“Enzyme Cocktail” to Fermentable Sugar Powers Biofuel Production

The study by Researchers at the Brazilian Center for Research in Energy and Materials (CNPEM) have genetically engineered a fungus to produce a cocktail of enzymes that break down the carbohydrates in biomass, such as sugarcane trash (tops and leaves) and bagasse, into fermentable sugar for industrially efficient conversion into biofuel. The development of low-cost enzyme cocktails is one of the main challenges in producing second-generation ethanol.

Second-generation biofuels are manufactured from various kinds of nonfood biomass, including agricultural residues, wood chips and waste cooking oil. The CNPEM research group’s process paves the way for optimized use of sugarcane residues to produce biofuels. Some 633 million tons of cane are processed per harvest in Brazil, annually generating 70 million metric tons of cane trash (dry mass). This waste is underutilized for fuel ethanol production.

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 enhance its productivity as a biofactory for the enzyme cocktail in question, the researchers introduced six genetic modifications into RUT-C30, a publicly available strain of the fungus. They patented the process and reported it in an article published in the journal Biotechnology for Biofuels.

“The fungus was rationally modified to maximize production of these enzymes of biotechnological interest. Using the CRISPR/Cas9 gene-editing technique, we modified transcription factors to regulate the expression of genes associated with the enzymes, deleted proteases that caused problems with the stability of the enzyme cocktail, and added important enzymes the fungus lacks in nature. As a result, we were able to allow the fungus produce a large amount of enzymes from agroindustrial waste, a cheap and abundant feedstock in Brazil,” Mario T. Murakami, Scientific Director of CNPEM’s Biorenewables Laboratory (LNBR), told Agência FAPESP.

The novel enzymes belong to the glycoside hydrolase (GH) family. These enzymes have significant potential for applications not just in the field of biofuels but also in medicine, food processing and textiles, for example. The enzymes will inspire novel industrial processes by leveraging the different ways in which nature decomposes 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 cereals, bacteria and fungi, and a large fraction of the world’s available biomass, indicating the enzymes’ potential use in food preservatives and textiles. In the case of biofuels, the key property is their capacity to digest material rich in vegetable fibers.

Murakami stressed that practically all the enzymes used in Brazil to decompose biomass are imported from a few foreign producers that keep the technology under trade secret protection. In this context, the imported enzyme cocktail can represent as much as 50% of a biofuel’s production cost.

“Under the traditional paradigm, decades of studies were needed to develop a competitive enzyme cocktail production platform,” he said. “Moreover, the cocktails couldn’t be obtained solely by synthetic biology techniques from publicly available strains because the producers used different methods to develop them, such as adaptive evolution, exposing the fungus to chemical reagents, and inducing genomic mutations in order to select the most interesting phenotype.

Now, however, thanks to advanced gene editing tools such as CRISPR/Cas9, we’ve succeeded in establishing a competitive platform with just a few rational modifications in two and a half years.”The bioprocess developed by the CNPEM researchers produced 80 grams of enzymes per liter, the highest experimentally supported titer so far reported for T. reesei from a low-cost sugar-based feedstock. 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 wasn’t confined to the lab,” Murakami said. “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 was customized for the production of cellulosic ethanol from sugarcane residues, he added, it can break down other kinds of biomass, and advanced sugars can be used to produce other biorenewables such as plastics and intermediate chemicals.

The novel enzymes belong to the glycoside hydrolase (GH) family. According to Murakami, these enzymes have significant potential for applications not just in the field of biofuels but also in medicine, food processing and textiles, for example. The enzymes will inspire novel industrial processes by leveraging the different ways in which nature decomposes 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 cereals, bacteria and fungi, and a large fraction of the world’s available biomass, indicating the enzymes’ potential use in food preservatives and textiles. In the case of biofuels, the key property is their capacity to digest material rich in vegetable fibers.