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Study enables the creation of modified yeast to produce second-generation ethanol



Brazilian research opens the way to increase efficiency in the production of second generation (2G) ethanol with the discovery of new targets for metabolic engineering towards a more robust industrial yeast strain. An article with the results of the work was recently published in Scientific Reports.

All of the work's databases are available to the scientific community in the repository at the State University of Campinas (Unicamp), which is part of the international Dataverse initiative, supported by FAPESP.

First-generation ethanol (1G) is produced from sources rich in carbohydrates (such as sucrose), mainly sugarcane, in the case of Brazil. Sugarcane processing generates a large amount of fibrous waste, such as bagasse, which can be used to generate steam and electricity in the mills. But this residue is rich in cellulose and hemicellulose, types of polymeric carbohydrates found in plants and trees, which give them rigidity. Thus, they can also be used to produce more ethanol, called 2G, as they can be converted into smaller molecules to be fermented by yeast and other microorganisms.

Porém, o maior desafio para a produção de etanol 2G é a eficiência da conversão da celulose and hemicellulose in ethanol, as they are polymers that are difficult to decompose (hydrolysis). The process requires the removal of lignin, a resistant polymer that makes up fibrous waste, and the hydrolysis of cellulose and hemicellulose into simple sugars, which can be converted into ethanol by yeast. These processes are expensive, consume a lot of energy and can generate highly inhibitory by-products that disrupt the fermentative capacity of the yeasts responsible for producing this alcohol.

“The production of 2G ethanol still needs to be optimized to increase its efficiency. One of the necessary approaches for this optimization is to identify yeasts that can resist the 'spoil' of inhibitory molecules derived from the processing of these residues", explains biologist Marcelo Mendes Brandão, a researcher at the Center for Molecular Biology and Genetic Engineering (CBMEG) at Unicamp. “It is already known that some strains of industrial yeast have higher levels of tolerance to these compounds. A well-documented example is the industrial yeast Saccharomyces cerevisiae SA-1, a Brazilian industrial strain of fuel ethanol that demonstrated high resistance to the inhibitors produced by the pre-treatment of cellulosic complexes and the focus of our study published in Scientific Reports”, he adds.

The analyzes are in line with the proposal of the FAPESP Thematic Project “An integrated approach to explore a novel paradigm for biofuel production from lignocellulosic feedstocks”. , coordinated by Telma Franco, from Unicamp's Faculty of Chemical Engineering. The group's work has also received funding through four other projects


Methodology



The experiments were carried out by Dielle Pierotti and Felipe Ciamponi, PhD students at the time, in a collaboration between the laboratories coordinated by Thiago Olitta Basso, from the Department of Chemical Engineering at the Polytechnic School of the University of São Paulo (USP), and Brandão, from CBMEG- Unicamp.

“To put this work in context with research on 2G ethanol, we already knew that certain strains of S. cerevisiae were resistant to these molecules with inhibitory activity, but the molecular mechanism used by these yeasts to resist such inhibitors is complex, involving multiple processes and regulatory pathways”, details Basso.

According to the USP scientist, another point that supported the publication is that one of the main by-products resulting from the processing of sugarcane bagasse in the production of 2G ethanol is p-coumaric acid (pCA), one of the main inhibitors present in the bagasse after such processing . “Data available in the literature indicate that this chemical generally inhibits the growth of S. cerevisiae and, as a result, decreases its performance in ethanol production.”

The team decided to use a bioinformatics approach to integrating “multiomics” data in the study. In other words, it brought together data from transcriptomics – the study of the set of mRNA, messenger ribonucleic acid, produced by an organism at a given time – with data from quantitative physiology. “This allowed us to better understand how this yeast responded to the culture environment”, says Brandão. With the data in hand, the study focused on the molecular and physiological characterization of the general response of the yeast to a relevant inhibitor for the process that uses sugarcane bagasse as raw material for the production of 2G ethanol.

The biological experiments were conducted by Pierotti and Ciamponi at the Bioprocess Laboratory (BELa) of the Department of Chemical Engineering at Poli-USP. The experiments were carried out using continuous cultivations in bioreactors (chemostats). Such cultures guarantee a very well-controlled and defined environment for the evaluated microorganisms, where it is possible to study the effect of the inhibitor on the physiological and genetic aspects of these microorganisms, without the interference of other variables that make it difficult to interpret alterations in the expression/transcription of genes in these microorganisms. microorganisms. Thus, the SA-1 yeast was cultivated in anaerobic chemostats, in the presence and absence of the inhibitor (pCA). From these cultures, samples were collected at steady state for determination of physiological parameters and part of the biological material was sent to Taiwan for RNA sequencing.

The results were analyzed at the Laboratory of Integrative and Systemic Biology at CBMEG. They show that the biological mechanisms used by S. cerevisiae SA-1 to survive under the influence of these inhibitors are much more intricate than previously understood. Quantitative physiological data suggest that pCA stress can induce greater cellular activity in the SA-1 strain under anaerobic conditions (relevant for the industrial process), with an increase in the rate of sugar uptake and ethanol production.

Brazil has advanced in research for a better use of the biomass available in its biodiversity for the production of bioproducts, those consumer goods that can be built/assembled/produced from the transformation of part of an organism, as in the case of tissues and vegetable fibres, or as a result of the metabolism of these living beings. “In the latter case, we can mention the production of fuel alcohol, a commodity with a great impact on the national economy”, points out Brandão.

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