemical bonds and architectures are recognized by each enzyme. For this reason, it took a multidisciplinary study, combining structural and functional data supported by analyzes using mass spectrometry, spectroscopy, mutagenesis and diffraction experiments to elucidate the atomic structure. “

In the “News and Views” section of the same issue of Chemical Nature Biology, Professor Paul Walton, Chair of Bioorganic Chemistry at York University in the UK, called the glycoside hydrolase study a “biochemical tour de force” for its innovative approach and praised its ” enormous knowledge “, adding that the researchers were” able to express and isolate copies of each class [of enzymes] examine whether the sequence differences between the classes were reflected in their structures and activities.