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Low cost second generation ethanol production powered by genetically modified enzyme cocktails

Publicado em 19 agosto 2020

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

Researchers from the Brazilian Center for Research in Energy and Materials (CNPEM) have genetically engineered a mushroom to produce a cocktail of enzymes that break down carbohydrates in biomass, such as sugarcane junk (buds and leaves) and bagasse, in fermentable sugar for industrial use efficient conversion into biofuel.

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

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

The mushroom Trichoderma reesei is one of the most prolific producers of enzymes that degrade plant cell walls and is widely used in the biotech industry. To increase its productivity as a biofactory for the enzyme cocktail in question, the researchers introduced six genetic modifications in 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 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 enzyme-associated genes, erased proteases that caused problems with the stability of the enzymatic 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 agro-industrial waste, a cheap and abundant raw material in Brazil, “Mario T. Murakami, Scientific Director of the Laboratory of Biorenewables (LNBR), told Agência FAPESP of the CNPEM.

According to the National Food Supply Company (CONAB), approximately 633 million tons of cane are processed per crop in Brazil, generating 70 million tons of cane waste (dry mass) annually. These wastes are underused for the production of combustible ethanol.

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

“According to the traditional paradigm, it took decades of studies to develop a competitive enzyme cocktail manufacturing platform,”

; he said. “Furthermore, the cocktails could not be obtained solely with synthetic biology techniques from publicly available strains because the producers used different 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. Now, however, thanks to advanced genetic modification tools such as CRISPR / Cas9, we have been able to establish a competitive platform with few rational modifications in two and a half years. “

The bioprocess developed by CNPEM researchers produced 80 grams of enzymes per liter, the highest experimentally supported titer reported 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 mushroom (37 grams per liter).

“An interesting aspect of this research is that it wasn’t limited to the lab,” Murakami said. “We tested the bioprocess in a semi-industrial production environment, adapting it to a pilot plant to assess its economic feasibility.”

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

New class of enzymes

The process was the practical result (in terms of industrial application) of extensive research conducted by LNBR to develop enzymes capable of breaking down carbohydrates. In another study supported by FAPESP and published in Chemical biology of nature, the researchers revealed seven new classes of enzymes found mostly 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, for example. Enzymes will inspire new industrial processes by exploiting 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 cereals, bacteria and fungi, and a large fraction of the biomass available in the world, indicating the potential use of enzymes in food preservatives and tissues. In the case of biofuels, the key property is their ability to digest material 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 fields,” Murakami said. “In addition to the discovery of new enzymes, another important aspect of this research is the similarity network approach we use to produce a systematic and in-depth knowledge of this enzyme family. The approach allowed us to start from scratch 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 the progress DNA sequencing technology, we now have many known genetic sequences and an established ability to study and characterize molecules and enzymes in terms of functionality. As a result, we were able to refine the similarity network methodology and use it for the first time to study enzymes active on polysaccharides, ”Murakami said.

Using the similarity network approach, the team classified seven subfamilies of the enzymes based on functionality. By characterizing at least one member of each subfamily, the researchers systematically accessed the diversity of molecular strategies for degrading the beta-glucans contained in thousands of enzyme family members.

Biochemical tour de force

Phylogenetic analysis focuses on DNA regions that have been conserved over time, while function classification 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.

As the approach was new, the researchers performed several other studies to double-check and validate the classification method. From the seven groups of enzymes capable of degrading polysaccharides, 24 completely new structures were obtained, including various substrate-enzyme complexes, considered crucial in 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 affected carbohydrates. “The polysaccharides come in dozens of configurations and are capable of many types of chemical bonds,” Murakami said. “We wanted to look at exactly which chemical bonds and architectures are recognized by each enzyme. For this reason, it had to be a multidisciplinary 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 issue of Chemical biology of nature, Professor Paul Walton, president of bioinorganic chemistry at the University of York in the UK, classified the glycoside hydrolase study a biochemical “tour de force” for its innovative approach and praised its “extraordinary insights”, adding that the researchers were “able to express and isolate specimens from each class.[[[[of enzymes]to examine whether the differences in the sequences between the classes were reflected in their structures and activities “.


“Structural insights in ß-1,3-glucan cleavage by 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, May 25, 2020, Chemical biology of nature.

DOI: 10.1038 / s41589-020-0554-5

“Enzymes knuck down to the job” by Paul H. Walton June 17, 2020, Chemical biology of nature.

DOI: 10.1038 / s41589-020-0585-y

“Rational engineering of the Trichoderma reesei RUT-C30 strain in 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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