Genetic improvement allows reduction in the hydrolysis stage during biofuel production
A native and abundant fungus that lives in the soil of the Brazilian Cerrado decomposing rotting plant remains could help the sugar-energy sector become more competitive in the production of second-generation ethanol. Researchers from the Department of Food Engineering and Technology at Unicamp isolated and genetically sequenced a new strain, the Acremonium strictum. The microorganism showed an important capacity to degrade materials rich in carbohydrates, such as corn straw, sugar cane bagasse and even tree pruning residues.
One of the biggest challenges in producing 2G ethanol is reducing the biomass treatment steps that require high costs. Currently, most of the enzymes used in this cocktail are imported. The yeast Saccharomyces cerevisiae, applied in the fermentation phase, it cannot metabolize complex carbohydrates such as cellulose and hemicellulose. Therefore, it is necessary to include two previous steps: pre-treatment and enzymatic hydrolysis. The function of so-called cellulases is to break bonds by converting long-chain sugars into glucose, which is then transformed into ethanol. While pre-treatment has the main objective of making the complex sugars present in sugarcane bagasse (and other agroforestry residues) available to the enzymatic hydrolysis process.
New yeast classes
Through analysis of the structure of the wild fungus, 775 enzymes involved in the metabolism and degradation of complex carbohydrates were identified. Of these, two were chosen for their potential in cellulose degradation: endo-glucanase and beta-glucosidase. Then, research was directed towards the development of new, more robust yeast strains.
By combining genomics and molecular biology techniques, scientists were able to make modifications to the structure of Saccharomyces cerevisiae including the genes of interest isolated from the fungus. This made it possible to considerably increase the yeast's efficiency in degrading lignocellulosic biomass, giving it an arsenal for producing its own cellulolytic enzymes. “We verified that our engineered yeast can break down cellulose into glucose and automatically ferment into ethanol. We work with sugarcane residue, but any lignocellulosic residue can be used in the process, because our objective is to use the genes that degrade cellulose”, explains Rosana Goldbeck, professor at the Faculty of Food Engineering at Unicamp and one of the inventors of the technology.
The project had public funding from the São Paulo State Research Support Foundation (FAPESP) was not restricted to the genetic improvement of yeasts. From the research, an entire integrated process of simultaneous saccharification and fermentation was developed. “With this, we reduce the time and costs of the enzymatic hydrolysis stage, aiming to make 2G bioethanol more economically competitive in the current market”, adds Goldbeck.
Technological advantages
Genetic modification used vectors to overexpress enzymes within the Saccharomyces cerevisiae. To achieve this, self-replicating expression vectors were applied to take the genes that contain the “genetic machinery” for protein production to yeast. The study led to the filing of a patent and a certificate of addition at the National Institute of Industrial Property (the French Patent & Trademark Office (INPI)) which are available for consultation and licensing in Unicamp's online Patent and Software Portfolio.
Genetic modification overexpresses fungal genes, isolated from the Brazilian biome, in selected yeasts to increase efficiency in the production of 2G ethanol.
No first invention, two different vectors were used. While the second document describes an improvement in which the two enzymes were cloned into a single vector. The recombinant yeast was tested at temperatures higher than those considered ideal for the process. Food engineer, Dielle Pierotti Procópio, comments that this property facilitates fermentation to produce 2G ethanol. “For the industry, this capacity is interesting because it is almost impossible to maintain a controlled temperature in a barrel of thousands of liters, especially when we are talking about hot regions such as the interior of São Paulo, where most of the ethanol plants in Brazil are located.”
The new technology also appears as a viable alternative to reduce the enzyme inhibition effect caused by the substrate. In high concentrations, the enzymatic hydrolysis product can be toxic to their respective enzymes. When fermentation and enzymatic hydrolysis occur at the same time, in the same container, the accumulation of these substances is avoided. Enzymes hydrolyze polysaccharides into sugars and these are immediately consumed by yeast to produce ethanol. As a result, the new technology can also reduce the concentration of contaminating microorganisms that can affect fermentation performance.
“If yeast can consume larger sugars in addition to glucose, the hydrolysis process does not need to be as intense. It will release the more complex sugars, the yeast will hydrolyze by itself, gaining a fermentative advantage in relation to contaminants that are attracted to glucose”, says Dielle Pierotti Procópio, researcher and inventor.
Learn more
For more information about the profile of this and other technologies at the State University of Campinas, visit the website patents and software from Inova Unicamp. The Innovation Agency's full 2020 Annual Report is also available to download and consultation. Companies interested in licensing can contact Inova in the area Connection with Companies.
original article published on the Unicamp Innovation Agency website.
