WO2018018111A1 - Levure industrielle génétiquement modifiée lvy127 avec la voie oxydoréductive de conversion de xylose, cassettes d'expression génétique, procédé d'obtention d'éthanol 2g et utilisation de la levure lvy127 - Google Patents

Levure industrielle génétiquement modifiée lvy127 avec la voie oxydoréductive de conversion de xylose, cassettes d'expression génétique, procédé d'obtention d'éthanol 2g et utilisation de la levure lvy127 Download PDF

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WO2018018111A1
WO2018018111A1 PCT/BR2017/000064 BR2017000064W WO2018018111A1 WO 2018018111 A1 WO2018018111 A1 WO 2018018111A1 BR 2017000064 W BR2017000064 W BR 2017000064W WO 2018018111 A1 WO2018018111 A1 WO 2018018111A1
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gene
yeast
promoter
terminator
lvy127
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Leandro Vieira DOS SANTOS
Gonçalo Amarante Guimarães PEREIRA
Renan Augusto Siqueira PIROLLA
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Universidade Estadual de Campinas UNICAMP
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/22—Processes using, or culture media containing, cellulose or hydrolysates thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • 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.
  • the invention has application in the ethanol production sector.
  • 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.
  • 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). .
  • PE-2 and CAT-1 were used in approximately 150 distilleries, representing 60% of ethanol produced in Brazil (Basso et al., 2008).
  • 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.
  • PE-2 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).
  • 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.
  • 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.
  • NAD + or NADP +
  • NADPH or NADH
  • oxyreductases also called dehydrogenases
  • 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.
  • the second proton removed from the substrate is released into the aqueous solvent (Nelson & Cox, 2011).
  • NADH is generated primarily in cytosol by glycolysis and mitochondria by the cyclic acid cycle (TCA) (Vemuri et al., 2006).
  • TCA cyclic acid cycle
  • glucose is oxidized using NAD + as cofactor, which is simultaneously converted into its reduced form, NADH, in an equivalent amount.
  • 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.
  • 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).
  • NADH reoxidation in S. cerevisiae 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).
  • 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
  • NADPH may be generated mainly in the reactions catalyzed by two dehydrogenases in the oxidative phase of pentose phosphate (ZWF1, glucose-6- ⁇ dehydrogenase and GND1, 6-phosphogluconate dehydrogenase) in the isocitrate-catalyzed reaction.
  • ZWF1 pentose phosphate
  • GND1 6-phosphogluconate dehydrogenase
  • NADP + dependent dehydrogenase ⁇ 2
  • NADP * dependent acetaldehyde catalyzed reaction ALD6
  • MAE1 malic enzyme catalyzed reaction
  • the pentose phosphate pathway represents the major NADPH production pathway in yeast (Santos et al., 2004; Wang et al., 2013).
  • the pentose phosphate pathway is mainly enzyme-controlled, with NADPH and ATP competitively inhibiting glucose-6-dehydrogenase (ZNFD and 6-phosphogluconate dehydrogenase (GND1).
  • ZNFD and 6-phosphogluconate dehydrogenase GNF1
  • GNFD and 6-phosphogluconate dehydrogenase GNF1
  • 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).
  • 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.
  • TAL1 and TKL1 are also up-regulated (Celton et al., 2012a).
  • 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).
  • 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.
  • 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.
  • XR-XDH Xylcse Reductase - Xylitol Dehydrogenase
  • XI Xylose Isomerase
  • 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 *.
  • XR xylose reductase
  • XDH Xylitol dehydrogenase
  • NAD * is regenerated in the respiratory chain, with oxygen as the final electron acceptor.
  • 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).
  • 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).
  • 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.
  • 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- ⁇ 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.
  • US20130040353 relates to ethanol production in the Saccharomyces cerevisiae medium and alteration of XR to have greater affinity for NADH rather than NADPH.
  • 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.
  • 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.
  • it was used in an industrial strain, more tolerant to inhibitors present in the 2G process and with characteristics of industrial interest.
  • 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.
  • 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.
  • Gene Expression Cassette 1 comprises:
  • XR - xylose reductase
  • PGK1 3-phosphoglycerate kinase
  • XDH S. stipitis xylitol dehydrogenase
  • nucleotide sequence is represented by SKQ ID NO: 1.
  • Gene Expression Cassette 2 comprises:
  • ADH1 ADH1
  • ADHD 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.
  • Gene Expression Cassette 3 comprises:
  • S. cerevlsiae URA3 gene together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation;
  • the genetically modified yeast is Saccharomyces cerevislae DSM32120.
  • Yeast LVY127 (D8K32120) is applicable to any process involving xylose consumption.
  • Figure 1 represents the flowchart of the development of the LVY127 strain (D8M32120).
  • Figure 2 is a graph showing xylose consumption by LVY127 strain (DSH32120) compared to wild-type PE-2 derived strain.
  • 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.
  • Figure 4 represents the graph showing the metabolic pathway representing sugar consumption by the LVY127 strain (DSM32120), containing the S. stipitis XP and XDH.
  • 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.
  • 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.
  • Gene Expression Cassette 1 comprises:
  • SEQ ID NO: 1 SEQ ID NO: 1.
  • Cassette 1 is flanked by regions showing 516 bp homology to S. cerevisiae centromere five.
  • Gene Expression Cassette 2 comprises:
  • ADHD promoter and terminator (ADH1) of the gene encoding the S. cerevisiae xylokinase enzyme [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;
  • nucleotide sequence is represented by SEQ AD NO: 2.
  • 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. cerevisi ⁇ e URA3 gene together with its promoter and terminator, flanked by two loxP sites at each end and in the same orientation;
  • Cassette 3 refers to the second copy of the gene encoding xylitol dehydrogenase inserted at 454 bp from centromere three.
  • the genetically modified yeast is Saccharomyces cerevisi ⁇ e DSM32120.
  • xylose fermenter LVY127 (DSM32120) strain 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.
  • the next step was to increase the xylose flux to xylulose-5- ⁇ 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.
  • XR xylose reductase
  • 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.
  • 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 ⁇ derived from the diploid S. cerev ⁇ siae 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.
  • 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.
  • 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).
  • the concentrations of the compounds in the samples were determined by comparing the chromatographic peak areas obtained with the calibration curves.
  • 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.

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Abstract

La présente invention concerne une levure industrielle génétiquement modifiée LVY127 avec la voie oxydoréductive de conversion de xylose, des cassettes d'expression génétique, un procédé d'obtention d'éthanol 2G et l'utilisation de la levure LVY127.
PCT/BR2017/000064 2016-07-28 2017-06-26 Levure industrielle génétiquement modifiée lvy127 avec la voie oxydoréductive de conversion de xylose, cassettes d'expression génétique, procédé d'obtention d'éthanol 2g et utilisation de la levure lvy127 Ceased WO2018018111A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5866382A (en) * 1990-04-06 1999-02-02 Xyrofin Oy Xylose utilization by recombinant yeasts
US20090246857A1 (en) * 1996-05-06 2009-10-01 Purdue Research Foundation Stable recombinant yeasts for fermenting xylose to ethanol
US20120329104A1 (en) * 2011-06-27 2012-12-27 Seoul University Research and Business Foundation Modified microorganism having enhanced xylose utilization
BR102014014407A2 (pt) * 2014-06-12 2016-04-19 Biocelere Agroindustrial Ltda cassete de expressão para transformar célula eucariótica, micro-organismo geneticamente modificado com eficiente consumo de xilose, processo para produção de biocombustíveis e bioquímicos e biocombustível e/ou bioquímico assim produzido
BR102014027984A2 (pt) * 2014-11-07 2016-06-07 Biocelere Agroindustrial Ltda cassete de expressão, processo, micro-organismo, processo de produção de biocombustíveis e/ou bioquímicos e biocombustível e/ou bioquímico

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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