Inexpensive second generation ethanol production with genetically modified enzyme cocktail

Publicado em 19 agosto 2020

Brazilian researchers used genetic engineering to develop a low-cost platform for making enzymes that break down sugarcane waste and bagasse for conversion into biofuel. The new molecules have many potential industrial applications.

Researchers at the Brazilian Center for Energy and Materials Research (CNPEM) have genetically engineered a mushroom to create a cocktail of enzymes that industrially break down the carbohydrates in biomass such as sugarcane garbage (tips and leaves) and bagasse into fermentable sugars, efficiently converting them into biofuel.

Developing inexpensive enzyme cocktails is one of the major 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 edible oil waste. The method of the CNPEM research group paves the way for an optimized use of sugar cane residues for the production of biofuels.

The mushroom Trichoderma reesei is one of the most productive manufacturers of plant cell wall degrading enzymes and is widely used in the biotechnology industry. To increase its productivity as a bio-factory for the enzyme cocktail in question, the researchers introduced six genetic modifications into RUT-C30, a publicly available strain of fungus. They patented the process and reported on 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 modified transcription factors to regulate the expression of genes associated with the enzymes, deleted proteases that were causing problems with the stability of the enzyme cocktail, and added key enzymes that are found in the fungus in the Nature is absent. As a result, we were able to enable the fungus to produce a large amount of enzymes from agro-industrial waste, a cheap and abundant feedstock in Brazil, ”Mario T. Murakami, scientific director of CNPEM Biorenewables Laboratory (LNBR), told Agência FAPESP.

According to the National Food Supply Company (CONAB), around 633 million tons of sugar cane per harvest are processed in Brazil, which creates 70 million tons of sugar cane waste (dry matter) annually. This waste is not used sufficiently for the production of ethanol.

Murakami stressed that practically all of the enzymes used to decompose biomass in Brazil are imported from some foreign manufacturers who keep the technology under trade secret protection. In this context, the imported enzyme cocktail can account for up to 50% of the production costs of a biofuel.

“Under the traditional paradigm, it took decades of study to develop a competitive platform for making enzyme cocktails,”

; he said. “In addition, the cocktails could not be obtained from publicly available strains by synthetic biological techniques alone, as manufacturers used various methods to develop them, such as adaptive evolution, exposure of the fungus to chemical reagents, and induction of genomic mutations, to select the most interesting phenotype. Thanks to advanced gene editing tools like CRISPR / Cas9, however, we have now managed to create a competitive platform in two and a half years with just a few rational changes. “

The bioprocess developed by CNPEM researchers produced 80 grams of enzyme per liter, the highest experimentally supported titer reported to date T. reesei from an inexpensive starting material based on sugar. This is more than double the concentration previously reported for the mushroom in the scientific literature (37 grams per liter).

“One interesting aspect of this research is that it wasn’t confined to the laboratory,” Murakami said. “We tested the bioprocess in a semi-industrial production environment and enlarged it for a pilot plant to assess its economic feasibility.”

Although the platform has been adapted to make cellulosic ethanol from sugar cane residues, it can break down other types of biomass and advanced sugars can be used to make other bio-renewable materials such as plastics and intermediate chemicals.

Novel enzyme class

The process was the practical result (in the sense of an industrial application) of extensive research carried out by LNBR to develop enzymes that are able to break down carbohydrates. In another study supported by FAPESP and published in Natural chemical biologyThe researchers discovered seven new enzyme classes that are found primarily in fungi and bacteria.

The new enzymes belong to the glycoside hydrolase (GH) family. According to Murakami, these enzymes have considerable potential for applications not only in the field of biofuels, but also, for example, in medicine, food processing and textiles. The enzymes will inspire new industrial processes by taking advantage of the different ways nature breaks down 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 grain, bacteria and fungi, as well as a large portion of the biomass available worldwide, indicating the potential use of the enzymes in food preservatives and textiles. In the case of biofuels, the key property is their ability to digest material that is rich in vegetable fibers.

“We wanted to explore the diversity of nature in the degradation of polysaccharides and how that 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 similarity network approach, with which we gain systematic and in-depth knowledge about this enzyme family. The approach allowed us to start from scratch and, in a relatively short time, get to the best-studied family of enzymes previously active on beta-1,3-glucans, with information on specificity and mechanisms of action available. “

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 the advances in DNA With sequencing technology, we now have many known genetic sequences and a well-established ability to study and characterize molecules and enzymes for functionality. As a result, we were able to refine the similarity network method and use it for the first time to study enzymes that are active on polysaccharides, ”said Murakami.

Using the similarity network approach, the group classified seven subfamilies of enzymes based on functionality. Researchers characterized at least one member of each subfamily and systematically accessed the variety of molecular strategies for breaking down beta-glucans found in thousands of members of the enzyme family.

Biochemical test of strength

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. “This gave us efficiency and allowed us to group more than 1,000 sequences into just seven subgroups or classes with the same function,” said Murakami.

Since the approach was new, the researchers conducted several other studies to review and validate the classification method. From the seven groups of enzymes that can break down polysaccharides, they obtained 24 entirely new structures, including various substrate-enzyme complexes, which are considered to be crucial in providing information for understanding the mechanisms involved.

The study included functional and structural analyzes to understand how these enzymes affect the carbohydrates in question. “Polysaccharides come in dozens of configurations, and they can form many types of chemical bonds,” Murakami said. “We wanted to closely observe which chemical bonds and architectures are recognized by each enzyme. Therefore, it had to be a multidisciplinary study that combined structural and functional data backed up by analysis using mass spectrometry, spectroscopy, mutagenesis, and diffraction experiments to elucidate the structure of the atom. “

In the “News & Views” section of the same edition of Natural chemical biology, Professor Paul Walton, Chair of Bioinorganic Chemistry at the University of York in the UK, the glycoside hydrolase study rated its innovative approach a “biochemical tour de force” and praised its “tremendous findings”. The researchers were able to “express and isolate specimens from each class[[[[of enzymes]to investigate whether the differences in the sequences between the classes are reflected in their structures and activities. “


“Structural Insights into the Cleavage of ß-1,3-Glucan by a Glycoside Hydrolase Family” by Camila R. Santos, Pedro ACR Costa, Plínio S. Vieira, Sinkler and Gonzalez, Thamy LR Correa, Evandro A. Lima, Fernanda Mandelli , Renan AS Pirolla, Mariane N. Domingues, Lucelia Cabral, Marcele P. Martins, Rosa L. Cordeiro, Atílio T. Junior, Beatriz P. Souza, Érica T. Prates, Fabio C. Gozzo, Gabriela F. Persinoti, Munir S. . Skaf and Mario T. Murakami, May 25, 2020, Natural chemical biology.

DOI: 10.1038 / s41589-020-0554-5

“Enzymes Knuckle at Work” by Paul H. Walton June 17, 2020, Natural chemical biology.

DOI: 10.1038 / s41589-020-0585-y

“Rational engineering of the Trichoderma reesei RUT-C30 strain on an industrially relevant platform for cellulase production “by Lucas Miranda Fonseca, Lucas Salera Parreiras and Mario Tyago Murakami, May 22, 2020, Biotechnology for biofuels.

DOI: 10.1186 / s13068-020-01732-w

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