BR-102016017560-B1 - Genetically modified industrial yeast LVY127 with the oxidoreductive pathway of xylose conversion, gene expression cassettes, process for obtaining 2G ethanol and use of the LVY127 yeast.

BR102016017560B1BR 102016017560 B1BR102016017560 B1BR 102016017560B1BR-102016017560-B1

Abstract

The present invention relates to the genetically modified industrial yeast LVY127 with the oxidoreductive pathway of xylose conversion, gene expression cassettes, process for obtaining 2G ethanol and use of the yeast LVY127.

Inventors

LEANDRO VIEIRA DOS SANTOS
Gonçalo Amarante Guimarães Pereira
RENAN AUGUSTO SIQUEIRA PIROLLA

Assignees

UNIVERSIDADE ESTADUAL DE CAMPINAS - UNICAMP

Dates

Publication Date: 20260317
Application Date: 20160728

Claims (7)

1. Gene expression cassette 1 characterized by comprising: - S. stipitis xylose reductase (XR) under the action of the promoter and terminator of the gene encoding 3-phosphoglycerate kinase (PGK1) in S. cerevisiae; - S. cerevisiae URA3 gene, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation; - S. stipitis xylitol dehydrogenase gene under the action of the promoter and terminator of the gene encoding glyceraldehyde 3-phosphate dehydrogenase, isoenzyme 1 (TDH1) in S. cerevisiae, cloned into the pRS304 vector; and - nucleotide sequence represented by SEQ ID NO:1.
2. Gene expression cassette 2 characterized by comprising: - promoter (ADH1) and terminator (ADH1) of the gene encoding the xylulokinase enzyme in S. cerevisiae; - URA3 gene of S. cerevisiae, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation; and - nucleotide sequence represented by SEQ ID NO:2.
3. Gene expression cassette 3 characterized by comprising: - the xylitol dehydrogenase (XDH) gene of S. stipitis under the action of the promoter and terminator of the gene encoding Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDH1) in S. cerevisiae; - the URA3 gene of S. cerevisiae, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation; and - the nucleotide sequence represented by SEQ ID NO:3.
4. Genetically modified yeast characterized as Saccharomyces cerevisiae DSM32120.
5. Yeast, according to claim 4, characterized by being constructed with the insertion of 1 copy of the gene encoding xylose reductase (XR) from S. stipitis, 2 copies of the gene encoding xylitol dehydrogenase (XDH) from S. stipitis, deletion of aldose reductase GRE3 and insertion of 2 copies of XKS1.
6. Process for obtaining 2G ethanol characterized by being carried out with the yeast Saccharomyces cerevisiae DSM32120.
7. Use of yeast as described in claims 4 and 5, characterized by being for application in any process involving the consumption of xylose.

Description

FIELD OF THE INVENTION [1] The present invention relates to genetically modified industrial yeast LVY127 with the oxidoreductive pathway of xylose conversion, gene expression cassettes, process for obtaining 2G ethanol and use of yeast LVY127. [2] The invention has application in the 2G ethanol production sector. FUNDAMENTALS OF THE INVENTION [3] The United States and Brazil are the two main world producers of ethanol, responsible for 40 and 27 billion liters of ethanol produced in 2009, respectively (Perrone et al., 2010). Ethanol can be produced from different raw material sources, such as corn, beet, wheat, sugarcane, among others. The ethanol production process in Brazil essentially uses sugarcane as raw material. The high productive capacity of this crop and the appropriate climatic conditions for its planting in the country have allowed for a low-cost production model, making Brazil a world reference in ethanol production. [4] Ethanol is obtained via fermentation inside vats where must (sugarcane juice and molasses) and a high concentration of yeast cells (10-17% w/v) are added. The juice and molasses are used as substrates and ethanol concentrations of 8-11% (v/v) are achieved in a period of 6-11 hours at 32-35°C. After fermentation, the entire contents are centrifuged and the fermented must (wine) goes to the distillation towers. The cells collected by centrifugation are treated with sulfuric acid and reused repeatedly in new fermentation cycles, with at least 2 fermentations per day, over a period of 200-250 days. The cell recycling characteristic of the Brazilian ethanol production process utilizes a high concentration of cells at the beginning of fermentation, contributing to reduced growth and high ethanol yield (90-92% of the theoretical conversion yield) (Basso et al., 2008). [5] The stressful conditions of the production process, such as high ethanol concentration, high temperatures, osmotic stress, acidity, and bacterial contamination, resulted in the isolation of more adapted wild yeasts that replaced the starter strains of fermentation in short periods of 20-30 days of cell recycling (Silva-Filho et al., 2005). Basso (2008) analyzed 350 isolates for desirable industrial characteristics such as flocculation, yield, fermentation speed, growth rate, recycling capacity, foam production, and ability to be implemented in distilleries. Among the selected strains, PE-2, CAT-1, and BG-1 stood out for their efficient performance and ability to compete with native yeasts, surviving and dominating the industrial fermentation process. In 2008, PE-2 and CAT-1 were used in approximately 150 distilleries, representing 60% of the ethanol produced in Brazil (Basso et al., 2008). Given their high fermentative performance, the PE-2 and CAT-1 strains are being studied more thoroughly to understand the characteristics that differentiate them from others on an industrial scale. Argueso et al. (2009), observing that commercially available PE-2 stocks presented a wide variety of karyotypes, selected and characterized a single colony, named JAY270. Molecular analyses showed that the PE-2 genome is highly heterozygous (2 SNPs/kb), both structurally and at the nucleotide level, exhibiting structural polymorphisms between homologous chromosomes. The high adaptability of PE-2 to the stressful conditions imposed in a fermentation tank is directly linked to its heterogeneous genomic architecture, making such strains ideal for creating a new generation of industrial organisms, designed for new ethanol production technologies and other biotechnological processes (Argueso & Pereira, 2010). [6] Cofactors play an essential role in a large number of biochemical reactions and in the production of different compounds (Liu et al., 2006). They participate in a series of physiological functions, including the regulation of energy metabolism, adjustment of intracellular redox state, control of carbon flux, mitochondrial activity, regulation of the cell cycle, and modulation of virulence. In microorganisms, the cofactors NADH/NAD+ and NADPH/NADP+ are involved in 740 and 887 biochemical reactions and interact with 433 and 462 enzymes, respectively (Chen et al., 2014). NAD+ acts in oxidations, generally associated with catabolic processes, while NADPH is used in reductions, generally associated with anabolic processes. Reactions in which a hydride ion is incorporated by NAD+ (or NADP+) from a reduced substrate, or NADPH (or NADH) donates a hydride ion to an oxidized substrate, are known as oxidoreductases, also called dehydrogenases (Nelson & Cox, 2011). [7] Nicotinamide adenine dinucleotide (NAD+ in its oxidized form) and its analogue nicotinamide adenine nucleotide phosphate (NADP+) are formed by two nucleotides linked by their phosphate groups through a phosphoanhydride bond. Both coenzymes undergo reversible reduction of the nicotinamide ring. As a substrate molecule undergoes oxidation (dehydrogenation), releasing two hydrogen ato