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Low Cost Second Generation Ethanol Production Powered by Genetically Engineered Enzyme Cocktail

Publicado em 19 agosto 2020

Brazilian researchers used genetic engineering to develop a low-cost platform for the production of enzymes that break down sugar cane waste and bagasse for conversion to biofuels. The new molecules have many potential industrial applications.

Researchers at the Brazilian Center for Energy and Materials Research (CNPEM) have genetically engineered a fungus to produce a cocktail of enzymes that break down carbohydrates into biomass, such as cane waste (tops and leaves) and bagasse, into fermentable sugar for industrially efficient conversion to biofuels.

The development of cheap enzyme cocktails is one of the biggest challenges in producing second-generation ethanol.

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

The fungus Trichoderma reesei is one of the most productive producers of wall cell degrading enzymes and is widely used in the biotechnology industry. To improve its productivity as a biofactory for the enzyme cocktail in question, the researchers introduced six genetic modifications to RUT-C30, a widely 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 the production of these enzymes of biotechnological interest. Using the CRISPR / Cas9 gene editing method, 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 that the fungus lacks in nature. As a result, we were able to let the fungus produce a large amount of enzymes from agricultural waste, a cheap and abundant raw material in Brazil, said Mario T. Murakami, scientific director of CNPEM’s Biorenewables Laboratory (LNBR), Agência FAPESP.

About 633 million tons of sugar cane are processed per harvest in Brazil, which annually generates 70 million tons of sugar cane (dry pulp), according to the National Food Supply Company (CONAB). This waste is underused for the production of fuel ethanol.

Murakami stressed that virtually all enzymes used in Brazil to decompose biomass are imported from a few foreign producers who 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 study were needed to develop a competitive enzyme cocktail production platform,” he said. “In addition, the cocktails could not only be obtained through synthetic biology techniques from widely available strains because the producers used various methods to develop them, such as adaptive evolution, exposing the fungus to chemical reagents and inducing genomic mutations to select the most interesting phenotype. But now, thanks to advanced genre editing tools such as CRISPR / Cas9, we have managed to establish a competitive platform with only 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 to date for T. reesei from a low-cost raw material. This is more than twice as much as 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 laboratory,” Murakami said. “We tested the bioprocess in a semi-industrial production environment and scaled it up for a pilot plant to assess its economic viability.”

Although the platform was adapted for the production of cellulose ethanol from sugar residues, he added, it can break down other types of biomass, and advanced sugars can be used to produce other biorenewables such as plastics and intermediate chemicals.

Novel enzyme class

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

The new enzymes belong 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 medicine, food processing and textiles. The enzymes will inspire new industrial processes by utilizing the different ways that nature breaks down polysaccharides (carbohydrates that consist 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 world’s available biomass, indicating the enzymes’ potential use in food preservatives and textiles. When it comes to biofuels, the most important feature is their ability to melt materials rich in vegetable fibers.

“We intend to study the diversity of nature in degrading 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 similarity network method we use to produce systematic and in-depth knowledge of this enzyme family. The approach enabled us to start from the beginning and in a relatively short time 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 the classification of enzymes is usually phylogeny, ie the evolutionary history of the molecule, while CNPEM researchers focus on functionality.

“Thanks to the progress in DNA sequencing technology, we now have many known genetic sequences and a well-established ability to study and characterize molecules and enzymes with respect to their functionality. As a result, we have been able to refine the similarity network methodology and use it for the first time to study enzymes that are active on polysaccharides, ”said Murakami.

Using the similarity network method, the group classified seven subfamilies of the enzymes based on functionality. The researchers identified at least one member of each subfamily and, in systematic terms, gained access to the diversity of molecular strategies for degrading beta-glucans found in thousands of members of the enzyme family.

Biochemical tour de force

Phylogenetic analysis focuses on DNA regions 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,” Murakami said.

Because the approach was new, the researchers conducted several other studies to double-check and validate the classification method. From the seven groups of enzymes that can degrade polysaccharides, they obtained 24 completely new structures, including various substrate-enzyme complexes, which were considered essential for providing information to help understand the mechanisms of action involved.

The study included functional and structural analyzes to understand how these enzymes act on the carbohydrates involved. “Polysaccharides come in dozens of configurations and can have many types of chemical bonds,” Murakami said. “We wanted to see exactly what chemical bonds and architectures are recognized by each enzyme. For this reason, it must be an interdisciplinary study that combines structural and functional data supported by analysis with mass spectrometry, spectroscopy, mutagenesis and diffraction experiments to elucidate the atomic structure. “

In the section “News and opinions” in the same issue of Natural chemical biology, Professor Paul Walton, Chair of Bioinorganic Chemistry at University of York In the UK, the glycoside hydrolase study rated a “biochemical” tour de force “for its innovative strategy, praising its” enormous insight “and adding that researchers” could express and isolate examples from each class[[[[of enzymes]to investigate whether the differences in sequences between the classes were reflected in their structures and activities. “


“Structural insights into ß-1,3-glucan cleavage of a glycoside hydrolase family” by Camila R. Santos, Pedro ACR Costa, Plínio S. Vieira, Sinkler ET 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, 25 May 2020, Natural chemical biology.

DOI: 10.1038 / s41589-020-0554-5

“Enzymes Knuckled to 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 into an industrially relevant platform for cellulase production ”by Lucas Miranda Fonseca, Lucas Salera Parreiras and Mario Tyago Murakami, 22 May 2020, Biotechnology for biofuels.

DOI: 10.1186 / s13068-020-01732-w

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