WO2018018111A1

WO2018018111A1 - Industrial yeast lvy127 genetically modified via the oxy-reductive xylose conversion pathway, genetic expression cartridges, method for obtaining ethanol 2g and use of the yeast lvy127

Info

Publication number: WO2018018111A1
Application number: PCT/BR2017/000064
Authority: WO
Inventors: Leandro Vieira DOS SANTOS; Gonçalo Amarante Guimarães PEREIRA; Renan Augusto Siqueira PIROLLA
Original assignee: Universidade Estadual De Campinas - Unicamp
Priority date: 2016-07-28
Filing date: 2017-06-26
Publication date: 2018-02-01

Abstract

The present invention relates to industrial yeast LVY127 genetically modified via the oxy-reductive xylose conversion pathway, to genetic expression cartridges, to a method for obtaining ethanol 2G and to the use of the yeast LVY127.

Description

LVY127 GENETICALLY MODIFIED INDUSTRIAL Yeast WITH QXI-HEDUCTIVE XLLOSE CONVERSION, OENIC EXPRESSION CASSETS, ETHANOL 26 OBTAINING PROCESS, AND LVX127 USE

FIELD OF INVENTION

[1] The present invention relates to LVY127 genetically modified industrial yeast with the oxy-reductive xylose conversion pathway, gene expression cassettes, 2G ethanol production process and use of LVY127 yeast.

[2] The invention has application in the ethanol production sector.

2G.

BACKGROUND OF THE INVENTION

[3] The United States and Brazil are the world's two largest producers of ethanol, accounting respectively for 40 and 27 billion liters of ethanol produced in 2009 (Perrone et al., 2010). Ethanol can be produced from different sources of raw materials, such as corn, beet, wheat, sugar cane, among others. The ethanol production process in Brazil essentially uses sugar cane as its raw material. The high productive capacity of this crop and the appropriate climatic conditions for its cultivation in the country allowed to obtain a low cost production model, making Brazil a world reference in ethanol production.

[4] Ethanol is obtained through the n-inland fermentation pathway of dornas where the wort (sugarcane juice and molasses) and a high concentration of yeast cells (10-17% w / v) are added. . Broth and molasses are used as substrates and ethanol concentrations of 8-11% (v / v) are achieved over a period of 6-1 hours at 32-35 ° C. After fermentation, all contents are centrifuged and fermented must (wine) goes to the distillation towers. Cells collected by centrifugation are treated with acid sulfuric acid and reused repeatedly in new fermentative 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 low growth and high ethanol yield (90-92% of theoretical conversion yield) (Basso et al., 2008). .

[51 Stressful conditions in 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 fermentation starters in short periods of 20-30 days of cellular recycling (Siiva-Filho et al., 2005). Basso (2008) analyzed 350 isolates for desirable characteristics of industrial interest such as flocculation, yield, fermentation speed, growth rate, recycle capacity, foam production and distillery deployment capacity. 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 ethanol produced in Brazil (Basso et al., 2008). In view of their high fermentative performance, strains PE-2 and CAT-1 are being studied further in order to understand the characteristics that differentiate them from others on an industrial scale. Argueso et al. (2009), noting that commercially available PE-2 stocks had a wide variety of karyotypes, selected and characterized a single colony called JAY270. Molecular analyzes have shown that the PE-2 genome is highly heterozygous (2 SNPs / kb), both structurally and at the nucleotide level, showing structural polymorphisms between homologous chromosomes. The high adaptability of PE-2 to the stressful conditions imposed on a fermentation vessel is directly linked to its heterogeneous genomic architecture, making such strains ideal for creating a new generation of industrial organisms, idealized for new production technologies. ethanol 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 number of physiological functions, including energy metabolism regulation, intracellular redox state adjustment, carbon flow control, mitochondrial activity, cell cycle regulation, and virulence modulation. In microorganisms, NADH / NAD + and NADPH / NADP + - cofactors are involved in 740 and 887 biochemical reactions and interact with 433 and 462 enzymes, respectively (Chen et al., 2014). NAD + acts on oxidations, usually associated with catabolic processes, while NADPH is used in reductions, usually associated with anabolic processes. Reactions where NAD + (or NADP +) incorporates an ion hydride from a reduced substrate, or NADPH (or NADH) donates an ion hydride to an oxidized substrate are known as oxyreductases, also called dehydrogenases (Nelson & Cox, 2011).

[7] Nicotinamide adenine dinucleotide (MAD + in its oxidized form) and its analog nicotinamide adenine nucleotide phosphate (NADP +) are formed by two nucleotides linked by their phosphate groups via a phosphohydride bond. Both coenzymes had been reversibly reduced in the nicotinamide ring. As a substrate molecule undergoes oxidation (dehydrogenation), releasing two hydrogen atoms, the oxidized form of the nucleotide (NAD + or NADP +) receives one hydride ion (: H-, the equivalent of one proton and two electrons), being transformed into its reduced form (NADH or NADPH). The second proton removed from the substrate is released into the aqueous solvent (Nelson & Cox, 2011).

[8] In S. cezeviaiae there is increased complexity in balancing stubbornness between formation and consumption of cofactors, as there is no activity of a transhydrogenase that can convert NADH directly into NADPH. In addition, since metabolism is compartmentalized and there is no cofactor flow between mitochondria and cytosol, the formation and consumption of NADH and NADPH cofactors must be balanced in each compartment, which imposes carbon flow restrictions. Therefore, there must be distinct mechanisms to re-oxidate cofactors in cytosol and mitochondria (Santos et al., 2004; Vemuri et al., 2006).

[9] NADH is generated primarily in cytosol by glycolysis and mitochondria by the cyclic acid cycle (TCA) (Vemuri et al., 2006). In cytosol, glucose is oxidized using NAD + as cofactor, which is simultaneously converted into its reduced form, NADH, in an equivalent amount. Since NADH is a cofactor highly used in various metabolic reactions, any change in NADH / NAD + rate leads to major changes in metabolism. NADH / NAD * balance is an important factor in maintaining glycolytic flow. Therefore, NAD + depletion may force flow to cease. In order to increase glycolytic activity, NADH produced in glycolysis, fatty acid oxidation and the citric acid cycle must be oxidized to NAD * to achieve a redox equilibrium (Liu et al., 2006).

[10] There are at least five mechanisms for NADH reoxidation in S. cerevisiae: alcoholic fermentation; glycerol production; intramitochondrial NADH oxidation via NADH internal raitochondrial dehydrogenase; and cytosolic regeneration via external mitochondrial NADH dehydrogenases or via glycerol-3-phosphate during respiration (Bakker et al., 2001).

[11] Yeast regeneration of NAD * from NADH can occur in aerobiosis, the electron transport chain, with oxygen as the final electron acceptor, and with the production of large amounts of ATP. Mitochondrial NADH is oxidized by a membrane-bound internal mitochondrial NADH dehydrogenase encoded by the NDI1 gene. Or it may occur under anaerobic conditions in fermentation, primarily for acetaldehyde as electron acceptor in a reaction where & Alcohol dehydrogenase catalyzes the oxidation of NADH to NAD ^+, to produce ethanol and carbon dioxide from pyruvate. Cytosolic NADH is oxidized by two membrane-bound external (cytosolic) mitochondrial NADH dehydrogenases encoded by the NDE1 and NDE2 genes with catalytic sites in contact with the cytosol. Additionally, glycerol-3-phosphate dehydrogenases

(encoded by GPD1 and GPD2) oxidize cytosolic NADH with concomitant glycerol formation. Under anaerobic conditions, NADH originating from the production of organic acids, biomass, among others, is re-oxidized to NAD * by glycerol formation, as respiration is not possible and ethanol formation is a redox neutral process (Bro et al., 2006; Liu et al., 2006; Vemuri et al., 2006).

[12] In S. cerevisiae, NADPH may be generated mainly in the reactions catalyzed by two dehydrogenases in the oxidative phase of pentose phosphate (ZWF1, glucose-6-Ρ dehydrogenase and GND1, 6-phosphogluconate dehydrogenase) in the isocitrate-catalyzed reaction. NADP + dependent dehydrogenase (ΙΌΡ2), the NADP * dependent acetaldehyde catalyzed reaction (ALD6) and the malic enzyme catalyzed reaction (MAE1). Of all cited above, the pentose phosphate pathway represents the major NADPH production pathway in yeast (Santos et al., 2004; Wang et al., 2013).

[13] The pentose phosphate pathway is mainly enzyme-controlled, with NADPH and ATP competitively inhibiting glucose-6-dehydrogenase (ZNFD and 6-phosphogluconate dehydrogenase (GND1). Coordinated regulation of genes involved with NADPH metabolism , including most PPP genes, has been reported under conditions of oxidative stress.NADPH-dependent gene activation involves STB5, also responsible for suppressing PGI expression, which encodes phosphoglycosis isomerase at the junction between the glycolytic pathway and the pathogen. This transcription factor plays a key role in redirecting carbon flow to provide additional NADPH in response to oxidative stress, for example, and is responsible for maintaining the basal flow of PPP under anaerobic conditions (Celton et al., 2012b).

[14] Celton (2012) has shown that yeast cells respond to increased NADPH demand by increasing flow through the pentose phosphate and acetate formation pathways, corresponding to 80 and 20% of NADPH demand, respectively. . Some pentose phosphate genes are up-regulated as demand for NADPH increases. GND1 and SOL3 are induced when a moderate concentration of NADPH is required by the addition of acetoin. When this demand increases, two genes from the non-oxidative phase of pentose phosphate, TAL1 and TKL1 are also up-regulated (Celton et al., 2012a).

[15] Redox balance occurs when cofactor production and consumption are approximately equal. Unbalancing the oxyductive potential can cause cell damage, energy and carbon expenditure and disrupt all cellular metabolism. The number and availability of cofactors in the cell may become a limiting step, being essential in various metabolic reactions and in the production of different compounds. Therefore, manipulations in the redox balance and the amount of cofactors produced by the cell can be a powerful tool in improving yeast fermentative performance (Liu et al., 2006; Chen et al., 2014). Redox balance can be regulated by different approaches, such as regulation of the metabolic pathway by adjusting gene expression, protein engineering involving cofactor binding site, genome restructuring by removal of redundant genes that use cofactors, coenzyme regeneration by protein restructuring. metabolic pathways, among others (Chen et al., 2014).

[16] In addition to the alternatives described above, one method that can lead to advances in ethanol production by 5. cerevisiaet and which does not involve direct genetic manipulation is the application of an electric current to stimulate yeast metabolism in electrode-modified fermenters called bioelectric reactors (Thrash and Coates, 2008). In this modified reactor, microorganisms are grown in a chamber containing an electrode that receives the electric current from a circuit coupled to a source, transferring electrons to the cells. The electrons received by the cells can then be used in metabolism reactions involving substrate reduction, providing reducing power to the cells, reducing the generation of byproducts and consequently increasing the ethanol production.

[17] Second generation ethanol or cellulosic ethanol consists of converting polymers that form the plant cell wall into ethanol. These polymers constitute cellulose, hemicellulose and lignin, and their hydrolysis provides fermentable sugars, represented by hepheres and pentose, which can be converted to ethanol by 5. cerevisiae. Hexoses are usually used by S. ceravlsiae. However, wild strains of these yeasts cannot metabolize the xylose and arablnose pentoses present in the biorass. Given their significant share in the constitution of biomass, the full use of these compounds would increase the yield and the viability of the second generation ethanol production process.

[18] Metabolic engineering of yeasts for introducing metabolic pathways of xylose consumption focuses on two major pathways: the Xylcse Reductase - Xylitol Dehydrogenase (XR-XDH) pathway and the Xylose Isomerase (XI) pathway. The XR-XDH pathway, present in eukaryotic microorganisms, consists of two ozone reduction reactions, where xylose is reduced to xylitol by the action of the enzyme xylose reductase (XR), a NADPH / NADH mediated reaction and then , xylitol is oxidized to xylulose by the enzyme Xylitol dehydrogenase (XDH) mediated exclusively by NAD *. The difference in specificity by the cofactors generates the redox imbalance. To generate NADPH for the xylose reductase reaction, part of the xylose carbon must be directed through the oxidative phase of pentose phosphate, involving the glucose-6-phosphate dehydrogenase and 6-phosphogluconate reactions. Although allowing efficient NADPH regeneration, carbon loss occurs in the form of CO: which impacts the yield of ethanol from xylose (van Maris et al., 2007).

[19] In aerobiosis, NAD * is regenerated in the respiratory chain, with oxygen as the final electron acceptor. Under limited oxygen concentrations, the reoxidation of excess NADH generated by the reaction of xylitol dehydrogenase is carried out through lower xylose compounds such as xylitol and glycerol by-products of fermentation. Xylitol production occurs via xylose reductase, which due to the dual specificity of coenzyme can also use NADH. Since this mechanism involves the consumption of one xylose for each NADH generated, it has a high impact. negative on xylose ethanol yield (Hahn-Hagerdal et al., 2001; Aguiar et al., 2002; Kuyper et al., 2004; van Maria et al., 2007).

[20] Although S. cerevisiae possess genes encoding a NADH dependent aldose reductase IGRE3) and xylitol dehydrogenase (XYL2) _R & they will not permit their efficient growth in xylose. Overexpression of these genes using endogenous promoters allowed growth in shakers, with growth rate of 0.01 h-1 in D-xylose and 55% xylitol yield. Under anaerobic conditions, these strains were unable to grow (van Maris et al., 2007).

[21] The XR and XDH most commonly used in studies involving the oxy-reductive pathway of xylose conversion belong to Scheffersomyces stlpitis yeast. Due to the preference of S. stipitis XR over NADPH, whereas XDH produces only NADH, the resulting xylitol accumulation and low ethanol yield have been attributed to such cofactor unbalance. Several studies have focused on strategies to solve this problem. Cofactor binding sites in xylose reductases are the target of protein engineering techniques to increase NADH affinity for NADPH. However, the increase in the NADH / NADPH rate in these studies is due to a decrease in the catalytic efficiency of NADPH binding by XR, while the actual increase in NADH binding affinity is quite low (Liang et al., 2007; Watanabe). et al., 2007; Bengtsson et al., 2009; Runquist et al., 2010; Cai et al., 2012).

[22] In a similar approach, Watanabe (2005) completely altered the affinity of the enzyme xylitol dehydrogenase from 5. NAD * to NADP 'stypotypes by site-directed mutagenesis. Double-mutated enzymes (D207A / I203R and D207A / F209S) had increased affinity for NADP, but preference for NAD + was still higher. Triple mutated (D207A / I203R / F209S) and quadruple enzymes (D207A / I208R / F209S / N211P.) Showed values greater than 4500 times in kcat / Kra with NADP + relative to wild enzyme, reaching values comparable to kcat / Km with NAD + relative to wild enzyme. In a later study, the triple mutation enzyme was introduced into a S. cerevisiae strain, replacing the wild-type S. stipitis XDH. The Y-ARSdR strain produced 86% less xylitol and consequently 41% more ethanol compared to the parent strain (Watanabe et 2007). The MA-N5 strain with the NADP + dependent mutant XOH showed a high yield of 0.49 g / g from total lignocellulosic hydrolyzate present sugars (Matsushika et al., 2009). In order to improve the cofactor balance in these strains, Khattab (2013) used site-directed mutagenesis strategies to produce a series of XRs that only use NADPH, which upon oxidation provides NADP + for the dependent NADP + XDH. The resulting strains showed a reduction of 34.4 to 54.7% xylitol compared to the control strains, as well as 10 and 20% increases in ethanol production in two of the strains constructed with the new xylose reductases.

[23] Unlike the approaches described above, which rationally or randomly modify the enzymes of the xylose conversion pathway, other strategies have been described in literature * where alteration occurs in the central sugar consumption pathways aiming at alteration and adjustment in cofactor balance. Decreased NADPH production by inactivation of the oxidative phase genes of the pentose phosphate pathway, GND1, which encodes 6-phosphogluconate dehydrogenase or ZWFl, which encodes glucose 6-phosphate dehydrogenase, resulted in reduced xylitol and increased yield of ethanol obtained from xylose. However, such strains had a significant decrease in the rate of xylose consumption, explained by the reduction of this sugar happening mainly mediated by NADH, by the action of the enzyme xylose reductase (XR), with specificity for both cofactors. Another fact observed was the increase in the amount of acetate produced, suggesting that acetate production from acetaldehyde has become the new source of NADPH supply to XP. (Jeppsson et al., 2002; Cai et al., 2012).

124] To facilitate the regeneration of NADPH, the expression of a KDP (dependent IGDP-dependent Kluyveromyces lactis NADP * glyceraldehyde-3-phosphate dehydrogenase) associated with deletion of ZWF1 resulted in a lineage with higher ethanol yield from xylose. The regeneration of NADPH through this gene is not associated with CO: production, as it happens in the pentose phosphate pathway, justifying the best performance in relation to the strain that does not have the expressed GOP. Such an approach allows the creation of a non-carbon loss cofactor regeneration system through the production of CO2, which results in a higher amount of ethanol produced (Verho et al., 2003).

[25] Another strategy for introducing an alternative NADH re-oxidation pathway was by introducing the alternative phosphocetolase pathway commonly found in prokaryotes. The heterologous expression of Bacillus subtilis phosphotransacetylase and iSntamoeba histolytica acetaldehyde dehydrogenase aims at a deviation in the pentose phosphate pathway by the conversion of xylulose-5-Ρ into glyceraldehyde-3-glic, and acetyl-P, which can be converted by phosphotransacetylase to acetyl-CoA, which is reduced to acetaldehyde by acetaldehyde dehydrogenase, using NADH as cofactor. The introduction of these genes into S. cerevislae resulted in a 25% increase in lineage in ethanol production due to less formation of the xylitol byproduct (Sonderegger et al., 2004}. [26] Despite efforts in strategies to regulate and adjust the redox balance in strains containing the oxidoredoxyl conversion pathway, other unknown reasons besides redox imbalance are responsible for the high amount of xylitol producing and low ethanol productivity. under anaerobic conditions of xylose consumption. Other efforts such as the development of a metabolic and regulatory network, taking into account the entire redox metabolism of the cell must be considered to solve this problem (Cai et al., 2012).

[27] Xiong, M. et al. The document refers to altering xylose reductase to increase ethanol production in Saccharomyces cezevisiae, more specifically altering XR to have greater affinity for NADH than NADPH.

[28] Khathaab, 8.M.R. et al. The document relates to bioethanol production using combinations of Saccharomyces cezevisiae, Xylose reductase and Xylitol dehydrogenase, more specifically in altering site specific XR to have affinity for NADPH only.

[29] US20130040353 relates to ethanol production in the Saccharomyces cerevisiae medium and alteration of XR to have greater affinity for NADH rather than NADPH.

[30] The present invention differs from the above documents in that it does not alter the XR sequence. An XR that naturally already has higher affinity for NADH instead of NADPH was used, decreasing redox imbalance and increasing ethanol production.

[31] US20120329104 relates to the combination of microorganisms for ethanol production and describes the creation of a lineage using the S. stipitls oxidative pathway. No strategy was used to regulate the redox balance of the strain. In the present invention it was used in an industrial strain, more tolerant to inhibitors present in the 2G process and with characteristics of industrial interest. In addition, various strategies have been employed to regulate the redoz balance of the strain and to improve ethanol production. The cassettes used also differ in promoter and terminator, insertion region and marker used.

[32] Given the above, it would be helpful if the technique had expression cassettes and yeasts capable of regulating the redoz balance: an S. cerevisiae industrial strain, widely used in the 2G ethanol sector, containing the oxy-reductive pathway. xylose consumption.

BRIEF DESCRIPTION OF THE INVENTION

[33] The present invention relates to LVY127 genetically modified industrial yeast with the oxy-reductive xylose conversion pathway, gene expression cassettes, 2G ethanol production process and use of LVY127 yeast.

[34] Gene Expression Cassette 1 comprises:

- xylose reductase (XR) of S. stipitis under the action of the promoter and terminator of the gene encoding 3-phosphoglycerate kinase (PGK1) in S. cerevisiae;

the 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 (XDH) gene under the action of the promoter and terminator of the gene encoding Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDHD in S. cerevisiae, cloned into pRS304 vector;

nucleotide sequence is represented by SKQ ID NO: 1.

[35] Gene Expression Cassette 2 comprises:

promoter (ADH1) and terminator (ADHD of the gene encoding xylulokinase enzyme 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; and nucleotide sequence represented by SEQ ID NO: 2.

[36] Gene Expression Cassette 3 comprises:

- S. stlpitis xylitol dehydrogenase (XDH) gene under the action of the promoter and terminator of the gene encoding Glyceraldehyde 3-Phosphate Dehydrogenase / isoenzyme 1 (TDH1) in S. cerevisiae;

S. cerevlsiae URA3 gene, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation; and

- nucleotide sequence is represented by SEQ AD NO: 3.

[37] The genetically modified yeast is Saccharomyces cerevislae DSM32120.

[38] The process for obtaining 2G ethanol is carried out with the genetically modified yeast is Sacchromyces cerevísiae DSM32120.

[39] Yeast LVY127 (D8K32120) is applicable to any process involving xylose consumption.

BRIEF DESCRIPTION OF DRAWINGS

[40] Figure 1 represents the flowchart of the development of the LVY127 strain (D8M32120).

[41] Figure 2 is a graph showing xylose consumption by LVY127 strain (DSH32120) compared to wild-type PE-2 derived strain.

[42] Figure 3 represents the overall fermentative performance graph of the LVY127 strain (DSM32120) in medium containing a mixture of glucose and xylose as carbon sources. The sugars and main products of fermentation are detailed in the caption.

[43] Figure 4 represents the graph showing the metabolic pathway representing sugar consumption by the LVY127 strain (DSM32120), containing the S. stipitis XP and XDH.

[44] Figure 5 is the representative schematic of plasmid pSsXRXDH, containing the 5. stipitis XR and XDH. The scheme was built using SnapGene software. Viewer the fragment containing the 5. stipitis XR and XDH genes was amplified from the pSsXRXDH vector and used for the 5. cerevisiae transformation.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to genetically modified industrial yeast LVY127 with the oxy-reductive zylose conversion pathway, gene expression cassettes, 2G ethanol production process and use of LVT127 yeast.

[46] Gene Expression Cassette 1 comprises:

- xylose reductase (XR) of $. stipitis under the action of the promoter and terrainator of the gene encoding 3-phosphoglycerate kinase iPGK1) in S. cerevisiae;

- S. stipitis xylitol dehydrogenase (XDH) gene under the action of the promoter and terminator of the gene encoding Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDH1) in 5. cerevisiae, cloned into pRS304 vector; and

- nucleotide sequence is represented by SEQ ID NO: 1.

[47] Cassette 1 is flanked by regions showing 516 bp homology to S. cerevisiae centromere five.

148] Gene Expression Cassette 2 comprises:

[ADHD promoter and terminator (ADH1) of the gene encoding the S. cerevisiae xylokinase enzyme;

S. cerevisiae URA3 gene, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation; and

nucleotide sequence is represented by SEQ AD NO: 2.

[491 For the construction of the DSM 32120 microorganism the first insertion of cassette 2 occurred at 288 bp from centromere two and the second copy is 228 bp from centromere eight. 150] Gene Expression Cassette 3 comprises: S. stipltls xylitol dehydrogenase (XDH) gene under the action of the promoter and terminator of the gene encoding Giiceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDH1) in s. cerevisi Mom;

S. cerevisiãe URA3 gene, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation; and

- nucleotide sequence is represented by SEQ AD NO: 3.

[51] Cassette 3 refers to the second copy of the gene encoding xylitol dehydrogenase inserted at 454 bp from centromere three.

[52] The genetically modified yeast is Saccharomyces cerevisiãe DSM32120.

[53] The construction steps of LVY127 (DSM32120) involved the insertion of 1 copy of the S. stipitis xylose reductase (XP.) Gene, 2 copies of the S. stipitis xylitol dehydrogenase (XOH) gene. , GRE3 aldose reductase deletion and insertion of 2 copies of XKS1 (Figure 1).

[54] For the construction of the xylose fermenter LVY127 (DSM32120) strain, a spore of the industrial strain PE-2, widely used in the first generation ethanol industry, was used. The PE-2 strain presents high fermentative performance and is highly tolerant to various industrial process stresses, becoming a robust platform for the introduction of the xylose conversion pathway.

[55] The genes expressing the 5. stipitis XR and XDH enzymes were cleaved, respectively, under the action of the PGK1 and TDH1 promoters, and inserted at 516 bp from centromere five, resulting in the LVY103 strain. The strain was able to consume all xylose present in culture medium, producing ethanol. Using only these modifications, however, most of the xylose was converted to xylitol. Overexpression of the XKS1 gene under the action of the ADR1 promoter gave rise to the LVY123 strain and allowed for an improvement. performance, reaching 34% ethanol yield from total sugars (51 * being theoretical yield), and 19% xylitol and 5.5H glycerol yield. The cassette containing the XKS1 gene was inserted near centromere two.

[56] Since the yield is still far from theoretic, the next step was to increase the xylose flux to xylulose-5-Ρ by doubling the integrated copy number of the XDH and XKS1 genes (inserted near centromeres three and eight). , respectively), in addition to deletion of the gene encoding an aldose reductase, GRE3. This gene has the same function as xylose reductase (XR), reducing xylose to xylitol, using exclusively NADPH as cofactor. The XR gene from $. Stipltis used in transformation uses both NADPH and NADH, although it has a higher affinity for the former. GRE3 deletion is a described strategy aimed at decreasing xylitol, since its production would be exclusively via s XR. stipltis, which performs the oxidation of NADH to NAD * allowing increased flow by the dependent NAD * XDH, near the pathway gene. Deletion of the GRE3 gene (LVY124) and increased copy number of XDH (LVY125) and XKS1 resulted in the LVY127 strain, object of the present invention, which showed 36% ethanol yield and decreased yields of xylitol t glycerol byproducts. with 8% and 8% respectively.

Example of embodiment

Used strains

[57] For plasmid cloning and multiplication experiments the Escherichia coli DH5μ bacterial strain (Stratagene®) was used. When necessary, the strain was activated at 37 ° C by depletion in LB (Luria-Bertaini) culture medium. For the construction of the strains was used the industrial strain of S. cerevisiae PE-2, maintained in YEPD medium. Expression cassette construction

[58] For the construction of cassettes, the technique described by Gibson et al. (2009), which consists of fusing DNA fragments with a mixture of T5 exonuclease, Taq ligase and DNA polymerase enzymes in a single reaction. For the selection process, the Cre / loxP system was used (Gueldener et al., 2002). The URA3 gene was PCR amplified, along with its promoter and terminator, and loxP sequences were added in the same orientation / at both ends, allowing this marker to be used in the transformation cassettes. Homology sequences at the insertion site were added at the ends of the cassettes.

Txmnmtormmm dm lmvmdaxm m pxoemmmo dm selection

(59} The transformation of yeast is done using the lithium acetate method described by Gietz and Schiestl (2007) .The strain used for the transformation is a haploid spore mat α derived from the diploid S. cerevísiae PE-2. cassette introductions, the strain had the URA3 gene removed to be used as an auxotrophic tag.The transformants were selected on YNB medium (without uracil) .The removal of the marker can then be performed with plasmid pSH65 (Gueldener et al., 2002). , which contains the gene encoding Cre recombinase under the action of the galactose-inducible promoter.

Fermentation test

Erlenmeyer cultivation

160] The fermentative assay was performed in YPDX medium ⁽ 20 g / l glucose and 50 g / l xylose) in 125 ml conical flasks and 80 ml working volume, starting the culture with approximately 1.0 GD. The temperature and stirring rate were kept constant at 30 ° C and 80 rpm respectively. Samples were taken to measure OD and for further analysis by high performance liquid chromatography (HPLC). Bioreactor cultivation [61] The fermentative assay was performed in YPDX medium (20 g / L glucose and 50 g / L xylose) in 2.5 L bioreactors, Labfors (Infors HT). The working volume used was 1 L, with pH kept constant during cell growth at 5.5 by the addition of 6 mol / L aqueous NaOH solution, starting the culture with approximately 1/0 OD. The temperature and stirring speed were kept constant at 30 ^and C and 150 rpm respectively. To ensure anaerobic condition, prior to inoculation, the bioreactor culture medium and atmosphere were saturated with a nitrogen gas flow of 3 LN / min (normal liters per minute) for 10 minutes. Samples were taken to measure OD and for further analysis by high performance liquid chromatography (HPLC).

Quantification of fermentation products

[62] Quantification of glucose, xylose, zylitol, glycerol, acetic acid and ethanol was performed by High Performance Liquid Chromatography (HPLC) using the Alliance HT (Waters) refractive index detector chromatograph (Waters 2414). Samples were analyzed by HPLC-RI using ion exclusion HPX-87H column (300mm κ 7,8mm, BioRadS '), heated in furnace 35 ^and C ₂ H ₂ SO mM (pH 2.3) as mobile phase at a flow rate of 0.6 raL / min. A standard curve with known concentrations of compounds of interest was also analyzed by the same procedure. The concentrations of the compounds in the samples were determined by comparing the chromatographic peak areas obtained with the calibration curves. The growth of the LVY127 strain, modified with the xylose conversion pathways object of the present invention, was compared to the wild type PE-2 strain, identified in figure 2 as WT ("wild-type"). The fermentative assay was conducted in medium containing a mixture of glucose and xylose at concentrations of 10 and 30 g / l, respectively. The figure 2 demonstrates that the LVY127 strain is now able to consume all the xylose present in the medium, whereas the wild strain was unable to consume this sugar. The experiment was conducted in triplicate. LVY127 (D8M32120) yeast has application in any process involving the consumption of xylose.

[63] The fermentative performance of the LVY127 strain (DSM32120) was evaluated under semi-anaerobic conditions in medium containing xylose and glucose as carbon sources, starting with low cell volume with

approximately 1.0 (0.25 g DW / L). The lineage showed an ethanol yield of 36%, besides a decrease in the yields of the xylitol and glycerol byproducts in relation to the predecessor lines, with 6% and 8%, respectively.

Claims

1. Gene expression cassette 1 comprising:

- xylose reductase (XR) of S. stipitís under the action of the promoter and terminator of the gene encoding 3-phosphoglycerate kinase (PGK1) in S. cerevislae;

- S. cerevísiae URA3 gene, together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation;

- S. stipitís xylitol dehydrogenase gene under the action of the promoter and terminator of the gene encoding Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 ITDH1) in S. cerevisiae, cloned into vector pRS304; and

- nucleotide sequence represented by SEQ AD NO: 1.

2. Gene expression cassette 2 comprising:

promoter (ADH1) and terminator (ADK1) of the gene encoding the enzyme xylulokinase in S. cerevislae;

- S. cerevísiae URA3 gene, together with its promoter and terminator, flanked by two lo / P sites at each end and in the same orientation; and

- nucleotide sequence represented by SEQ AD NO: 2.

3. Gene expression cassette 3 comprising:

S. stipitis xylitol dehydrogenase (XDH) gene under the action of the promoter and terminator of the gene encoding Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDH1) in S. cerevísiae;

- S. cerevísiae URA3 gene, 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 AD NO: 3.

4. Genetically Modified Yeast

Sacchaxomyces cerevisiae DSM32120.

Yeast according to claim A, characterized in that it is constructed with the insertion of 1 copy of the gene encoding S. xplose reductase (XR), 2 copies of the gene encoding xylitol dehydrogenase (XOH). stipitls, GRE3 aldose reductase deletion and insertion of 2 copies of XKS1.

6. Process for obtaining 2G ethanol characterized by being carried out with the yeast Saccharomyces cerevisiae DSM32120.

Use of the yeast as described in claims 4 and 5 for use in any process involving the consumption of xylose.