Low-cost second-generation ethanol production driven by genetically engineered enzyme cocktails


Trichoderma reci fungus RUT-C30 strain, engineered to produce high-yielding enzymes. Sincerely: LNBR-CNPEM

Brazilian researchers used genetic engineering to develop a low-cost platform for the production of the enzyme that breaks down sugarcane waste and Bagas for conversion to bioengineering. Novel molecules have many potential industrial applications.

Researchers at the Brazilian Center for Research in Energy and Materials (CNPEM) have genetically produced a fungus that produces a cocktail of enzymes that break down carbohydrates in biomass, such as sugarcane waste (tops and leaves) and Industrially in fermented sugar for sugar. Efficient conversion to biofuels.

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

Second generation biofuels are manufactured from a variety of non-edible biomass, including agricultural residues, wood chips, and waste cooking oil. The CNPEM research group’s process paves the way for the optimized use of sugarcane residues for the production of biofuels.

Mold Trichoderma Reasi The plant cell is one of the most prolific producers of wall-degrading enzymes and is widely used in the biotechnology industry. To increase its productivity as a biochemical for the enzyme cocktail in question, researchers introduced six genetic modifications to RUT-C30, a publicly available strain of fungi. He patented the process and reported it in an article published in the magazine Biotechnology for Biofuels.

“The fungus was arguably modified to produce maximum of these enzymes of biochemical interest. Using the CRISPR / Cas9 gene-editing technique, we modified transcription factors to regulate the expression of enzyme-associated genes, removing proteases that cause problems with the stability of the enzyme cocktail, and important enzymes. Is called lack of fungus in nature. As a result, we were able to allow Fungus to produce a large amount of enzymes from a cheap and abundant feedstroke from Brazil, “Mario T. Murakami, Scientific Director of CNPEM’s Biorenvouratory Laboratory (LNBR), told Agnesia FAPESP .

According to the National Food Supply Company (CONAB), some 633 million tons of sugarcane are harvested annually in Brazil, generating 70 million metric tons of sugarcane waste (dry mass) annually. This waste is intended for fuel ethanol production.

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

“Under the traditional paradigm, decades of study were needed to develop a competitive enzyme cocktail production platform,” he said. “Furthermore, cocktails cannot be obtained only from publicly available strains by synthetic biology techniques because producers used various methods to grow them, such as adaptive growth, fungi for chemical reagents. To uncover and select genomic stimuli. The most interesting phenotype. Now, however, thanks to advanced gene editing tools such as CRISPR / Cas9, we have been able to establish a competitive platform with some rational modifications in two and a half years. ”

Bioprocess developed by CNPEM researchers produced 80 grams of enzyme per liter, the most experimentally supported titer to date T. Racy From low cost sugar based feedstock. This is more than twice the concentration previously reported in scientific literature for fungi (37 grams per liter).

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

Although the platform was adapted for the production of cellulosic ethanol from sugarcane residues, he said that it could break down other types of biomass, and that elevated sugars be used to produce other biorenewables such as plastics and intermediate chemicals Can.

Novel enzyme class

The process had practical results (in terms of industrial application) under extensive research conducted by LNBR to develop enzymes capable of breaking down carbohydrates. Another study supported by FAPESP and published in Nature Chemical BiologyResearchers revealed seven novel enzyme classes present above all in fungi and bacteria.

The novel enzyme belongs to the glycoside hydrolase (GH) family. According to Murakami, these enzymes have significant potential for applications not only in the field of biofuels, but also in the pharmaceutical, food processing and textile sectors. Enzymes will induce novel industrial processes by the liver in various ways in which nature decomposes polysaccharides (carbohydrates made from 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 a large proportion of the world’s available biomass, indicating the potential use of enzymes in food preservatives and textiles Huh. In the case of biofuels, the important property is their ability to digest material rich in vegetable fibers.

“We set out to study the diversity of nature in degrading polysaccharides and how this knowledge can be applied in various industries,” Murakami said. “In addition to the discovery of novel enzymes, another important aspect of this research is the similarity network approach we use to produce systematic and in-depth knowledge of this enzyme family. The approach enabled us to start from scratch and relatively In a short time, it reached the most studied family of enzymes active on beta-1,3-glucans with available information on specificity and mechanism 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 in advance DNA Sequencing techniques, we now have many well-known genetic sequences and a well-established ability to study and characterize molecules and enzymes in terms of their functionality. As a result, we are able to refine the similarity network method and use it for the first time to study enzymes active on polysaccharides, ”Murakami said.

Using the similarity network approach, the group classified seven subfamilies of enzymes based on functionality. Characterizing at least one member of each subfamily, the researchers systematically accessed a diversity of molecular strategies to degrade beta-glucans comprised of thousands of members of the enzyme family.

Biochemical seizures de force

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

Because the approach was novel, the researchers conducted several other studies to double-check and verify the classification method. From seven groups of enzymes capable of reducing polysaccharides, they derived 24 completely novel structures, including various substrate-enzyme complexes, believed to be important in providing information to help understand the action mechanisms involved.

The study included functional and structural analyzes to understand how these enzymes act on related carbohydrates. Murakami said, “Polysaccharides come in dozens of configurations and are capable of a variety of chemical bonds.” “We really wanted to observe which chemical bonds and architectures are identified by each enzyme. For this reason, it was to be a multi-disciplinary study, combining structural and functional data supported by analysis using mass spectrometry, spectroscopy, mutagenesis, and diffraction experiments to elucidate the atomic structure. ”

In the “News and Views” section of the same Nature Chemical Biology, Professor Paul Walton, Chairman of Bioinorganic Chemistry University of York In the United Kingdom, glycoside hydrolases have been evaluated as a “biochemical ‘tour de force’ for their innovative approach and praising its” tremendous insights “, researchers say” each Were able to express and differentiate examples for the class. [of enzymes] To test whether differences in sequences between classes were reflected in their structures and activities. ”

References:

Camilla R. Santos, Pedro ACR Costa, Plinio s. Vieira, Sinkler ET Gonzalez, Thami LR Korea, Avandro A. “Structural insights into the 1,3-gluten crack” by a glycoside hydrolase family – Lima, Fernando Mandelli, by Renan Madisin. AS Pirola, Marianne Ann. Domingues, Lucelia Cabral, Marcel p. Martins, Rosa L. Cordeiro, Attleo T. Jr., Beatridge p. Souza, arica t. Pratts, Fabio C. Goose, Gabriella F. Persinotti, Munir S. Skaf s. And Mario T. Murakami, 25 May 2020, Nature Chemical Biology.
DOI: 10.1038 / s41589-020-0554-5

Paul H. 17 June 2020 by Walton, “Enzymes down for the job” Nature Chemical Biology.
DOI: 10.1038 / s41589-020-0585-y

“Rational Engineering” Trichoderma Reasi RUT-C30 strain in an industrially relevant platform for cellulase production on 22 May 2020 by Lucas Miranda Fonseca, Lucas Salera Parriras and Mario Tygo Murakami. Biotechnology for Biofuels.
DOI: 10.1186 / s13068-020-01732-w

Leave a Reply

Your email address will not be published.