WO2016065453A1 - Expression cassette for eukaryotic cell transformation, method for transforming eukaryotic cells, genetically modified microorganism, method for producing biofuels and/or biochemicals, and biofuel and/or biochemical thereby produced - Google Patents

Expression cassette for eukaryotic cell transformation, method for transforming eukaryotic cells, genetically modified microorganism, method for producing biofuels and/or biochemicals, and biofuel and/or biochemical thereby produced Download PDF

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WO2016065453A1
WO2016065453A1 PCT/BR2015/050198 BR2015050198W WO2016065453A1 WO 2016065453 A1 WO2016065453 A1 WO 2016065453A1 BR 2015050198 W BR2015050198 W BR 2015050198W WO 2016065453 A1 WO2016065453 A1 WO 2016065453A1
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seq
sequence
gene
xylose
promoter
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PCT/BR2015/050198
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French (fr)
Portuguese (pt)
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WO2016065453A9 (en
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Angela Luzia DREZZA
Gonçalo Amarante Guimarães PEREIRA
Leandro Vieira DOS SANTOS
Maria Carolina De Barros Grassi
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Biocelere Agroindustrial Ltda.
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Priority to BR112017008873A priority Critical patent/BR112017008873A2/en
Publication of WO2016065453A1 publication Critical patent/WO2016065453A1/en
Publication of WO2016065453A9 publication Critical patent/WO2016065453A9/en

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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, e.g. protozoa; 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
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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
    • 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 biofuels, biochemicals and processes for obtaining them. More specifically, the present invention provides technical solutions for the production of second generation fuels based on the conversion of sugars present in plant biomass, preferably from plant cell wall polymers.
  • the present technology describes an expression cassette for eukaryotic cell transformation and a genetically modified microorganism, with efficient fermentative performance in the conversion of sugars present in plant biomass, mainly pentoses, and among pentoses, mainly xylose, in biochemical and / or biofuels.
  • the genetically modified microorganism by cassette insertion is able to consume xylose when compared to unmodified microorganisms, thus favoring its performance on an industrial scale.
  • a process for obtaining biofuels and / or biochemicals and the products thus obtained are also described.
  • Second-generation fuels for example ethanol
  • This process consists of converting polymers that form plant biomass, especially those present in the cell wall such as cellulose, hemicellulose and lignin, into biofuels and / or biochemicals.
  • Plant biomass is a complex mixture of chemically distinct compounds that can be fractionated into components with specific applications.
  • microorganisms naturally capable of consuming sugars present in the cellulose and hemicellulose chains are generally not capable of industrial use efficiently.
  • Wild S. cerevisae strains are not naturally capable of fermenting pentoses, such as xylose, present in biomass.
  • pentoses such as xylose
  • several studies have already performed metabolic engineering procedures in S. cerevisiae introducing in these organisms the metabolic pathways for xylose consumption, focusing on two main pathways: Xylose Reductase - Xylitol Dehydrogenase (XR-XDH) and Xylose Isomerase ( XI).
  • xylose isomerase xylose isomerase
  • the XR-XDH pathway common in eukaryotic microorganisms, has higher initial productivity by allowing ethanol to be produced more rapidly, only by inserting the genes responsible for xylose conversion.
  • This pathway consists of two oxy-reduction reactions. In the first, xylose is reduced to xylitol by the action of the enzyme xylose reductase (XR), in 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
  • the NADPH cofactor is mainly regenerated in the oxidative phase of the pentose phosphate pathway with CO 2 production.
  • NAD + is regenerated mainly in the respiratory chain, with O 2 as the final electron acceptor.
  • O 2 the final electron acceptor.
  • complete NAD + reoxidation does not occur, resulting in redox imbalance and xylitol accumulation, which directly impacts the final ethanol yield [Biochemical Engineering Journal, Amsterdam, v.12, n.1, pp. 49-59, 2002].
  • another formed byproduct is glycerol [FEMS Yeast Research, Delft, v.4, n.6, p.655-664, 2004].
  • xylose metabolism carried out via the xylose isomerase (XI) pathway is more common in prokaryotes and occurs in a single step, thus avoiding redox imbalance and allowing less formation of byproducts that decrease ethanol yield. Additionally, the XI pathway may result in higher ethanol yield by accumulating fewer fermentation by-products [Microbial Celi Factories, London, v.6, n.5, p.1-10, 2007].
  • WO2010074577 describes the existence of some domains that would be conserved in proteins with xylose isomerase function, indicating that the presence of these domains in the peptide, using alignment with The Piromyces sp E2 XI sequence would ensure that the protein would be functionally expressed in Saccharomyces.
  • xylose isomerase genes from various other organisms that have the domains indicated in WO2010074577 have also been inserted into yeast but have no functional activity [Appl Eviron Microbiol. 2009; 75 (8): 2304-11; Enzyme and Microbial Technology, Vol. 32, pp. 252-259, 2003; Applied and Environmental Microbiology, Vol. 62, No. 12, pp. 4,648-4,651, Dec. 1996; Biotechnology and Bioengineering Symposium, No. 13, pp. 245-250, 1983].
  • thermophilus does not have the domains advocated by WO2010074577 as being necessary for the protein xylose isomerase to be functional in Saccharomyces, also observed in Figure 1.
  • WO2011078262 also describes 7 functional xylose isomerases, wherein: 4 are from Reticulitermes speratus (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7) and 3 xylose isomerases from Mastotermes darwiniensis (SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13). Comparison of these 7 sequences with the Piromyces sp E2 sequence reveals 2 groups with very distinct identity values, ranging from 50% (Reticulitermes speratus) to 73% (Mastotermes darwiniensis), as can be seen in Figure 2, obtained from Figure 2. from the BlastX / NCBI tool (http://biast.ncbj.nim.nih.gov/) ["Basic local alignment search tool.” J. Mol. Biol. 215: 403-410, 1990].
  • the microorganism in order to be able to consume xylose, the microorganism must be at least genetically modified by the addition of the gene encoding xylose isomerase.
  • pentose phosphate pathway genes such as transaldolase, transcetolase, xylulokinase, ribose 5-phosphate isomerase and ribose 5-phosphate epimerase, all contribute to the conversion of xylose to ethanol and are therefore necessary for the consumption of this sugar to be efficient.
  • Saccharomyces cerevisiae yeasts described in the state of the art which have been genetically modified for xylose consumption invariably have the genetic modifications described above.
  • the differential of each of these microorganisms is the combination of these genes and the promoters by which they are regulated, as well as the gene that encodes the protein with xylose isomerase function, since this is the main gene that enables the consumption of xylose by each microorganism. -modified organism.
  • microorganisms that produce proteins with xylose isomerase activity from the insertion in their genome of another microorganism gene that are capable of consuming xylose as the sole carbon source and, mainly, that Being able to produce biochemicals and / or biofuels is not a trivial process.
  • the microorganism described in the present invention proves to be efficient in converting sugars present in lignocellulosic plant biomass, mainly pentoses and, among pentoses, mainly xylose, biofuels and / or biochemicals, mainly ethanol. .
  • the present invention describes, among other objects, a genetically modified microorganism with efficient fermentative performance in the conversion of sugars such as those contained in lignocellulosic plant biomass, e.g. the genetic modifications described herein.
  • the genetically modified microorganism described in the present invention refers to a eukaryotic cell. genetically transformed, preferably a yeast or filamentous fungus, preferably a yeast of the genus Saccharomyces.
  • a microorganism of the species Saccharomyces cerevisiae more efficient in converting pentoses present in the lignocellulosic material into alcohols and / or biochemicals such as, for example, ethanol, succinic acid, acid is described herein.
  • the pentose preferably used by the microorganism for conversion to alcohols and / or biochemicals above is xylose, but is not restricted to it.
  • SEQ ID NO: 1 represents a peptide with xylose isomerase characteristic which, when expressed in eukaryotic cell, favors the xylose isomerization into xylulose.
  • SEQ ID NO: 2 comprises nucleotide sequence capable of encoding protein with xylose isomerase function.
  • the microorganism described in the present invention is genetically modified by introducing an expression cassette comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1, which peptide may optionally be expressed in a eukaryotic cell in its active and functional form.
  • xylose isomerase capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1, which peptide may optionally be expressed in a eukaryotic cell in its active and functional form.
  • the expression cassette of the invention is characterized in that it comprises: - nucleotide sequence SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding a xylose function peptide isomerase comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1; at least one promoter for said coding nucleotide sequence; and - a nucleotide sequence selected from: a transcription terminator nucleotide sequence; a selection marker; one or more nucleotide sequence (s) coding for another enzyme; combinations thereof or a plasmid comprising such sequences, with at least one of the nucleotide sequences defined above being heterologous.
  • One or more expression cassettes may be used in eukaryotic cell transformation according to the invention.
  • the cassette comprising SEQ ID NO: 2, capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1, is inserted into the host cell flanked preferably by the promoter and terminator region of the Glyceraldehyde 3 gene.
  • -Phosphate dehydrogenase, isoenzyme 1 (TDH1) described in SEQ ID NO: 18 and SEQ ID NO: 3, respectively.
  • the expression cassette of the invention also comprises sequences selected from the group comprising the coding sequences of the enzymes Xylulokinase (SEQ ID NO: 9), Transaldolase (SEQ NO ID: 5), Transcetolase (SEQ ID NO: 11) , Ribose 5-Phosphate Isomerase (SEQ ID NO: 7) and / or Ribose 5-Phosphate Epimerase (SEQ ID NO: 12), operably linked to promoters and terminators, which promoters are preferably constitutive of the cell into which the cassette will be inserted. or naturally inducible.
  • Such sequences may be comprised of the same or different expression cassettes.
  • the present invention describes eukaryotic host cell / microorganism being yeast of the Saccharomyces cerevisiae species, but it is emphasized that any eukaryotic cell can be transformed with one or more expression cassettes of the invention comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding peptide with function xylose isomerase comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1.
  • genes encoding the xylulokinase enzymes (XKS1, EC 2.7.1 .17), whose nucleotide sequence is represented herein by SEQ ID NO: 9, are inserted.
  • Transaldolase (TAL1, EC 2.2.1 .2), represented by the sequence SEQ NO ID: 5, Transcetolase (TKL1, EC 2.2.1 .1), whose nucleotide sequence is represented by SEQ ID NO: 11, Ribose 5- Phosphate Isomerase (RKI1, EC 5.3.1 .6), the nucleotide sequence of which is represented by SEQ ID NO: 7; and Ribose 5-Phosphate Epimerase (RPE1, EC 5.1 .3.1), whose nucleotide sequence is represented by SEQ ID NO: 12.
  • the present invention describes host cells comprising one or more expression cassettes containing endogenous enzyme genes from the non-oxidative phase of the pentose phosphate pathway, which are preferably constructed using strong and constitutive promoters of the cell into which they will be inserted.
  • All expression cassettes with the pentose phosphate metabolic pathway genes that favor xylose consumption are inserted into the target chromosome region located between the centromere and the first gene adjacent to it, preferably in the region of the first 5,000 base pairs counted. from the centromere in either upstream or downstream direction, and may even be upstream only, downstream only, or both simultaneously.
  • the present invention also describes the deletion or inactivation of the GRE3 gene (represented in SEQ ID NO: 14), which encodes an aldose reductase that favors xylitol production from xylose. Xylitol production decreases the total yield of ethanol that can be obtained, as well as being an inhibitor of the action of the enzyme xylose isomerase.
  • the microorganism described in the present invention is preferably of industrial lineage and differentially exhibits the characteristics of being non-flocculant, permitting low glycerol and xylitol formation, having high viability, high growth rate, not producing foam, among others.
  • the present invention also discloses and comprises a process for producing biofuels and / or biochemicals from plant biomass, preferably the lignocellulosic portion of plant biomass.
  • the biofuel and / or biochemical production process described in the present invention utilizes the microorganism of the invention for biofuel and / or biochemical production.
  • the said process comprises the following steps:
  • the lignocellulosic material may, optionally be subjected to pretreatment and hydrolysis. This stage may occur, preferably under anaerobic conditions, but this is not a restrictive condition for the process;
  • the process of the invention provides for the production of biofuels comprising predominantly alcohols, especially ethanol.
  • the process of the invention provides for the production of biochemicals. selected from the group including, but not limited to: succinic acid, malic acid, 1,3-propanediol, 1,2-propanediol, butanol, isobutanol, biodiesel, 1,4-butanediol, 2,3-butanediol and / or PHB - poly (butyrate hydroxide).
  • the present invention describes biofuels, preferably ethanol, and biochemicals produced by the process utilizing the microorganism comprising the genetic modifications described in this invention.
  • inventive concept claimed herein is not limited to the embodiments exemplified above, which are to be construed as: evidence of the material existence of the invention; and as an informational medium that readily enables a person skilled in the art to reproduce it - both in the specific forms here exemplified and in others legally within the full scope of the disclosed inventive concept and claimed objects.
  • the microorganism comprising the genetic modifications described in the present invention is, by itself, one of the objects of the invention, being particularly efficient in the conversion into alcohols and / or sugar acids of constituent material.
  • lignocellulosic mainly pentoses, among them stands out the xylose.
  • Said microorganism is efficient in converting sugars, including pentoses, including xylose, present in lignocellulosic material, such as that previously subjected to hydrolysis process.
  • Figure 1 shows the alignment between the Thermus thermophilus, and Piromyces sp E2 sequences, both functional in Saccharomyces, which is only 28.1% and was obtained from the Clustal Omega alignment tool (http: // www. ebi.acukTools / msa. / clustaJo /).
  • the Clustal Omega alignment tool http: // www. ebi.acukTools / msa. / clustaJo /.
  • Figure 2 shows comparisons between the sequences of a) Mastotermes darwiniensis and b) Reticulitermes speratus against Piromyces sp E2 xylose isomerase showing the percentages of identity of 73% and 50% for a) and b), respectively.
  • M represents the marker 1 kb ladder
  • 1a the XKS1 gene cassette inserted near centromere 2
  • 1 b the blank of reaction 1
  • 2a is the XKS1 gene cassette inserted near centromere 8
  • 2b is the blank of reaction 2
  • 3a is the cassette of the TAL1 and RKI1 genes inserted near centromere 12
  • 3b is the blank of reaction 3
  • 4a is the cassette of the TKL1 and RKI1 genes inserted near centromere 13
  • 4b is the blank of reaction 4
  • 5b is the blank of the reaction.
  • Figure 4 shows the PCR reaction result that was made with S. cerevisiae strain DNA transformed with the xylose isomerase plasmid SEQ ID NO: 2. Amplifications confirm that the plasmid was inserted into the yeast.
  • A the agarose gel with TDH1 promoter region amplification and xylose isomerase SEQ ID NO: 2 is shown which has been cloned into a plasmid.
  • B we observed xylose isomerase amplification SEQ ID NO: 2 along with the TDH1 terminator region that was cloned into plasmid.
  • the present invention describes, among other objects, a genetically modified microorganism with efficient fermentative performance in the conversion of sugars such as those contained in lignocellulosic plant biomass, for example pentoses, into biofuels and / or biochemicals when compared to their untreated version. the genetic modifications described herein.
  • the genetically modified microorganism described in the present invention refers to a genetically transformed eukaryotic cell, preferably a yeast or filamentous fungus.
  • yeast are considered to be any individual of the Eumycotina group, i.e. unicellularly growing true fungi which preferentially perform anaerobic fermentation such as Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Candida, Kluyveromyces. , Schizosaccharomyces, Brettanomyces, Hansenula and Yarrowia.
  • Filamentous fungi are those characterized by presenting vegetative mycelium and growing from the elongation of the hyphae, besides performing aerobic respiration, such as Aspergillus, Penicillium, Fusarium, Trichoderma, Moniliophthora and Acremonium.
  • the microorganism is a yeast of the genus selected from the group consisting of: Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Candida, Kluyveromyces, Schizosaccharomyces, Brettanomyces, Hansenula and Yarrowia.
  • the present invention describes a genetically modified microorganism, preferably a yeast of the genus Saccharomyces.
  • a microorganism of the species Saccharomyces cerevisiae more efficient in converting pentoses, such as xylose, present in lignocellulosic material into alcohols and / or biochemicals such as, for example, is described in the present invention.
  • the pentose preferably used by the microorganism for conversion to alcohols and / or biochemicals indicated above is xylose, but is not restricted to it.
  • Ribose 5-Phosphate Isomerase SEQ I D NO: 7
  • ADH1 Enzyme Promoter Alcohol Dehydrogenase 1 SEQ I D NO: 8
  • XKS1 SEQ I D NO: 9
  • Alcohol Dehydrogenase Terminator SEQ I D NO: 10
  • RPE1 Ribose 5-Phosphate Epimerase SEQ I D NO: 12
  • TSH1 Glyceraldehyde 3-Phosphate Dehydrogensase Promoter, isoenzyme 1 (TDH1) SEQ I D NO: 18
  • SEQ ID NO: 1 represents a peptide with xylose isomerase characteristic which, when expressed in a eukaryotic cell, favors the xylose isomerization in xylulose.
  • SEQ ID NO: 2 comprises nucleotide sequence capable of encoding protein with xylose isomerase function.
  • the microorganism described in the present invention is genetically modified by introducing an expression cassette comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1, which peptide may optionally be expressed in a eukaryotic cell in its active and functional form.
  • xylose isomerase capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1, which peptide may optionally be expressed in a eukaryotic cell in its active and functional form.
  • the expression cassette of the invention is characterized in that it comprises: - the nucleotide sequence SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and is capable of encoding a xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1; at least one promoter for said coding nucleotide sequence; and - a nucleotide sequence selected from: a transcription terminator nucleotide sequence; a selection marker; one or more nucleotide sequence (s) coding for another enzyme; combinations thereof or a plasmid comprising such sequences, with at least one of the nucleotide sequences defined above being heterologous.
  • One or more expression cassettes may be used in eukaryotic cell transformation according to the invention.
  • the cassette comprising SEQ ID NO: 2 capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 is inserted into the host cell flanked preferably by the promoter and terminator region of the Glyceraldehyde 3 gene.
  • -Phosphate dehydrogenase, isoenzyme 1 (TDH1) described in SEQ ID NO: 18 and SEQ ID NO: 3, respectively.
  • the expression cassette of the invention also comprises sequences selected from the group comprising the coding sequences for the enzymes Xylulokinase (SEQ ID NO: 9), Transaldolase (SEQ NO ID: 5), Transcetolase (SEQ ID NO: 11) , Ribose 5-Phosphate Isomerase (SEQ ID NO: 7) and / or Ribose 5-Phosphate Epimerase (SEQ ID NO: 12), operably linked to promoters and terminators, which promoters are preferably constitutive of the cell into which the cassette will be inserted. or naturally inducible.
  • Such sequences may be comprised of the same or different expression cassettes.
  • the present invention describes stable and high copy number integration of the cassette comprising SEQ ID NO: 2 or any sequence of at least 80% identity with SEQ ID NO: 2 and capable of encoding peptide.
  • xylose isomerase function comprising SEQ ID N: 1, or any sequence of at least 80% identity with SEQ ID NO: 1 in the host cell genome.
  • the insertion of at least 5 copies of the gene in question is considered high number, being preferred the insertion of at least 20 copies.
  • the present invention describes eukaryotic host cell / microorganism being yeast of the Saccharomyces cerevisiae species, but it is noted that any eukaryotic cell can be transformed with one or more expression cassettes of the invention comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1 .
  • the present invention describes eukaryotic host cell, yeast or filamentous fungi, preferably yeast of the species Saccharomyces cerevisiae, transformed with one or more expression cassettes of the invention, wherein at least one cassette comprises SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1.
  • nucleotide sequence comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any A sequence of at least 80% identity with SEQ ID NO: 1 may be in single copy or preferably multiple copies of such a genome-inserted nucleotide sequence.
  • the genetically modified host cell described in the present invention additionally comprises pentose phosphate pathway genes, so that the expression cassette insert comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO. : 2 and capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1, may favor xylose isomerization into xylulose.
  • the present invention describes genetic modifications in that same cell aimed at enhancing metabolic flow through the pentose phosphate pathway, but such modifications are not, however. restriction factor for transformation of the expression cassette host cell comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO : 1 or any sequence with at least 80% identity with SEQ ID NO: 1.
  • genes encoding the xylulokinase enzymes (XKS1, EC 2.7.1 .17), whose nucleotide sequence is represented herein by SEQ ID NO: 9, are inserted.
  • Transaldolase (TAL 1, EC 2.2.1 .2), represented by the sequence SEQ NO ID: 5, Transcetolase (TKL 1, EC 2.2.1 .1), whose nucleotide sequence is represented by SEQ ID NO: 11, Ribose 5-Phosphate Isomerase (RKI1, EC 5.3.1 .6), the nucleotide sequence of which is represented by SEQ ID NO: 7; and Ribose 5-Phosphate Epimerase (RPE1, EC 5.1 .3.1), whose nucleotide sequence is represented by SEQ ID NO: 12.
  • promoters may be constitutive or naturally inducible.
  • the present invention describes host cell comprising one or more expression cassettes containing endogenous enzyme genes of the non-oxidative phase of the pentose phosphate pathway, which are preferably constructed using strong and constitutive cell promoters in the cell. which will be inserted and stably integrated into the host cell genome. All expression cassettes with the pentose phosphate metabolic pathway genes that favor xylose consumption are inserted into the target chromosome region located between the centromere and the first gene adjacent to it, preferably in the region of the first 5,000 base pairs counted. from the centromere in either the upstream or downstream direction, and may even be upstream only, downstream only, or both simultaneously.
  • Upstream direction is considered to be that located prior to the starting point of the transcriptional unit of a DNA sequence, which starts at the promoter and ends at the terminator.
  • downstream is considered the region located after the start point of the transcription unit of a DNA sequence.
  • the present invention also describes deletion or inactivation of the GRE3 gene (depicted in SEQ ID NO: 14), which encodes an aldose reductase which favors xylitol production from xylose.
  • Xylitol production decreases the total yield of ethanol that can be obtained, as well as being an inhibitor of the action of the enzyme xylose isomerase.
  • the present invention describes stable and high copy-number integration of expression cassettes comprising SEQ ID NO: 2 or any sequence having at least 80% identity with SEQ ID NO: 2 and being capable of encoding xylose isomerase function peptide comprising SEQ ID N: 1, or any sequence of at least 80% identity with SEQ ID NO: 1) in the host cell genome.
  • this document describes a eukaryotic cell, preferably a microorganism of the species Saccharomyces cerevisiae, genetically modified containing in its genome at least one of the enzyme genes required to favor the non-oxidative part of the pentose phosphate pathway inserted preferably in high copy number and in the region between the centromere and its first adjacent gene, thus possessing all the metabolic pathway necessary for conversion into fuels and / or biochemicals, mainly ethanol, of sugars component of the lignocellulosic biomass, mainly pentoses, such as the xylose.
  • the microorganism described in the present invention is preferably of industrial lineage and differentially exhibits the characteristics of being non-flocculant, permitting low glycerol and xylitol formation, having high viability, high growth rate, not producing foam, among others.
  • the present invention also discloses and comprises a process for producing biofuels and / or biochemicals from the fermentation of material comprising sugars such as those present in biomass.
  • material comprising sugars such as those present in biomass.
  • lilgnocellulosic vegetable mainly pentoses and, among pentoses, mainly xylose.
  • the biofuel and / or biochemical production process described in the present invention utilizes the genetically modified microorganism described herein for biofuel and / or biochemical production.
  • Said process comprises the following steps:
  • the lignocellulosic material may, optionally be subjected to pretreatment and hydrolysis. This stage may occur, preferably under anaerobic conditions, but this is not a restrictive condition for the process;
  • the process of the invention provides for the production of biofuels comprising predominantly alcohols, especially ethanol.
  • the process of the invention provides for the production of biochemicals selected from the group comprising, but not limited to: succinic acid, malic acid, 1,3-propanediol, 1,2-propanediol, butanol, isobutanol, biodiesel, 1,4-butanediol 2,3-butanediol and / or PHB - poly (butyrate hydroxide).
  • the present invention describes biofuels, preferably ethanol, and biochemicals produced by the process using the microorganism comprising the genetic modifications described in this invention.
  • inventive concept claimed herein is not limited to the embodiments exemplified above, which are to be construed as: evidence of the material existence of the invention; and as an informational medium that readily enables a person skilled in the art to reproduce it - both in the specific forms here exemplified as in others legally within the full scope of the disclosed inventive concept and the claimed objects.
  • the microorganism comprising the genetic modifications described in the present invention is, by itself, one of the objects of the invention, being particularly efficient in the conversion to alcohols and / or sugar acids as those comprised.
  • lignocellulosic biomass mainly pentoses, and among pentoses, mainly xylose.
  • Said microorganism is efficient in the conversion of pentoses, including xylose, present in lignocellulosic material, such as that previously subjected to hydrolysis process.
  • xylose isomerases that could be functionally expressed in Saccharomyces
  • a public database available at NCBI, was searched for incomplete and fragmented genomes (draft genomes) that could contain genes coding for new genes. xylose isomerases and which, until then, would have unknown function.
  • the parameter used for the search was protein sequences of xylose isomerases that so far had been shown to be functional in Saccharomyces, such as Piromyces sp E2 and Orpinomyces.
  • sequence of xylose isomerase indicated in the databases as belonging to the fungus Melampsora pinitorqua was identified by comparing the protein sequence of xylose isomerase from Piromyces sp. E2 against NCBI's Whole-genome shotgun contigs (NCBI) database.
  • contig1668883_0 (GenBank ID: AUYS01012195.1) was returned because it contains a region similar to the Piromyces xylose isomerase protein, with only 62% identity, a fact that would not make its functional expression as xylose isomerase obvious, considering the precedents described in this document.
  • each gene was amplified by PCR of the S. cerevisiae genome and cloned into integrative expression cassettes. .
  • the URA3 gene flanked by two loxP regions in the same orientation was cloned, allowing that region to be removed by expression of Cre recombinase (SEQ ID NO: 15) and the URA3 auxotrophic marker could be deleted. used in all expression cassettes with the described genes.
  • the xylulokinase-encoding gene expression cassette for example, that gene was amplified by PCR of the S. cerevisiae genome and cloned adjacent to the promoter and terminator of the Alcohol dehydrogenase (ADH1) encoding gene. SED ID NO: 8 and SEQ ID NO: 10, respectively. After the terminator, the URA3 gene flanked by two loxP regions (SEQ ID NO: 4) was inserted in the same orientation. At the end of the cassette, homology regions were cloned near S. cerevisiae centromere two and eight.
  • ADH1 Alcohol dehydrogenase
  • Transaldolase TAL 1
  • Ribose 5-Phosphate Isomerase RKI1
  • genes were cloned under the action of the Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1, (TDH1) gene promoters and terminators represented by SEQ ID NO: 18 and 3-phosphoglycerate kinase, ⁇ PGK1) represented by SEQ ID NO: 3, respectively, separated by the URA3 marker flanked by the loxP sites, and properly inserted into the host cell chromosome.
  • TDH1 Glyceraldehyde 3-Phosphate Dehydrogenase
  • TDH1 isoenzyme 1 gene promoters and terminators represented by SEQ ID NO: 18 and 3-phosphoglycerate kinase
  • ⁇ PGK1 3-phosphoglycerate kinase
  • TTL1 Transcetolase
  • RPE1 Ribose 5-Phosphate Epimerase
  • SEQ ID NO: 12 Ribose 5-Phosphate Epimerase
  • these genes were cloned under the action of the Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDH1) and 3-phosphoglycerate kinase (PGK1) gene promoters and terminators, represented by SEQ ID NO: 13, respectively, separated by the URA3 marker flanked by the loxP sites, and properly inserted into the host cell chromosome.
  • 126 bp was cloned from each side with homology to a region close to the Saccharomyces cerevisiae chromosome five, allowing integration via homologous recombination in this region.
  • the cassette containing the SEQ ID NO: 2 gene capable of expressing the Xylose Isomerase peptide represented by SEQ ID NO: 1 has been modified with the inclusion, at the ends of the cassette, delta elements of the retrotransposon Ty1, (element present in high copy number in the S. cerevisiae genome and represented by SEQ ID NO: 16).
  • the URA3 marker flanked by the loxP regions is replaced in that plasmid by the marker LEU2 (SEQ ID NO: 17).
  • the LEU2 gene is deleted in a step of genetic manipulation. In this step, the URA3 gene is integrated, flanked by loxP regions adjacent to LEU2 promoter and terminator homology regions, resulting in deletion of this gene.
  • the XI cassette flanked by the Ty1 elements is inserted and using the auxotrophic marker LEU2 to select the transformants.
  • the deletion of the GRE3 gene which encodes an aldose reductase and is represented in SEQ ID NO: 14, was performed in two steps by genetic manipulation, aimed at decreasing xylitol production from xylose.
  • the URA3 gene was integrated, flanked by loxP regions adjacent to GRE3 gene promoter and terminator homology regions, resulting in deletion of this region.
  • the URA3 marker was removed by transient expression of Cre recombinase.
  • the genetically modified microorganism DNA was used as a template for the polymerase chain reaction using oligonucleotides that ring in a region outside the insertion site of gene expression cassettes. For each reaction, a pair of oligos specific for the external region of each inserted cassette was used.
  • FIG. 3 shows the electrophoresis gel obtained from the amplification of the external regions to the inserted cassettes, proving the integration with the yeasts.
  • M represents the marker 1 kb ladder
  • 1a the XKS1 gene cassette inserted near centromere 2
  • 1 b the blank of reaction 1
  • 2a is the XKS1 gene cassette inserted near centromere 8
  • 2b is the blank of reaction 2
  • 3a is the cassette of the TAL1 and RKI1 genes inserted near centromere 12
  • 3b is the blank of reaction 3
  • 4a is the cassette of the TKL1 and RKI1 genes inserted near centromere 13
  • 4b is the blank of reaction 4
  • 5b is the blank of reaction 5.
  • strains both containing the genetic modifications described in the present invention as well as those not containing them, were grown in minimal medium with glucose without uracil for approximately 16 hours at 30 ° C. C, 200 rpm.
  • the cells were washed two times. times with sterile distilled water and used for inoculation in minimal medium without uracil, containing only xylose as carbon source.
  • the inoculum was started with an OD of approximately 1, and 0 Fermentations were conducted in 250 mL Erlenmeyer flask with a volume of 100 ml work and incubated in a shaker at 30 Q C, 200 rpm. From time to time, aliquots were taken to observe cell growth.

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Abstract

The present invention describes an expression cassette for eukaryotic cell transformation, comprising the nucleotide sequence (SEQ ID NO: 2) encoding a xylose isomerase peptide (SEQ ID NO: 1), optionally also comprising other genes of the pentose phosphate pathway. The invention also describes a microorganism genetically modified by insertion of the described expression cassette, which is capable of consuming xylose and converting ethanol in comparison to its equivalent without such modifications. A method is also described for producing biofuels and biochemicals, preferably ethanol, from sugars that are abundant in the lignocellulosic portion of plant biomass, mainly pentoses, and, among the pentoses, mainly xylose. Biofuels, preferably ethanol, and biochemicals produced by the method of the invention are also described.

Description

CASSETE DE EXPRESSÃO PARA A TRANSFORMAÇÃO DE CÉLULA EUCARIÓTICA, PROCESSO PARA A TRANSFORMAÇÃO DE CÉLULA EUCARIÓTICA, MICRO-ORGANISMO GENETICAMENTE MODIFICADO, PROCESSO DE PRODUÇÃO DE BIOCOMBUSTÍVEIS E/OU BIOQUÍMICOS E BIOCOMBUSTÍVEL E/OU BIOQUÍMICO ASSIM PRODUZIDOS  EXPRESSION CASSETTE FOR EUCHARIOTIC CELL TRANSFORMATION, PROCESS FOR EUCHARIOTIC CELL TRANSFORMATION, GENETICALLY MODIFIED MICRO-ORGANISM, PROCESS FOR PRODUCING BIOFUEL AND BIOQUIUS AND BIOQUIOSI BIOKYBIOS
CAMPO DA INVENÇÃO  FIELD OF INVENTION
[0001] A presente invenção se relaciona a biocombustíveis, bioquímicos e a processos para sua obtenção. Mais especificamente, a presente invenção proporciona soluções técnicas para a produção de combustíveis de segunda geração baseados na conversão de açúcares presentes na biomassa vegetal, preferencialmente a partir de polímeros da parede celular vegetal. Dentre outros objetos, a presente tecnologia descreve um cassete de expressão para transformação de células eucarioticas e um micro-organismo geneticamente modificado, com eficiente performance fermentativa na conversão dos açúcares presentes na biomassa vegetal, principalmente pentoses, e dentre as pentoses, principalmente xilose, em bioquímicos e/ou biocombustíveis. O micro-organismo geneticamente modificado pela inserção do cassete é capaz de consumir xilose quando comparado com micro-organismos não modificados, favorecendo assim seu desempenho em escala industrial. São também descritos um processo para a obtenção de biocombustíveis e/ou bioquímicos e os produtos assim obtidos. [0001] The present invention relates to biofuels, biochemicals and processes for obtaining them. More specifically, the present invention provides technical solutions for the production of second generation fuels based on the conversion of sugars present in plant biomass, preferably from plant cell wall polymers. Among other objects, the present technology describes an expression cassette for eukaryotic cell transformation and a genetically modified microorganism, with efficient fermentative performance in the conversion of sugars present in plant biomass, mainly pentoses, and among pentoses, mainly xylose, in biochemical and / or biofuels. The genetically modified microorganism by cassette insertion is able to consume xylose when compared to unmodified microorganisms, thus favoring its performance on an industrial scale. Also described are a process for obtaining biofuels and / or biochemicals and the products thus obtained.
FUNDAMENTOS DA INVENÇÃO  BACKGROUND OF THE INVENTION
[0002] A necessidade da substituição da matriz mundial de combustíveis baseada em fontes fósseis por alternativas renováveis tornou a produção de combustíveis de segunda geração, por exemplo, etanol, uma das mais promissoras tecnologias em fase de desenvolvimento. Este processo consiste na conversão de polímeros que formam a biomassa vegetal, principalmente aqueles presentes na parede celular como celulose, hemicelulose e lignina, em biocombustíveis e/ou bioquímicos. [0003] A biomassa vegetal é uma mistura complexa de compostos quimicamente distintos que podem ser fracionados gerando componentes com aplicações específicas. Assim, do mesmo modo que uma refinaria petroquímica produz uma grande variedade de produtos derivados do petróleo bruto, os mesmos princípios podem ser aplicados a biorrefinarias, ou seja, refinarias baseadas em biomassa (Santos, L. V.; Pereira, G. A. G. Petroquímica verde - Anais do Simpósio Microrganismos em Agroenergia: da Prospecção aos Bioprocessos. Editora Embrapa. ISSN 2177-4439, 2013). The need to replace the world's fossil-based fuel matrix with renewable alternatives has made the production of second-generation fuels, for example ethanol, one of the most promising technologies under development. This process consists of converting polymers that form plant biomass, especially those present in the cell wall such as cellulose, hemicellulose and lignin, into biofuels and / or biochemicals. Plant biomass is a complex mixture of chemically distinct compounds that can be fractionated into components with specific applications. Thus, just as a petrochemical refinery produces a wide variety of crude oil products, the same principles can be applied to biorefineries, ie biomass-based refineries (Santos, LV; Pereira, GAG Green Petrochemicals - Anais do Symposium Microorganisms in Agroenergy: From Prospecting to Bioprocesses (Embrapa Publishing House ISSN 2177-4439, 2013).
[0004] A utilização da biomassa vegetal como fonte de açúcares fermentescíveis é uma alternativa promissora e sustentável, entretanto, alguns desafios ainda precisam ser superados, como a disponibilização dos açúcares da parede celular vegetal. Esse procedimento pode ser feito através da ação de enzimas hidrolíticas (celulases e hemicelulases), as quais disponibilizam os monômeros de açúcares (hexoses e pentoses) que, por sua vez, são posteriormente metabolizados por micro-organismos para geração de bioquímicos e biocombustíveis. The use of plant biomass as a source of fermentable sugars is a promising and sustainable alternative, however, some challenges still need to be overcome, such as the availability of plant cell wall sugars. This procedure can be done through the action of hydrolytic enzymes (cellulases and hemicellulases), which provide the sugar monomers (hexoses and pentoses) which, in turn, are later metabolized by microorganisms to generate biochemicals and biofuels.
[0005] Entretanto, micro-organismos naturalmente capazes de consumir açúcares presentes nas cadeias de celulose e hemicelulose não são geralmente passíveis de utilização em escala industrial de forma eficiente. Assim, torna-se necessário o desenvolvimento de micro-organismos com capacidade de utilizar esses açúcares da parede celular vegetal de forma eficientemente, como o descrito na presente invenção.  However, microorganisms naturally capable of consuming sugars present in the cellulose and hemicellulose chains are generally not capable of industrial use efficiently. Thus, it is necessary to develop microorganisms capable of using such plant cell wall sugars efficiently as described in the present invention.
[0006] A utilização de micro-organismos como plataformas eficazes na conversão de açúcares da biomassa em produtos de alto valor agregado é amplamente descrita. Nesse contexto, a levedura Saccharomyces cerevisiae tem recebido papel de destaque devido sua robustez e tolerância em condições industriais de fermentação. A facilidade de manipulação genética desse organismo e o uso de ferramentas de engenharia metabólica, em sinergia com biologia de sistemas e biologia sintética tem possibilitado a inclusão de novas rotas metabólicas para a produção de combustíveis e químicos como etanol, biobutanol, biodiesel, 1 ,2-propanediol, ácido succínico, ácido pirúvico, entre outros [Cellular and Molecular Life Sciences, 69(16): 2671 -90, 2012]. The use of microorganisms as effective platforms for converting sugars from biomass into high value added products is widely described. In this context, Saccharomyces cerevisiae yeast has been given a prominent role due to its robustness and tolerance in industrial fermentation conditions. The ease of genetic manipulation of this organism and the use of metabolic engineering tools, in synergy with systems biology and synthetic biology, has enabled the inclusion of new metabolic pathways for the production of fuels and chemicals such as ethanol. biobutanol, biodiesel, 1,2-propanediol, succinic acid, pyruvic acid, among others [Cellular and Molecular Life Sciences, 69 (16): 2671-90, 2012].
[0007] Linhagens selvagens de S. cerevisae não são naturalmente capazes de fermentar pentoses, como, por exemplo, a xilose, presente na biomassa. Entretanto, diversos trabalhos já realizaram procedimentos de engenharia metabólica em S. cerevisiae introduzindo nesses organismos as vias metabólicas para consumo de xilose, tendo como foco duas vias principais: a via Xilose Redutase - Xilitol Desidrogenase (XR-XDH) e a via Xilose Isomerase (XI). Wild S. cerevisae strains are not naturally capable of fermenting pentoses, such as xylose, present in biomass. However, several studies have already performed metabolic engineering procedures in S. cerevisiae introducing in these organisms the metabolic pathways for xylose consumption, focusing on two main pathways: Xylose Reductase - Xylitol Dehydrogenase (XR-XDH) and Xylose Isomerase ( XI).
[0008] É descrito no estado da técnica que a introdução de gene que codifica a enzima xilose isomerase (XI) permite que a cepa apresente maior rendimento na produção de álcool e/ou ácidos do que quando ela é modificada com outros genes, como por exemplo, gene que codifica a enzima xilose redutase ou xilitol desidrogenase, visto que o metabolismo utilizando xilose isomerase permite menor acúmulo de subprodutos não desejáveis, como xilitol e glicerol [2004, FEMS Yeast Res. 4: 655-664].  It is described in the prior art that the introduction of a gene encoding the enzyme xylose isomerase (XI) allows the strain to yield higher yields of alcohol and / or acids than when it is modified with other genes, such as for example, gene encoding the enzyme xylose reductase or xylitol dehydrogenase, since metabolism using xylose isomerase allows less accumulation of undesirable byproducts such as xylitol and glycerol [2004, FEMS Yeast Res. 4: 655-664].
[0009] A via XR-XDH, comum em micro-organismos eucariotos, tem maior produtividade inicial por permitir que o etanol seja produzido mais rapidamente, apenas com a inserção dos genes responsáveis pela conversão da xilose. Essa via consiste em duas reações de oxi-redução, sendo que na primeira, a xilose é reduzida a xilitol pela ação da enzima xilose redutase (XR), em reação mediada por NADPH/NADH e, em seguida, o xilitol é oxidado a xilulose por meio da enzima xilitol desidrogenase (XDH), mediada exclusivamente por NAD+. O cofator NADPH é principalmente regenerado na fase oxidativa da via das pentoses fosfato, com produção de CO2. Porém, NAD+ é regenerado principalmente na cadeia respiratória, com o O2 como aceptor final de elétrons. Sob limitadas concentrações de oxigénio, não ocorre a completa reoxidação de NAD+, resultando em um desbalanço redox e no acúmulo de xilitol, o que impacta diretamente no rendimento final de etanol [Biochemical Engineering Journal, Amsterdam, v.12, n.1 , p.49-59, 2002]. Além do xilitol, outro subproduto formado é o glicerol [FEMS Yeast Research, Delft, v.4, n.6, p.655-664, 2004]. The XR-XDH pathway, common in eukaryotic microorganisms, has higher initial productivity by allowing ethanol to be produced more rapidly, only by inserting the genes responsible for xylose conversion. This pathway consists of two oxy-reduction reactions. In the first, xylose is reduced to xylitol by the action of the enzyme xylose reductase (XR), in a NADPH / NADH-mediated reaction, and then xylitol is oxidized to xylulose. by the enzyme xylitol dehydrogenase (XDH), mediated exclusively by NAD + . The NADPH cofactor is mainly regenerated in the oxidative phase of the pentose phosphate pathway with CO 2 production. However, NAD + is regenerated mainly in the respiratory chain, with O 2 as the final electron acceptor. Under limited oxygen concentrations, complete NAD + reoxidation does not occur, resulting in redox imbalance and xylitol accumulation, which directly impacts the final ethanol yield [Biochemical Engineering Journal, Amsterdam, v.12, n.1, pp. 49-59, 2002]. In addition to xylitol, another formed byproduct is glycerol [FEMS Yeast Research, Delft, v.4, n.6, p.655-664, 2004].
[0010] Por sua vez, o metabolismo de xilose realizado através da via xilose isomerase (XI) é mais comum em procariotos e ocorre em um único passo, evitando assim o desbalanço redox e permitindo menor formação de subprodutos que diminuem o rendimento de etanol. Adicionalmente, a via da XI pode resultar em um maior rendimento de etanol por acumular menor quantidade de subprodutos da fermentação [Microbial Celi Factories, Londres, v.6, n.5, p.1-10, 2007].  In turn, xylose metabolism carried out via the xylose isomerase (XI) pathway is more common in prokaryotes and occurs in a single step, thus avoiding redox imbalance and allowing less formation of byproducts that decrease ethanol yield. Additionally, the XI pathway may result in higher ethanol yield by accumulating fewer fermentation by-products [Microbial Celi Factories, London, v.6, n.5, p.1-10, 2007].
[0011] Um dos primeiros relatos de xilose isomerase funcional em Saccharomyces foi a do micro-organismo eucariótico Piromyces sp E2, descrita por FEMS Yeast Research, Delft, v.4, n.1 , p.69-78, 2003 e por WO03062430, os quais descrevem mutantes capazes de crescer em xilose como única fonte de carbono, produzir etanol, obter menor produção e acúmulo de metabolitos intermediários e uma menor repressão catabólica em meio contendo glicose e xilose [FEMS Yeast Research, Delft, v.4, n.6, p.655-664, 2004; FEMS Yeast Research, Delft, v.5, n.4, p.399-409, 2005a; FEMS Yeast Research, Delft, v.5, n.10, p.925-934, 2005b].  One of the first reports of functional xylose isomerase in Saccharomyces was that of the eukaryotic microorganism Piromyces sp E2, described by FEMS Yeast Research, Delft, v.4, n.1, p.69-78, 2003 and by WO03062430. , which describe mutants capable of growing on xylose as the sole carbon source, producing ethanol, achieving lower production and accumulation of intermediate metabolites, and lower catabolic repression in glucose and xylose containing medium [FEMS Yeast Research, Delft, v.4, n .6, p.655-664, 2004; FEMS Yeast Research, Delft, v.5, no. 4, p.399-409, 2005a; FEMS Yeast Research, Delft, v.5, no.10, p.925-934, 2005b].
[0012] O documento W09624667, por sua vez, já sugeria que xilose isomerase eucariótica seria mais eficientemente expressa em leveduras, em comparação com xilose isomerase de bactéria, devido à similaridade evolutiva entre os organismos. Entretanto, existem exemplos na literatura de xilose isomerases de eucariotos que não são funcionalmente expressos em Saccharomyces [Applied and environmental microbiology, Apr. 2009, p. 2304-2311]. Adicionalmente, Haranghi (Arch Microbiol, 180: 134-141 , 2003) e Mandhavan (Appl Microbiol Biotechnol, 82:1067-1078, 2009) acabaram demonstrando que a xilose isomerase de Piromyces sp E2, seria mais intimamente relacionada com xilose isomerase de procariotos do que eucariotos, possivelmente por um evento de transferência horizontal de bactéria para fungo. Esse argumento seria ainda reforçado pelo documento WO 2006009434 A1 , o qual descreve que a porcentagem de identidade entre sequências de xilose isomerase em relação à sequência já descrita de Piromyces sp E2 é que seria o fator responsável por conferir a habilidade das sequências nucleotídicas codificarem proteínas com função de xilose isomerase ativas em Saccharomyces. Document W09624667, in turn, already suggested that eukaryotic xylose isomerase would be more efficiently expressed in yeast compared to bacterial xylose isomerase due to evolutionary similarity between organisms. However, there are examples in the literature of eukaryotic xylose isomerases that are not functionally expressed in Saccharomyces [Applied and environmental microbiology, Apr. 2009, p. 2304-2311]. Additionally, Haranghi (Arch Microbiol, 180: 134-141, 2003) and Mandhavan (Appl Microbiol Biotechnol, 82: 1067-1078, 2009) eventually demonstrated that Piromyces sp E2 xylose isomerase would be more closely related to prokaryotic xylose isomerase. than eukaryotes, possibly due to a horizontal bacterial to fungal transfer event. This argument would be further reinforced by the document WO 2006009434 A1, which discloses that the percent identity between xylose isomerase sequences relative to the already described Piromyces sp E2 sequence is the factor responsible for conferring the ability of nucleotide sequences to encode Saccharomyces active xylose isomerase proteins .
[0013] Na tentativa de reforçar ainda mais essa argumentação, o documento WO2010074577, por exemplo, descreve a existência de alguns domínios que seriam conservados em proteínas com função xilose isomerase, indicando que a presença desses domínios no peptídeo, usando como referência o alinhamento com a sequência de XI de Piromyces sp E2, garantiria que a proteína seria expressa de maneira funcional em Saccharomyces. Essas análises basearam-se em sequências de aminoácidos de xiloses isomerases de Piromyces sp E2, previamente descrita em WO03062430, Cyllamyces aberensi e Bacteroides thetaitaomicron, descritas em US7943366 e Clostridium phylofermentans, descrita por Brat, D e colaboradores [Applied and Environmental Microbiology, vol. 75, No. 8, pp. 2304-2311 , Apr. 2009].  In an attempt to further reinforce this argument, WO2010074577, for example, describes the existence of some domains that would be conserved in proteins with xylose isomerase function, indicating that the presence of these domains in the peptide, using alignment with The Piromyces sp E2 XI sequence would ensure that the protein would be functionally expressed in Saccharomyces. These analyzes were based on amino acid sequences from Piromyces sp E2 xylose isomerases, previously described in WO03062430, Cyllamyces aberensi and Bacteroides thetaitaomicron, described in US7943366 and Clostridium phylofermentans, described by Brat, D and colleagues [Applied and Environmental Microbiology, vol. 75, No. 8, pp. 2304-2311, Apr. 2009].
[0014] Entretanto, genes de xiloses isomerases de vários outros organismos que possuem os domínios indicados no documento WO2010074577 também foram inseridas em leveduras, mas não apresentaram atividade funcional [Appl Eviron Microbiol. 2009; 75(8): 2304-11 ; Enzyme and Microbial Technology, vol. 32, pp. 252-259, 2003.; Applied and Environmental Microbiology, vol. 62, No. 12, pp. 4648-4651 , Dec. 1996; Biotechnology and Bioengineering Symposium, No. 13, pp. 245-250, 1983]. However, xylose isomerase genes from various other organisms that have the domains indicated in WO2010074577 have also been inserted into yeast but have no functional activity [Appl Eviron Microbiol. 2009; 75 (8): 2304-11; Enzyme and Microbial Technology, Vol. 32, pp. 252-259, 2003; Applied and Environmental Microbiology, Vol. 62, No. 12, pp. 4,648-4,651, Dec. 1996; Biotechnology and Bioengineering Symposium, No. 13, pp. 245-250, 1983].
[0015] A partir dessas informações, é de extrema relevância salientar-se que a porcentagem de identidade entre a sequência de xilose isomerase de Thermus thermophilus [Applied and envirnmental microbiology 62(12) (1996) 4648-4651], e Piromyces sp E2, ambas funcionais em Saccharomyces, é de apenas 28.1 %, como pode ser observado na Figura 1 , obtida a partir da ferramenta de alinhamento Clustal OmegaFrom this information, it is of utmost relevance to note that the percent identity between the Thermus thermophilus xylose isomerase sequence [Applied and envirnmental microbiology 62 (12) (1996) 4648-4651], and Piromyces sp E2 , both functional in Saccharomyces, is only 28.1%, as can be seen in Figure 1, obtained from the Clustal Omega alignment tool.
(http://www.ebi.ac.uk/Tools/msa/clustalo/). Adicionalmente, é também relevante salientar que Thermus thermophilus não apresenta os domínios defendidos por WO2010074577, como sendo necessários para que a proteína xilose isomerase seja funcional em Saccharomyces, também observado na Figura 1. (http://www.ebi.ac.uk/Tools/msa/clustalo/). Additionally, it is also relevant Note that Thermus thermophilus does not have the domains advocated by WO2010074577 as being necessary for the protein xylose isomerase to be functional in Saccharomyces, also observed in Figure 1.
[0016] Adicionalmente, Gárdonyi e colaboradores [Enzyme and Microbial Technology 32 (2003) 252-259], fizeram a comparação entre xilose isomerases de duas bactérias, Streptomyces rubiginosus e Thermus Thermophilus, pois ambas apresentam alta taxa de homologia entre si, sendo 60% de identidade e 80% de alinhamento, além de tamanho e propriedades físico químicas similares. Segundo suas análises, a xilose isomerase de Thermus thermophilus é expressa de maneira funcional em Saccharomyces, enquanto a de Streptomyces rubiginosus é expressa inativamente. Additionally, Gárdonyi and colleagues [Enzyme and Microbial Technology 32 (2003) 252-259] compared xylose isomerases of two bacteria, Streptomyces rubiginosus and Thermus Thermophilus, as both have a high homology rate, with 60 % identity and 80% alignment plus similar size and physical chemical properties. According to their analysis, Thermus thermophilus xylose isomerase is functionally expressed in Saccharomyces, while Streptomyces rubiginosus is inactively expressed.
[0017] O documento de Gárdonyi e colaboradores [Enzyme and Microbial Technology 32 (2003) 252-259], ao comparar xilose isomerases de Streptomyces rubiginosus e Thermus Thermophilus mostra ainda que, mesmo quando comparadas proteínas com alta taxa de homologia e provenientes de micro-organismos evolutivamente próximos, não é possível se obter respostas claras de porque a primeira seria inativamente expressa, enquanto a segunda é expressa em sua forma ativa de XI. Adicionalmente, já havia sido descrito que a xilose isomerase de S. rubiginosus seria expressa com sucesso em organismos eucariotos, como por exemplo, batata. The paper by Gárdonyi et al. [Enzyme and Microbial Technology 32 (2003) 252-259] comparing xylose isomerases of Streptomyces rubiginosus and Thermus Thermophilus further shows that even when compared to proteins with high homology and from micro evolutionarily close organisms, it is not possible to get clear answers as to why the former would be inactively expressed while the latter is expressed in its active form of XI. Additionally, it has already been described that S. rubiginosus xylose isomerase would be successfully expressed in eukaryotic organisms such as potatoes.
[0018] Gárdonyi e colaboradores sugerem, então, que a não formação do tetrâmero (xilose isomerase é uma proteína tetramérica) poderia ser a principal causa da não funcionalidade de XI em S. cerevisiae. A presença da combinação correta de aminoácidos na interface do tetrâmero gerando pontes de sulfeto e alto número de ligações iónicas, além de condições fisiológicas como pH interno da célula diferente do hospedeiro original, ou uma somatória desses motivos, poderiam ser as principais razões da não funcionalidade das xiloses isomerases quando expressas em S. cerevisiae. Entretanto, nenhuma conclusão foi obtida, fazendo com que essas informações não sejam afirmadas com certeza e, portanto, não permitindo a correta determinação de porquê as xilose isomerases heterólogas tornam-se ou não ativas em Saccharomyces [Appl Environ Microbiol 1987;53(9):1996-2000]. Gárdonyi and collaborators then suggest that non-formation of tetramer (xylose isomerase is a tetrameric protein) could be the main cause of non-functionality of XI in S. cerevisiae. The presence of the correct combination of amino acids at the tetramer interface generating sulfide bridges and high number of ionic bonds, as well as physiological conditions such as cell internal pH different from the original host, or a summation of these reasons could be the main reasons for non-functionality of xylose isomerases when expressed in S. cerevisiae. However, no conclusion has been reached, making this information unclear with certainty and therefore not allowing the correct determination as to why Heterologous xylose isomerases become or are not active in Saccharomyces [Appl Environ Microbiol 1987; 53 (9): 1996-2000].
[0019] Finalmente, o documento WO2011078262 também descreve 7 xilose isomerases funcionais, sendo que: 4 são provenientes de Reticulitermes speratus (SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5 e SEQ ID NO: 7) e 3 xilose isomerases de Mastotermes darwiniensis (SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13). A comparação dessas 7 sequências com a sequência de Piromyces sp E2 revela 2 grupos com valores de identidade bem distintos, variando entre 50% {Reticulitermes speratus) e 73% {Mastotermes darwiniensis) de identidade, como pode ser observado na Figura 2, obtida através da ferramenta BlastX/NCBI (http://biast.ncbj.nim.nih.gov/) ["Basic local alignment search tool." J. Mol. Biol. 215:403-410, 1990].  Finally, WO2011078262 also describes 7 functional xylose isomerases, wherein: 4 are from Reticulitermes speratus (SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5 and SEQ ID NO: 7) and 3 xylose isomerases from Mastotermes darwiniensis (SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13). Comparison of these 7 sequences with the Piromyces sp E2 sequence reveals 2 groups with very distinct identity values, ranging from 50% (Reticulitermes speratus) to 73% (Mastotermes darwiniensis), as can be seen in Figure 2, obtained from Figure 2. from the BlastX / NCBI tool (http://biast.ncbj.nim.nih.gov/) ["Basic local alignment search tool." J. Mol. Biol. 215: 403-410, 1990].
[0020]Cada um desses documentos previamente apresentados, nos mostram que ainda não existe qualquer padrão em proteínas com atividade xilose isomerase que garanta que a inserção de seu gene codificante em Saccharomyces irá gerar proteína com atividade xilose isomerase funcional. Each of these previously presented documents show us that there is as yet no standard in proteins with xylose isomerase activity that ensures that the insertion of its coding gene into Saccharomyces will generate protein with functional xylose isomerase activity.
[0021] A partir dessas informações acima apresentadas, a busca por micro-organismos, sejam eles procariotos ou eucariotos, que contenham em seu genoma genes que codifiquem proteínas com função xilose isomerase, não garante que essas proteínas sejam funcionalmente expressas quando inseridas em Saccharomyces. Bibliotecas de metagenômica que permitem a prospecção de xiloses isomerases nos mais diferentes ambientes tornam-se cada vez mais comuns, como por exemplo, a biblioteca de metagenômica do fluido de rumen bovino, que permitiu a obtenção da xilose isomerase descrita no documento US 2012/0225452 A1. From this information presented above, the search for microorganisms, whether prokaryotes or eukaryotes, containing in their genome genes encoding proteins with xylose isomerase function, does not guarantee that these proteins will be functionally expressed when inserted into Saccharomyces. Metagenomic libraries that allow the prospecting of xylose isomerases in different environments become increasingly common, such as the bovine rumen fluid metagenomic library, which allowed the obtention of xylose isomerase described in US 2012/0225452 TO 1.
[0022] Adicionalmente, como descrito no presente documento, para que seja capaz de consumir xilose, é necessário que o micro-organismo seja, no mínimo, geneticamente modificado com a adição do gene que codifica a xilose isomerase. Entretanto, é descrito na literatura que o aumento da expressão dos genes da via das pentoses fosfato, como transaldolase, transcetolase, xiluloquinase, ribose 5-fosfato isomerase e ribose 5-fosfato epimerase, contribuem no favorecimento da conversão de xilose a etanol, sendo, então, necessário para que o consumo desse açúcar seja eficiente. Additionally, as described herein, in order to be able to consume xylose, the microorganism must be at least genetically modified by the addition of the gene encoding xylose isomerase. However, it is described in the literature that increased expression of the pentose phosphate pathway genes, such as transaldolase, transcetolase, xylulokinase, ribose 5-phosphate isomerase and ribose 5-phosphate epimerase, all contribute to the conversion of xylose to ethanol and are therefore necessary for the consumption of this sugar to be efficient.
[0023] Dessa forma, leveduras do tipo Saccharomyces cerevisiae descritas no estado da técnica, que foram geneticamente modificadas para consumo de xilose, invariavelmente possuem as modificações genéticas descritas acima. Basicamente, o diferencial de cada um desses microorganismos é a combinação desses genes e dos promotores pelos quais estão regulados, bem como o gene que codifica a proteína com função xilose isomerase, pois é esse o principal gene que possibilita o consumo de xilose por cada micro-organismo modificado. Thus, Saccharomyces cerevisiae yeasts described in the state of the art which have been genetically modified for xylose consumption invariably have the genetic modifications described above. Basically, the differential of each of these microorganisms is the combination of these genes and the promoters by which they are regulated, as well as the gene that encodes the protein with xylose isomerase function, since this is the main gene that enables the consumption of xylose by each microorganism. -modified organism.
[0024] Portanto, a obtenção de micro-organismos que produzam proteínas com atividade de xilose isomerase a partir da inserção em seu genoma de gene de outro micro-organismo, que sejam capazes de consumir xilose como única fonte de carbono e, principalmente, que sejam capazes de produzir bioquímicos e/ou biocombustíveis, não é um processo trivial.  Therefore, obtaining microorganisms that produce proteins with xylose isomerase activity from the insertion in their genome of another microorganism gene that are capable of consuming xylose as the sole carbon source and, mainly, that Being able to produce biochemicals and / or biofuels is not a trivial process.
[0025] Assim, o micro-organismo descrito na presente invenção mostra- se eficiente na conversão de açúcares presentes na biomassa vegetal lignocelulósica, principalmente pentoses e, entre as pentoses, principalmente xilose, em biocombustíveis e/ou bioquímicos, dentre eles, principalmente etanol. Thus, the microorganism described in the present invention proves to be efficient in converting sugars present in lignocellulosic plant biomass, mainly pentoses and, among pentoses, mainly xylose, biofuels and / or biochemicals, mainly ethanol. .
BREVE DESCRIÇÃO DA INVENÇÃO  BRIEF DESCRIPTION OF THE INVENTION
[0026] A presente invenção descreve, dentre outros objetos, um microorganismo geneticamente modificado com eficiente performance fermentativa na conversão de açúcares como aqueles contidos na biomassa vegetal lignocelulósica, por exemplo, pentoses, em biocombustíveis e/ou bioquímicos, quando comparado à sua versão sem as modificações genéticas descritas no presente documento. The present invention describes, among other objects, a genetically modified microorganism with efficient fermentative performance in the conversion of sugars such as those contained in lignocellulosic plant biomass, e.g. the genetic modifications described herein.
[0027] De forma mais específica, o micro-organismo geneticamente modificado descrito na presente invenção refere-se uma célula eucariótica transformada geneticamente, preferencialmente uma levedura ou fungo filamentoso, preferencialmente uma levedura do género Saccharomyces. More specifically, the genetically modified microorganism described in the present invention refers to a eukaryotic cell. genetically transformed, preferably a yeast or filamentous fungus, preferably a yeast of the genus Saccharomyces.
[0028] De forma mais específica, é descrito na presente invenção um micro-organismo da espécie Saccharomyces cerevisiae mais eficiente na conversão de pentoses presentes no material lignocelulósico em álcoois e/ou bioquímicos, tais como, por exemplo, etanol, ácido succínico, ácido málico, 1 ,3- propanediol, 1 ,2-propanediol, butanol, isobutanol, biodiesel, 1 ,4-butanediol, 2,3- butanediol, PHB - poli(hidróxido butirato), sem entretanto restringir-se a eles, quando comparado com sua versão sem as modificações genéticas contidas no presente documento. More specifically, a microorganism of the species Saccharomyces cerevisiae more efficient in converting pentoses present in the lignocellulosic material into alcohols and / or biochemicals such as, for example, ethanol, succinic acid, acid is described herein. malic, 1,3-propanediol, 1,2-propanediol, butanol, isobutanol, biodiesel, 1,4-butanediol, 2,3-butanediol, PHB - poly (butyrate hydroxide), but not limited to them as compared version without the genetic modifications contained in this document.
[0029] A pentose preferencialmente utilizada pelo micro-organismo para conversão em álcoois e/ou bioquímicos acima indicados é xilose, sem, entretanto, restringir-se a ela.  The pentose preferably used by the microorganism for conversion to alcohols and / or biochemicals above is xylose, but is not restricted to it.
[0030] A sequência de aminoácidos apresentada em SEQ ID NO:1 , representa um peptídeo com característica de xilose isomerase que, quando expresso em célula eucariótica, favorece a isomerização de xilose em xilulose. Por sua vez, SEQ ID NO:2 compreende sequência de nucleotídeo capaz de codificar proteína com função xilose isomerase. Assim, o micro-organismo descrito na presente invenção é geneticamente modificado pela introdução de cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade em relação à SEQ ID NO:2 e que seja capaz de codificar peptídeo com função xilose isomerase que compreenda SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , sendo que esse peptídeo pode, opcionalmente, ser expresso em uma célula eucariótica em sua forma ativa e funcional de xilose isomerase.  The amino acid sequence shown in SEQ ID NO: 1 represents a peptide with xylose isomerase characteristic which, when expressed in eukaryotic cell, favors the xylose isomerization into xylulose. In turn, SEQ ID NO: 2 comprises nucleotide sequence capable of encoding protein with xylose isomerase function. Thus, the microorganism described in the present invention is genetically modified by introducing an expression cassette comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1, which peptide may optionally be expressed in a eukaryotic cell in its active and functional form. xylose isomerase.
[0031] O cassete de expressão da invenção é caracterizado por compreender: - a sequência de nucleotídeos SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar um peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 ; - ao menos um promotor para a referida sequência nucleotídica codificante; e - uma sequência nucleotídica selecionada dentre: uma sequência nucleotídica terminadora de transcrição; um marcador de seleção; uma ou mais sequência(s) nucleotídica(s) codificante(s) de outra enzimas; combinações dos mesmos ou ainda um plasmídeo compreendo tais sequências, sendo heteróloga ao menos uma das sequências nucleotídicas acima definidas. Um ou mais cassetes de expressão podem ser usados na transformação de células eucarióticas de acordo com a invenção. The expression cassette of the invention is characterized in that it comprises: - nucleotide sequence SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding a xylose function peptide isomerase comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1; at least one promoter for said coding nucleotide sequence; and - a nucleotide sequence selected from: a transcription terminator nucleotide sequence; a selection marker; one or more nucleotide sequence (s) coding for another enzyme; combinations thereof or a plasmid comprising such sequences, with at least one of the nucleotide sequences defined above being heterologous. One or more expression cassettes may be used in eukaryotic cell transformation according to the invention.
[0032] Em uma concretização o cassete que compreende SEQ ID NO:2, capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 , é inserido na célula hospedeira flanqueado, preferencialmente, pela região promotora e terminadora do gene Gliceraldeído 3-Fosfato Desidrogenase, isoenzima 1 ( TDH1), descrito em SEQ ID NO:18 e SEQ ID NO:3, respectivamente.  In one embodiment the cassette comprising SEQ ID NO: 2, capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1, is inserted into the host cell flanked preferably by the promoter and terminator region of the Glyceraldehyde 3 gene. -Phosphate dehydrogenase, isoenzyme 1 (TDH1), described in SEQ ID NO: 18 and SEQ ID NO: 3, respectively.
[0033] Opcionalmente, o cassete de expressão da invenção compreende também sequências selecionadas do grupo que compreende as sequências codificantes das enzimas Xiluloquinase (SEQ ID NO:9), Transaldolase (SEQ NO ID:5), Transcetolase (SEQ ID NO:11 ), Ribose 5-Fosfato Isomerase (SEQ ID NO:7) e/ou Ribose 5-Fosfato Epimerase (SEQ ID NO:12), operacionalmente ligados a promotores e terminadores, sendo que esses promotores são preferencialmente constitutivos da célula onde o cassete será inserido ou naturalmente induzíveis. Essas sequencias podem estar compreendidas no mesmo cassete ou cassetes de expressão diferentes.  Optionally, the expression cassette of the invention also comprises sequences selected from the group comprising the coding sequences of the enzymes Xylulokinase (SEQ ID NO: 9), Transaldolase (SEQ NO ID: 5), Transcetolase (SEQ ID NO: 11) , Ribose 5-Phosphate Isomerase (SEQ ID NO: 7) and / or Ribose 5-Phosphate Epimerase (SEQ ID NO: 12), operably linked to promoters and terminators, which promoters are preferably constitutive of the cell into which the cassette will be inserted. or naturally inducible. Such sequences may be comprised of the same or different expression cassettes.
[0034] Assim, a presente invenção descreve célula hospedeira eucariótica/micro-organismo sendo levedura da espécie Saccharomyces cerevisiae, porém salienta-se que qualquer célula eucariótica pode ser transformada com um ou mais cassetes de expressão da invenção, que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreenda SEQ ID NO: 1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 . Thus, the present invention describes eukaryotic host cell / microorganism being yeast of the Saccharomyces cerevisiae species, but it is emphasized that any eukaryotic cell can be transformed with one or more expression cassettes of the invention comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding peptide with function xylose isomerase comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1.
[0035] Para aumento de fluxo da via das pentose fosfato na célula hospedeira, são inseridos genes que codificam as enzimas Xiluloquinase {XKS1, EC 2.7.1 .17), cuja sequência de nucleotídeos é representada neste documento por SEQ ID NO:9, a Transaldolase { TAL1, EC 2.2.1 .2), representada pela sequência SEQ NO ID:5, Transcetolase { TKL1, EC 2.2.1 .1 ), cuja sequência de nucleotídeos é representada por SEQ ID NO:11 , Ribose 5- Fosfato Isomerase {RKI1, EC 5.3.1 .6), cuja sequência de nucleotídeos é representada por SEQ ID NO:7; e Ribose 5-Fosfato Epimerase {RPE1, EC 5.1 .3.1 ), cuja sequência de nucleotídeos é representada por SEQ ID NO:12.  For increased flow of the pentose phosphate pathway into the host cell, genes encoding the xylulokinase enzymes (XKS1, EC 2.7.1 .17), whose nucleotide sequence is represented herein by SEQ ID NO: 9, are inserted. Transaldolase (TAL1, EC 2.2.1 .2), represented by the sequence SEQ NO ID: 5, Transcetolase (TKL1, EC 2.2.1 .1), whose nucleotide sequence is represented by SEQ ID NO: 11, Ribose 5- Phosphate Isomerase (RKI1, EC 5.3.1 .6), the nucleotide sequence of which is represented by SEQ ID NO: 7; and Ribose 5-Phosphate Epimerase (RPE1, EC 5.1 .3.1), whose nucleotide sequence is represented by SEQ ID NO: 12.
[0036] Ao menos um dos genes que codificam as enzimas apresentadas, compreendidas na via das pentose-fosfato, bem como cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , deve apresentar-se superexpresso e, preferencialmente, ligado a promotores constitutivos ou naturalmente induzíveis. Assim, a presente invenção descreve célula hospedeira compreendendo um ou mais cassetes de expressão contendo genes endógenos de enzimas da fase não oxidativa da via das pentoses fosfato, os quais são, preferencialmente, construídos utilizando-se promotores fortes e constitutivos da célula na qual serão inseridos e estavelmente integrados ao genoma da célula hospedeira. Todos os cassetes de expressão com os genes da via metabólica das pentoses fosfato que favorecem o consumo de xilose são inseridos na região do cromossomo alvo localizada entre o centrômero e o primeiro gene adjacente a ele, preferencialmente na região dos 5 mil primeiros pares de bases contados a partir do centrômero tanto na direção upstream quanto downstream, podendo inclusive ser apenas upstream, apenas downstream ou ambos simultaneamente. [0037] Adicionalmente à inserção dos cassetes de expressão, a presente invenção descreve também a deleção ou inativação do gene GRE3 (representado em SEQ ID NO:14), o qual codifica uma aldose redutase que favorece a produção de xilitol a partir de xilose. A produção de xilitol diminui o rendimento total de etanol que pode ser obtido, além de ser um inibidor da ação da enzima xilose isomerase. At least one of the genes encoding the enzymes shown, comprised in the pentose phosphate pathway, as well as an expression cassette comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1, must be overexpressed and preferably linked to constitutive or naturally inducible promoters. . Thus, the present invention describes host cells comprising one or more expression cassettes containing endogenous enzyme genes from the non-oxidative phase of the pentose phosphate pathway, which are preferably constructed using strong and constitutive promoters of the cell into which they will be inserted. and stably integrated into the host cell genome. All expression cassettes with the pentose phosphate metabolic pathway genes that favor xylose consumption are inserted into the target chromosome region located between the centromere and the first gene adjacent to it, preferably in the region of the first 5,000 base pairs counted. from the centromere in either upstream or downstream direction, and may even be upstream only, downstream only, or both simultaneously. In addition to the insertion of expression cassettes, the present invention also describes the deletion or inactivation of the GRE3 gene (represented in SEQ ID NO: 14), which encodes an aldose reductase that favors xylitol production from xylose. Xylitol production decreases the total yield of ethanol that can be obtained, as well as being an inhibitor of the action of the enzyme xylose isomerase.
[0038] O micro-organismo descrito na presente invenção é, preferencialmente, de linhagem industrial e apresenta diferencialmente as características de ser não floculante, permitir baixa formação de glicerol e xilitol, ter alta viabilidade, alta taxa de crescimento, não produzir de espuma, entre outros.  The microorganism described in the present invention is preferably of industrial lineage and differentially exhibits the characteristics of being non-flocculant, permitting low glycerol and xylitol formation, having high viability, high growth rate, not producing foam, among others.
[0039] A presente invenção também revela e compreende um processo de produção de biocombustíveis e/ou bioquímicos a partir de biomassa vegetal, preferencialmente a porção lignocelulosica da biomassa vegetal. O processo de produção de biocombustíveis e/ou bioquímicos descrito na presente invenção utiliza o micro-organismo da invenção para produção de biocombustíveis e/ou bioquímicos.  The present invention also discloses and comprises a process for producing biofuels and / or biochemicals from plant biomass, preferably the lignocellulosic portion of plant biomass. The biofuel and / or biochemical production process described in the present invention utilizes the microorganism of the invention for biofuel and / or biochemical production.
[0040] O referido processo compreende as seguintes etapas:  The said process comprises the following steps:
colocar o micro-organismo que compreende as modificações genéticas descritas na presente invenção em contato com material que compreende açúcares como aqueles provenientes de biomassa vegetal lignocelulosica, os quais, por sua vez, compreendem pentoses, como a xilose, sendo que o material lignocelulósico pode, opcionalmente ser submetido a prévio pré- tratamento e hidrólise. Essa etapa pode ocorrer, preferencialmente em condições anaeróbias, não sendo esta, entretanto, uma condição restritiva para o processo; e  bringing the microorganism comprising the genetic modifications described in the present invention into contact with material comprising sugars such as those from lignocellulosic plant biomass, which in turn comprise pentoses such as xylose, the lignocellulosic material may, optionally be subjected to pretreatment and hydrolysis. This stage may occur, preferably under anaerobic conditions, but this is not a restrictive condition for the process; and
opcionalmente, fazer o posterior recolhimento do composto gerado.  optionally make further collection of the generated compound.
[0041] O processo da invenção proporciona a produção de biocombustíveis que compreendem predominantemente álcoois, especialmente o etanol. O processo da invenção proporciona a produção de bioquímicos selecionados do grupo que compreende, mas não se limita a: ácido succínico, ácido málico, 1 ,3-propanediol, 1 ,2-propanediol, butanol, isobutanol, biodiesel, 1 ,4-butanediol, 2,3-butanediol e/ou PHB - poli(hidróxido butirato). The process of the invention provides for the production of biofuels comprising predominantly alcohols, especially ethanol. The process of the invention provides for the production of biochemicals. selected from the group including, but not limited to: succinic acid, malic acid, 1,3-propanediol, 1,2-propanediol, butanol, isobutanol, biodiesel, 1,4-butanediol, 2,3-butanediol and / or PHB - poly (butyrate hydroxide).
[0042] A presente invenção descreve, finalmente, os biocombustíveis, preferencialmente etanol, e bioquímicos produzidos através do processo que utiliza o micro-organismo que compreende as modificações genéticas descritas nesta invenção. Finally, the present invention describes biofuels, preferably ethanol, and biochemicals produced by the process utilizing the microorganism comprising the genetic modifications described in this invention.
[0043] Os versados na arte/técnica imediatamente compreenderão, a partir da presente descrição, que o conceito inventivo ora reivindicado não se limita às concretizações exemplificadas acima, que devem ser interpretadas: como prova da existência material da invenção; e como meio informacional que prontamente permite que um técnico no assunto consiga reproduzi-lo - tanto nas formas específicas ora exemplificadas como em outras legalmente abrangidas pelo escopo completo do conceito inventivo revelado e dos objetos reivindicados.  Those skilled in the art / art will immediately understand from this disclosure that the inventive concept claimed herein is not limited to the embodiments exemplified above, which are to be construed as: evidence of the material existence of the invention; and as an informational medium that readily enables a person skilled in the art to reproduce it - both in the specific forms here exemplified and in others legally within the full scope of the disclosed inventive concept and claimed objects.
[0044] Salienta-se, então, que o microrganismo que compreende as modificações genéticas descritas na presente invenção é, por si só, um dos objetos da invenção, sendo ele particularmente eficiente na conversão em álcoois e/ou ácidos de açúcares constituintes de material lignocelulosico, principalmente pentoses, entre elas destaca-se a xilose. Referido microrganismo é eficiente na conversão açúcares, entre eles pentoses, incluindo xilose, presentes no material lignocelulosico, como aquele previamente submetido a processo de hidrólise.  It is noted, then, that the microorganism comprising the genetic modifications described in the present invention is, by itself, one of the objects of the invention, being particularly efficient in the conversion into alcohols and / or sugar acids of constituent material. lignocellulosic, mainly pentoses, among them stands out the xylose. Said microorganism is efficient in converting sugars, including pentoses, including xylose, present in lignocellulosic material, such as that previously subjected to hydrolysis process.
BREVE DESCRIÇÃO DAS FIGURAS  BRIEF DESCRIPTION OF THE FIGURES
[0045] Na Figura 1 mostra o alinhamento entre as sequências de Thermus thermophilus, e Piromyces sp E2, ambas funcionais em Saccharomyces, que é de apenas 28.1 % e foi obtida a partir da ferramenta de alinhamento Clustal Omega (http://www.ebi.a.c.ukTools/msa./clustaJo/). Nesta figura, vemos a comparação entre as sequências de Thermus thermophilus e a xilose isomerase de Piromyces sp E2 evidenciando a não conservação dos domínios. [0045] In Figure 1 shows the alignment between the Thermus thermophilus, and Piromyces sp E2 sequences, both functional in Saccharomyces, which is only 28.1% and was obtained from the Clustal Omega alignment tool (http: // www. ebi.acukTools / msa. / clustaJo /). In this figure we see the comparison between the Thermus thermophilus sequences and the xylose isomerase from Piromyces sp E2 showing the non conservation of the domains.
[0046] A Figura 2 mostra as comparações entre as sequencias de a) Mastotermes darwiniensis e b) Reticulitermes speratus contra a xilose isomerase de Piromyces sp E2 mostrando as porcentagens de identidade de 73% e 50% para a) e b), respectivamente.  Figure 2 shows comparisons between the sequences of a) Mastotermes darwiniensis and b) Reticulitermes speratus against Piromyces sp E2 xylose isomerase showing the percentages of identity of 73% and 50% for a) and b), respectively.
[0047] Na Figura 3, mostra-se o gel de eletroforese obtido a partir da amplificação das regiões externas aos cassetes inseridos, comprovando a integração às leveduras. Na presente figura, M representa omarcador 1 kb ladder; 1 a, o cassete do gene XKS1 inserido próximo ao centrômero 2; 1 b, o branco da reação 1 ; 2a é o cassete do gene XKS1 inserido próximo ao centrômero 8; 2b é o branco da reação 2; 3a é o cassete dos genes TAL1 e RKI1 inseridos próximo ao centrômero 12; 3b é o branco da reação 3; 4a é o cassete dos genes TKL1 e RKI1 inseridos próximo ao centrômero 13; 4b é o branco da reação 4; e 5b é o branco da reação.  In Figure 3, the electrophoresis gel obtained from the amplification of the external regions to the inserted cassettes is shown, proving the integration to the yeasts. In the present figure, M represents the marker 1 kb ladder; 1a, the XKS1 gene cassette inserted near centromere 2; 1 b, the blank of reaction 1; 2a is the XKS1 gene cassette inserted near centromere 8; 2b is the blank of reaction 2; 3a is the cassette of the TAL1 and RKI1 genes inserted near centromere 12; 3b is the blank of reaction 3; 4a is the cassette of the TKL1 and RKI1 genes inserted near centromere 13; 4b is the blank of reaction 4; and 5b is the blank of the reaction.
[0048] Na Figura 4 é mostrado o resultado de reação PCR que foi feito com o DNA da linhagem de S. cerevisiae transformada com o plasmídeo que contém a xilose isomerase SEQ ID NO:2. As amplificações confirmam que o plasmídeo foi inserido na levedura. Em (A), é mostrado o gel de agarose com amplificação da região promotora do TDH1 e a xilose isomerase SEQ ID NO:2 que foi clonada em plasmídeo. Enquanto em (B), observamos a amplificação de xilose isomerase SEQ ID NO:2 juntamente com a região terminadora do TDH1 que foi clonada em plasmídeo.  [0048] Figure 4 shows the PCR reaction result that was made with S. cerevisiae strain DNA transformed with the xylose isomerase plasmid SEQ ID NO: 2. Amplifications confirm that the plasmid was inserted into the yeast. In (A), the agarose gel with TDH1 promoter region amplification and xylose isomerase SEQ ID NO: 2 is shown which has been cloned into a plasmid. While in (B), we observed xylose isomerase amplification SEQ ID NO: 2 along with the TDH1 terminator region that was cloned into plasmid.
[0049] Na Figura 5 observamos a comparação entre as taxas de crescimento da linhagem selvagem (A ), em relação à linhagem geneticamente modificada (■), com inserção do cassete de expressão que compreende SEQ ID NO:2, utilizando xilose como única fonte de carbono. Sendo Densidade Óptica (OD) medida em 600nm apresentada no eixo vertical e tempo (h) no eixo horizontal. [0050] Na Figura 6 observamos o consumo de xilose comparando-se a linhagem sem as modificações genéticas descritas no presente documento ( A ), em relação à linhagem geneticamente modificada (■), com inserção do cassete de expressão que compreende SEQ ID NO:2. Sendo concentração de xilose (g/L) expresso no eixo vertical e tempo (h) no eixo horizontal. In Figure 5 we observe the comparison between growth rates of wild strain (A), relative to genetically modified strain (■), with expression cassette insert comprising SEQ ID NO: 2, using xylose as sole source of carbon. Being Optical Density (OD) measured at 600nm displayed on the vertical axis and time (h) on the horizontal axis. In Figure 6 we observe the xylose consumption comparing the strain without the genetic modifications described herein (A), relative to the genetically modified strain (■), with insertion of the expression cassette comprising SEQ ID NO: 2. Being xylose concentration (g / L) expressed in the vertical axis and time (h) in the horizontal axis.
[0051] Na Figura 7 é possível verificar-se a produção de etanol pelo micro-organismo com as modificações genéticas descritas no presente documento (barra 2), em comparação com o micro-organismo sem as modificações genéticas (barra 1 ). In Figure 7 it is possible to verify the production of ethanol by the microorganism with the genetic modifications described in this document (bar 2) compared to the microorganism without the genetic modifications (bar 1).
DESCRIÇÃO DETALHADA DA INVENÇÃO  DETAILED DESCRIPTION OF THE INVENTION
[0052] A presente invenção descreve, dentre outros objetos, um microorganismo geneticamente modificado com eficiente performance fermentativa na conversão de açúcares como aqueles contidos na biomassa vegetal lignocelulósica, por exemplo, pentoses, em biocombustíveis e/ou bioquímicos, quando comparado à sua versão sem as modificações genéticas descritas no presente documento. The present invention describes, among other objects, a genetically modified microorganism with efficient fermentative performance in the conversion of sugars such as those contained in lignocellulosic plant biomass, for example pentoses, into biofuels and / or biochemicals when compared to their untreated version. the genetic modifications described herein.
[0053] De forma mais específica, o micro-organismo geneticamente modificado descrito na presente invenção refere-se uma célula eucariotica transformada geneticamente, preferencialmente uma levedura ou fungo filamentoso.  More specifically, the genetically modified microorganism described in the present invention refers to a genetically transformed eukaryotic cell, preferably a yeast or filamentous fungus.
[0054] Na presente invenção, leveduras são consideradas como qualquer indivíduo do grupo Eumycotina, ou seja, fungos verdadeiros, que crescem de modo unicelular e que façam preferencialmente fermentação anaeróbia, como por exemplo, Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Cândida, Kluyveromyces, Schizosaccharomyces, Brettanomyces, Hansenula e Yarrowia.  In the present invention, yeast are considered to be any individual of the Eumycotina group, i.e. unicellularly growing true fungi which preferentially perform anaerobic fermentation such as Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Candida, Kluyveromyces. , Schizosaccharomyces, Brettanomyces, Hansenula and Yarrowia.
[0055] Fungos filamentosos, por sua vez, são aqueles caracterizados por apresentarem micélio vegetativo e crescerem a partir da elongação das hifas, além de realizarem respiração aeróbia, como por exemplo, Aspergillus, Penicillium, Fusarium, Trichoderma, Moniliophthora e Acremonium. [0056] Em uma concretização, o micro-organismo é uma levedura do género selecionado do grupo consistindo de: Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Cândida, Kluyveromyces, Schizosaccharomyces, Brettanomyces, Hansenula e Yarrowia. Filamentous fungi, in turn, are those characterized by presenting vegetative mycelium and growing from the elongation of the hyphae, besides performing aerobic respiration, such as Aspergillus, Penicillium, Fusarium, Trichoderma, Moniliophthora and Acremonium. In one embodiment, the microorganism is a yeast of the genus selected from the group consisting of: Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Candida, Kluyveromyces, Schizosaccharomyces, Brettanomyces, Hansenula and Yarrowia.
[0057] De forma ainda mais específica, a presente invenção descreve um micro-organismo geneticamente modificado, preferencialmente uma levedura do género Saccharomyces. Even more specifically, the present invention describes a genetically modified microorganism, preferably a yeast of the genus Saccharomyces.
[0058] De forma mais específica, é descrito na presente invenção um micro-organismo da espécie Saccharomyces cerevisiae mais eficiente na conversão de pentoses, como por exemplo xilose, presentes no material lignocelulósico em álcoois e/ou bioquímicos, tais como, por exemplo, etanol, ácido succínico, ácido málico, 1 ,3-propanediol, 1 ,2-propanediol, butanol, isobutanol, biodiesel, 1 ,4-butanediol, 2,3-butanediol, PHB - poli(hidróxido butirato), sem entretanto restringir-se a eles, quando comparado com sua versão sem as modificações genéticas contidas no presente documento.  More specifically, a microorganism of the species Saccharomyces cerevisiae more efficient in converting pentoses, such as xylose, present in lignocellulosic material into alcohols and / or biochemicals such as, for example, is described in the present invention. ethanol, succinic acid, malic acid, 1,3-propanediol, 1,2-propanediol, butanol, isobutanol, biodiesel, 1,4-butanediol, 2,3-butanediol, PHB - poly (butyrate hydroxide), but without compared to their version without the genetic modifications contained herein.
[0059] A pentose preferencialmente utilizada pelo micro-organismo para conversão em álcoois e/ou bioquímicos acima indicados é xilose, sem, entretanto, restringir-se a ela. The pentose preferably used by the microorganism for conversion to alcohols and / or biochemicals indicated above is xylose, but is not restricted to it.
[0060] Na presente invenção são feitas referências a diversas sequências gênicas, todas listadas na seção Listagem de Sequências. Para breve referência e facilidade de compreensão, suas funções ou genes respectivos são indicados na tabela 1 a seguir.  In the present invention references are made to various gene sequences, all listed in the Sequence Listing section. For brief reference and ease of understanding, their respective functions or genes are shown in table 1 below.
Tabela 1 - Sequências referidas na presente invenção e respectivos nomes/funções. Table 1 - Sequences referred to in the present invention and their names / functions.
SEQUÊNCIAS / FUNÇÃO REFERÊNCIASEQUENCES / REFERENCE FUNCTION
Xilose Isomerase - Peptídeo SEQ I D NO:lXylose Isomerase - Peptide SEQ I D NO: 1
Xilose Isomerase - Nucleotídeos SEQ I D NO:2Xylose Isomerase - Nucleotides SEQ I D NO: 2
Terminador Gliceraldeído 3-Fosfato Desidrogenase, isoenzima 1 (TDH1) SEQ I D NO:3 Glyceraldehyde 3-Phosphate Dehydrogenase Terminator, Isoenzyme 1 (TDH1) SEQ I D NO: 3
URA3 e loxp SEQ I D NO:4 URA3 and loxp SEQ I D NO: 4
Transaldolase (TAL1) SEQ I D NO:5Transaldolase (TAL1) SEQ I D NO: 5
Promotor 3-Fosfato Quinase (PGK1) SEQ I D NO:6Promoter 3-Phosphate Kinase (PGK1) SEQ I D NO: 6
Ribose 5-Fosfato Isomerase (RKI1) SEQ I D NO:7Ribose 5-Phosphate Isomerase (RKI1) SEQ I D NO: 7
Promotor da enzima Álcool Desidrogenase 1 (ADH1) SEQ I D NO:8 Enzyme Promoter Alcohol Dehydrogenase 1 (ADH1) SEQ I D NO: 8
Xiluloquinase (XKS1) SEQ I D NO:9 Xylulokinase (XKS1) SEQ I D NO: 9
Terminador de Álcool Desidrogenase (ADH 1) SEQ I D NO:10 Alcohol Dehydrogenase Terminator (ADH 1) SEQ I D NO: 10
Transcetolase (TKL1) SEQ I D NO:ll Transketolase (TKL1) SEQ I D NO: ll
Ribose 5-Fosfato Epimerase (RPE1) SEQ I D NO:12Ribose 5-Phosphate Epimerase (RPE1) SEQ I D NO: 12
Terminador 3-Fosfato Quinase (PGK1) SEQ I D NO:133-Phosphate Kinase Terminator (PGK1) SEQ I D NO: 13
Aldose Redutase (GRE3) SEQ I D NO:14Aldose Reductase (GRE3) SEQ I D NO: 14
Recombinase CRE SEQ I D NO:15Recombinase CRE SEQ I D NO: 15
LTR do retrotransposon Tyl SEQ I D NO:16Retrotransposon Tyl LTR SEQ I D NO: 16
LEU2 (ORF + promotor e terminador) SEQ I D NO:17LEU2 (ORF + promoter and terminator) SEQ I D NO: 17
Promotor Gliceraldeído 3-Fosfato Desidrogensase, isoenzima 1 (TDH1) SEQ I D NO:18 Glyceraldehyde 3-Phosphate Dehydrogensase Promoter, isoenzyme 1 (TDH1) SEQ I D NO: 18
[0061] A sequência de aminoácidos apresentada em SEQ ID NO:1 , representa um peptídeo com característica de xilose isomerase que, quando expresso em célula eucariótica, favorece a isomerisação de xilose em xilulose. Por sua vez, SEQ ID NO:2 compreende sequência de nucleotídeo capaz de codificar proteína com função xilose isomerase. Assim, o micro-organismo descrito na presente invenção é geneticamente modificado pela introdução de cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade em relação à SEQ ID NO:2 e que seja capaz de codificar peptídeo com função xilose isomerase que compreenda SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , sendo que esse peptídeo pode, opcionalmente, ser expresso em uma célula eucariótica em sua forma ativa e funcional de xilose isomerase. The amino acid sequence shown in SEQ ID NO: 1 represents a peptide with xylose isomerase characteristic which, when expressed in a eukaryotic cell, favors the xylose isomerization in xylulose. In turn, SEQ ID NO: 2 comprises nucleotide sequence capable of encoding protein with xylose isomerase function. Thus, the microorganism described in the present invention is genetically modified by introducing an expression cassette comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1, which peptide may optionally be expressed in a eukaryotic cell in its active and functional form. xylose isomerase.
[0062] O cassete de expressão da invenção é caracterizado por compreender: - a sequência de nucleotídeos SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar um peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 ; - ao menos um promotor para a referida sequência nucleotídica codificante; e - uma sequência nucleotídica selecionada dentre: uma sequência nucleotídica terminadora de transcrição; um marcador de seleção; uma ou mais sequência(s) nucleotídica(s) codificante(s) de outra enzimas; combinações dos mesmos ou ainda um plasmídeo compreendo tais sequências, sendo heteróloga ao menos uma das sequências nucleotídicas acima definidas. Um ou mais cassetes de expressão podem ser usados na transformação de células eucarióticas de acordo com a invenção. The expression cassette of the invention is characterized in that it comprises: - the nucleotide sequence SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and is capable of encoding a xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1; at least one promoter for said coding nucleotide sequence; and - a nucleotide sequence selected from: a transcription terminator nucleotide sequence; a selection marker; one or more nucleotide sequence (s) coding for another enzyme; combinations thereof or a plasmid comprising such sequences, with at least one of the nucleotide sequences defined above being heterologous. One or more expression cassettes may be used in eukaryotic cell transformation according to the invention.
[0063] Em uma concretização o cassete que compreende SEQ ID NO:2, capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 , é inserido na célula hospedeira flanqueado, preferencialmente, pela região promotora e terminadora do gene Gliceraldeído 3-Fosfato Desidrogenase, isoenzima 1 ( TDH1), descrito em SEQ ID NO:18 e SEQ ID NO:3, respectivamente.  In one embodiment the cassette comprising SEQ ID NO: 2 capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 is inserted into the host cell flanked preferably by the promoter and terminator region of the Glyceraldehyde 3 gene. -Phosphate dehydrogenase, isoenzyme 1 (TDH1), described in SEQ ID NO: 18 and SEQ ID NO: 3, respectively.
[0064] Opcionalmente, o cassete de expressão da invenção compreende também sequências selecionadas do grupo que compreende as sequências codificantes das enzimas Xiluloquinase (SEQ ID NO:9), Transaldolase (SEQ NO ID:5), Transcetolase (SEQ ID NO:11 ), Ribose 5-Fosfato Isomerase (SEQ ID NO:7) e/ou Ribose 5-Fosfato Epimerase (SEQ ID NO:12), operacionalmente ligados a promotores e terminadores, sendo que esses promotores são preferencialmente constitutivos da célula onde o cassete será inserido ou naturalmente induzíveis. Essas sequencias podem estar compreendidas no mesmo cassete ou cassetes de expressão diferentes.  Optionally, the expression cassette of the invention also comprises sequences selected from the group comprising the coding sequences for the enzymes Xylulokinase (SEQ ID NO: 9), Transaldolase (SEQ NO ID: 5), Transcetolase (SEQ ID NO: 11) , Ribose 5-Phosphate Isomerase (SEQ ID NO: 7) and / or Ribose 5-Phosphate Epimerase (SEQ ID NO: 12), operably linked to promoters and terminators, which promoters are preferably constitutive of the cell into which the cassette will be inserted. or naturally inducible. Such sequences may be comprised of the same or different expression cassettes.
[0065] Dessa maneira, a presente invenção descreve a integração estável e em alto número de cópias do cassete que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID N :1 , ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 no genoma da célula hospedeira. No presente documento, é considerado alto número de cópias a inserção de, no mínimo 5 cópias do gene em questão, sendo preferencial a inserção de pelo menos 20 cópias. Accordingly, the present invention describes stable and high copy number integration of the cassette comprising SEQ ID NO: 2 or any sequence of at least 80% identity with SEQ ID NO: 2 and capable of encoding peptide. xylose isomerase function comprising SEQ ID N: 1, or any sequence of at least 80% identity with SEQ ID NO: 1 in the host cell genome. In the present document, the insertion of at least 5 copies of the gene in question is considered high number, being preferred the insertion of at least 20 copies.
[0066] Assim, a presente invenção descreve célula hospedeira eucariótica/micro-organismo sendo levedura da espécie Saccharomyces cerevisiae, porém salienta-se que qualquer célula eucariótica pode ser transformada com um ou mais cassetes de expressão da invenção, que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreenda SEQ ID NO: 1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 .  Thus, the present invention describes eukaryotic host cell / microorganism being yeast of the Saccharomyces cerevisiae species, but it is noted that any eukaryotic cell can be transformed with one or more expression cassettes of the invention comprising SEQ ID NO: 2 or any sequence of at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence of at least 80% identity to SEQ ID NO: 1 .
[0067] Portanto, a presente invenção descreve célula hospedeira eucariótica, leveduras ou fungos filamentosos, preferencialmente levedura da espécie Saccharomyces cerevisiae, transformada com um ou mais cassetes de expressão da invenção, sendo que ao menos um cassete compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreenda SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO: 1. Adicionalmente, salienta-se que a sequência de nucleotídeos que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO: 1 pode apresentar-se em cópia única ou, preferencialmente, em múltiplas cópias dessa sequência de nucleotídeos inseridas no genoma.  Therefore, the present invention describes eukaryotic host cell, yeast or filamentous fungi, preferably yeast of the species Saccharomyces cerevisiae, transformed with one or more expression cassettes of the invention, wherein at least one cassette comprises SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1. In addition, it is noted that the nucleotide sequence comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO: 1 or any A sequence of at least 80% identity with SEQ ID NO: 1 may be in single copy or preferably multiple copies of such a genome-inserted nucleotide sequence.
[0068] A célula hospedeira geneticamente modificada descrita na presente invenção adicionalmente compreende genes da via das pentoses fosfato, para que a inserção de cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , possa favorecer a isomerização de xilose em xilulose. Portanto, adicionalmente à inserção na célula hospedeira de cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , a presente invenção descreve modificações genéticas nessa mesma célula que visam o favorecimento do fluxo metabólico através da via das pentoses fosfato, não sendo essas modificações, entretanto, um fator restritivo para transformação da célula hospedeira com cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e que seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 . The genetically modified host cell described in the present invention additionally comprises pentose phosphate pathway genes, so that the expression cassette insert comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO. : 2 and capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1, may favor xylose isomerization into xylulose. Therefore, in addition to insertion into the expression cassette host cell comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and being capable of encoding xylose isomerase peptide comprising SEQ ID NO : 1 or any sequence of at least 80% identity to SEQ ID NO: 1, the present invention describes genetic modifications in that same cell aimed at enhancing metabolic flow through the pentose phosphate pathway, but such modifications are not, however. restriction factor for transformation of the expression cassette host cell comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and capable of encoding xylose isomerase peptide comprising SEQ ID NO : 1 or any sequence with at least 80% identity with SEQ ID NO: 1.
[0069] Para aumento de fluxo da via das pentose fosfato na célula hospedeira, são inseridos genes que codificam as enzimas Xiluloquinase {XKS1, EC 2.7.1 .17), cuja sequência de nucleotídeos é representada neste documento por SEQ ID NO:9, a Transaldolase { TAL 1, EC 2.2.1 .2), representada pela sequência SEQ NO ID:5, Transcetolase { TKL 1, EC 2.2.1 .1 ), cuja sequência de nucleotídeos é representada por SEQ ID NO:11 , Ribose 5-Fosfato Isomerase {RKI1, EC 5.3.1 .6), cuja sequência de nucleotídeos é representada por SEQ ID NO:7; e Ribose 5-Fosfato Epimerase {RPE1, EC 5.1 .3.1 ), cuja sequência de nucleotídeos é representada por SEQ ID NO:12.  For increased flow of the pentose phosphate pathway into the host cell, genes encoding the xylulokinase enzymes (XKS1, EC 2.7.1 .17), whose nucleotide sequence is represented herein by SEQ ID NO: 9, are inserted. Transaldolase (TAL 1, EC 2.2.1 .2), represented by the sequence SEQ NO ID: 5, Transcetolase (TKL 1, EC 2.2.1 .1), whose nucleotide sequence is represented by SEQ ID NO: 11, Ribose 5-Phosphate Isomerase (RKI1, EC 5.3.1 .6), the nucleotide sequence of which is represented by SEQ ID NO: 7; and Ribose 5-Phosphate Epimerase (RPE1, EC 5.1 .3.1), whose nucleotide sequence is represented by SEQ ID NO: 12.
[0070] Ao menos um dos genes que codificam as enzimas apresentadas, compreendidas na via das pentose-fosfato, bem como cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID NO:1 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , deve apresentar-se superexpresso e, preferencialmente, ligado a promotores constitutivos ou naturalmente induzíveis. A superexpressão dos genes que codificam essas enzimas pode acontecer em decorrência do aumento do número de cópias da sequência de nucleotídeos que as codifica, expressão de genes epissomais presentes em vetores que podem ser inseridos na célula eucariótica hospedeira, através do uso de promotores heterólogos àquela sequência na qual ele encontra-se operativamente ligado, ou mesmo homólogos da célula onde foram inseridos, ou como endógenos na célula hospedeira, desde que eles sejam capazes de produzir um estado estável de transcrição mais elevado do que seria realizado pela célula em sua versão sem as presentes modificações genéticas, nas situações em que fontes de carbono como glicose e xilose estiverem disponíveis no meio. Esses promotores podem ser constitutivos ou naturalmente induzíveis. At least one of the genes encoding the enzymes shown, comprised in the pentose phosphate pathway, as well as an expression cassette comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2 and is capable of encoding xylose isomerase function peptide comprising SEQ ID NO: 1 or any sequence having at least 80% identity to SEQ ID NO: 1, should be overexpressed and preferably bound to constitutive or naturally inducible promoters. Overexpression of genes encoding these enzymes may be due to increased copy number of the nucleotide sequence encoding them, expression of episomal genes present in vectors that may be inserted into the host eukaryotic cell through the use of heterologous promoters to that sequence. in which it is operably linked, or even homologues of the cell into which it was inserted, or as endogenous in the host cell, provided that they are capable of producing a higher stable transcription state than would be achieved by the cell in its version without genetic modifications are present in situations where carbon sources such as glucose and xylose are available in the environment. Such promoters may be constitutive or naturally inducible.
[0071] Assim, a presente invenção descreve célula hospedeira compreendendo um ou mais cassetes de expressão contendo genes endógenos de enzimas da fase não oxidativa da via das pentoses fosfato, os quais são, preferencialmente, construídos utilizando-se promotores fortes e constitutivos da célula na qual serão inseridos e estavelmente integrados ao genoma da célula hospedeira. Todos os cassetes de expressão com os genes da via metabólica das pentoses fosfato que favorecem o consumo de xilose são inseridos na região do cromossomo alvo localizada entre o centromero e o primeiro gene adjacente a ele, preferencialmente na região dos 5 mil primeiros pares de bases contados a partir do centromero tanto na direção upstream quanto downstream, podendo inclusive ser apenas upstream, apenas downstream ou ambos simultaneamente.  Thus, the present invention describes host cell comprising one or more expression cassettes containing endogenous enzyme genes of the non-oxidative phase of the pentose phosphate pathway, which are preferably constructed using strong and constitutive cell promoters in the cell. which will be inserted and stably integrated into the host cell genome. All expression cassettes with the pentose phosphate metabolic pathway genes that favor xylose consumption are inserted into the target chromosome region located between the centromere and the first gene adjacent to it, preferably in the region of the first 5,000 base pairs counted. from the centromere in either the upstream or downstream direction, and may even be upstream only, downstream only, or both simultaneously.
[0072] Direção upstream é considerada aquela localizada anteriormente ao ponto de início da unidade de transcrição de uma sequência de DNA, a qual se inicia no promotor e encerra no terminador. Por sua vez, downstream é considerada a região localizada após o ponto de início da unidade de transcrição de uma sequência de DNA. Upstream direction is considered to be that located prior to the starting point of the transcriptional unit of a DNA sequence, which starts at the promoter and ends at the terminator. In turn, downstream is considered the region located after the start point of the transcription unit of a DNA sequence.
[0073] Adicionalmente à inserção dos cassetes de expressão, a presente invenção descreve também a deleção ou inativação do gene GRE3 (representado em SEQ ID NO:14), o qual codifica uma aldose redutase que favorece a produção de xilitol a partir de xilose. A produção de xilitol diminui o rendimento total de etanol que pode ser obtido, além de ser um inibidor da ação da enzima xilose isomerase.  In addition to the insertion of expression cassettes, the present invention also describes deletion or inactivation of the GRE3 gene (depicted in SEQ ID NO: 14), which encodes an aldose reductase which favors xylitol production from xylose. Xylitol production decreases the total yield of ethanol that can be obtained, as well as being an inhibitor of the action of the enzyme xylose isomerase.
[0074] Dessa maneira, a presente invenção descreve a integração estável e em alto número de cópias de cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2 e seja capaz de codificar peptídeo com função xilose isomerase que compreende SEQ ID N:1 , ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 ) no genoma da célula hospedeira.  Accordingly, the present invention describes stable and high copy-number integration of expression cassettes comprising SEQ ID NO: 2 or any sequence having at least 80% identity with SEQ ID NO: 2 and being capable of encoding xylose isomerase function peptide comprising SEQ ID N: 1, or any sequence of at least 80% identity with SEQ ID NO: 1) in the host cell genome.
[0075] O presente documento descreve, portanto, uma célula eucariótica, preferencialmente micro-organismo da espécie Saccharomyces cerevisiae, geneticamente modificada contendo em seu genoma pelo menos um dos genes das enzimas necessárias para favorecer a parte não oxidativa da via das pentoses fosfato, inseridos preferencialmente em alto número de cópias e na região entre o centrômero e seu primeiro gene adjacente, possuindo assim toda a via metabólica necessária para conversão em combustíveis e/ou bioquímicos, principalmente etanol, de açúcares componentes da biomassa lignocelulósica, principalmente pentoses, como por exemplo a xilose. Therefore, this document describes a eukaryotic cell, preferably a microorganism of the species Saccharomyces cerevisiae, genetically modified containing in its genome at least one of the enzyme genes required to favor the non-oxidative part of the pentose phosphate pathway inserted preferably in high copy number and in the region between the centromere and its first adjacent gene, thus possessing all the metabolic pathway necessary for conversion into fuels and / or biochemicals, mainly ethanol, of sugars component of the lignocellulosic biomass, mainly pentoses, such as the xylose.
[0076] O micro-organismo descrito na presente invenção é, preferencialmente, de linhagem industrial e apresenta diferencialmente as características de ser não floculante, permitir baixa formação de glicerol e xilitol, ter alta viabilidade, alta taxa de crescimento, não produzir de espuma, entre outros. The microorganism described in the present invention is preferably of industrial lineage and differentially exhibits the characteristics of being non-flocculant, permitting low glycerol and xylitol formation, having high viability, high growth rate, not producing foam, among others.
[0077] A presente invenção também revela e compreende um processo de produção de biocombustíveis e/ou bioquímicos a partir da fermentação de material que compreende açúcares como aqueles presentes na biomassa vegetal lilgnocelulósica, principalmente pentoses e, entre as pentoses, principalmente xilose. O processo de produção de biocombustíveis e/ou bioquímicos descrito na presente invenção utiliza o micro-organismo geneticamente modificado descrito no presente documento para produção de biocombustíveis e/ou bioquímicos. The present invention also discloses and comprises a process for producing biofuels and / or biochemicals from the fermentation of material comprising sugars such as those present in biomass. lilgnocellulosic vegetable, mainly pentoses and, among pentoses, mainly xylose. The biofuel and / or biochemical production process described in the present invention utilizes the genetically modified microorganism described herein for biofuel and / or biochemical production.
[0078] O referido processo compreende as seguintes etapas:  Said process comprises the following steps:
colocar o micro-organismo que compreende as modificações genéticas descritas na presente invenção em contato com material que compreende açúcares como aqueles provenientes de biomassa vegetal lignocelulosica, os quais, por sua vez, compreendem pentoses, como a xilose, sendo que o material lignocelulósico pode, opcionalmente ser submetido a prévio pré- tratamento e hidrólise. Essa etapa pode ocorrer, preferencialmente em condições anaeróbias, não sendo esta, entretanto, uma condição restritiva para o processo; e  bringing the microorganism comprising the genetic modifications described in the present invention into contact with material comprising sugars such as those from lignocellulosic plant biomass, which in turn comprise pentoses such as xylose, the lignocellulosic material may, optionally be subjected to pretreatment and hydrolysis. This stage may occur, preferably under anaerobic conditions, but this is not a restrictive condition for the process; and
opcionalmente, fazer o posterior recolhimento do composto gerado.  optionally make further collection of the generated compound.
[0079] O processo da invenção proporciona a produção de biocombustíveis que compreendem predominantemente álcoois, especialmente o etanol. O processo da invenção proporciona a produção de bioquímicos selecionados do grupo que compreende, mas não se limita a: ácido succínico, ácido málico, 1 ,3- propanediol, 1 ,2-propanediol, butanol, isobutanol, biodiesel, 1 ,4-butanediol, 2,3- butanediol e/ou PHB - poli(hidróxido butirato). The process of the invention provides for the production of biofuels comprising predominantly alcohols, especially ethanol. The process of the invention provides for the production of biochemicals selected from the group comprising, but not limited to: succinic acid, malic acid, 1,3-propanediol, 1,2-propanediol, butanol, isobutanol, biodiesel, 1,4-butanediol 2,3-butanediol and / or PHB - poly (butyrate hydroxide).
[0080] A presente invenção descreve, finalmente, os biocombustíveis, preferencialmente etanol, e bioquímicos produzidos através do processo que utiliza o micro-organismo que compreende as modificações genéticas descritas nesta invenção.  Finally, the present invention describes biofuels, preferably ethanol, and biochemicals produced by the process using the microorganism comprising the genetic modifications described in this invention.
[0081] Os versados na arte/técnica imediatamente compreenderão, a partir da presente descrição, que o conceito inventivo ora reivindicado não se limita às concretizações exemplificadas acima, que devem ser interpretadas: como prova da existência material da invenção; e como meio informacional que prontamente permite que um técnico no assunto consiga reproduzi-lo - tanto nas formas específicas ora exemplificadas como em outras legalmente abrangidas pelo escopo completo do conceito inventivo revelado e dos objetos reivindicados. Those skilled in the art / art will immediately understand from this disclosure that the inventive concept claimed herein is not limited to the embodiments exemplified above, which are to be construed as: evidence of the material existence of the invention; and as an informational medium that readily enables a person skilled in the art to reproduce it - both in the specific forms here exemplified as in others legally within the full scope of the disclosed inventive concept and the claimed objects.
[0082] Salienta-se, então, que o microrganismo que compreende as modificações genéticas descritas na presente invenção é, por si só, um dos objetos da invenção, sendo ele particularmente eficiente na conversão em álcoois e/ou ácidos de açúcares como aqueles compreendidos na biomassa lignocelulósica, principalmente pentoses, e entre as pentoses, principalmente xilose. Referido microrganismo é eficiente na conversão de pentoses, incluindo xilose, presentes no material lignocelulósico, como aquele previamente submetido a processo de hidrólise.  It is noted, then, that the microorganism comprising the genetic modifications described in the present invention is, by itself, one of the objects of the invention, being particularly efficient in the conversion to alcohols and / or sugar acids as those comprised. in lignocellulosic biomass, mainly pentoses, and among pentoses, mainly xylose. Said microorganism is efficient in the conversion of pentoses, including xylose, present in lignocellulosic material, such as that previously subjected to hydrolysis process.
EXEMPLOS  EXAMPLES
Exemplo 1 - SELEÇÃO DE SEQUÊNCIA DE NUCLEOTÍDEOS CAPAZ DE CODIFICAR PROTEÍNA COM FUNÇÃO XILOSE ISOMERASE.  Example 1 - SELECTION OF NUCLEOTIDE SEQUENCE ABLE TO ENCODE PROTEIN WITH XYLOSIS ISOMERASE FUNCTION.
[0083] Na tentativa de encontrar xilose isomerases que pudessem ser funcionalmente expressas em Saccharomyces, foi realizada busca em banco de dados públicos, disponíveis no NCBI, de genomas incompletos e fragmentados (genomas draft) de regiões não anotadas que poderiam conter genes codificantes de novas xilose isomerases e que, até então, teriam função desconhecida. O parâmetro utilizado para busca foram sequências proteicas de xilose isomerases que até o momento haviam se mostrados funcionais em Saccharomyces, como, por exemplo, Piromyces sp E2 e Orpinomyces. In an attempt to find xylose isomerases that could be functionally expressed in Saccharomyces, a public database, available at NCBI, was searched for incomplete and fragmented genomes (draft genomes) that could contain genes coding for new genes. xylose isomerases and which, until then, would have unknown function. The parameter used for the search was protein sequences of xylose isomerases that so far had been shown to be functional in Saccharomyces, such as Piromyces sp E2 and Orpinomyces.
[0084] Dessa forma, a sequência de xilose isomerase indicada nos bancos de dados como pertencente ao fungo Melampsora pinitorqua foi identificada através da comparação da sequência proteica da xilose isomerase de Piromyces sp. E2 contra o banco de dados de genomas não anotados do NCBI (Whole-genome shotgun contigs). Thus, the sequence of xylose isomerase indicated in the databases as belonging to the fungus Melampsora pinitorqua was identified by comparing the protein sequence of xylose isomerase from Piromyces sp. E2 against NCBI's Whole-genome shotgun contigs (NCBI) database.
[0085] Para tal foi utilizada a versão online do BLAST disponibilizada pelo NCBI, mais especificadamente o TBLASTN, com um E-value de corte de 1 e-5. Como resultado dessa busca, o contig1668883_0 (GenBank ID: AUYS01012195.1 ) foi retornado por conter uma região com similaridade com a proteína da xilose isomerase de Piromyces, apresentando apenas 62% de identidade, fato que não tornaria óbvia sua expressão funcional como xilose isomerase, considerando anterioridades descritas no presente documento. For this purpose the online version of BLAST provided by NCBI was used, more specifically TBLASTN, with a cut-off E-value of 1 and -5. As a result of this search, contig1668883_0 (GenBank ID: AUYS01012195.1) was returned because it contains a region similar to the Piromyces xylose isomerase protein, with only 62% identity, a fact that would not make its functional expression as xylose isomerase obvious, considering the precedents described in this document.
Exemplo 2 - CONSTRUÇÃO DOS CASSETES PARA DE EXPRESSÃO E INSERÇÃO DOS MESMOS NO GENOMA Example 2 - CONSTRUCTION OF CASSETS FOR EXPRESSION AND INSERTION OF THE SAME IN THE GENOME
[0086] Para construção de cada um dos cassetes que compreendiam genes da fase não oxidativa da via das pentoses fosfato, incluindo gene da Xilose Isomerase, cada um dos genes foi amplificado por PCR do genoma de S. cerevisiae e clonado em cassetes de expressão integrativos.  To construct each of the cassettes that comprised non-oxidative phase genes of the pentose phosphate pathway, including the Xylose Isomerase gene, each gene was amplified by PCR of the S. cerevisiae genome and cloned into integrative expression cassettes. .
[0087] Adjacente ao terminador de cada cassete, foi clonado o gene URA3 flanqueado por duas regiões loxP na mesma orientação, permitindo que essa região pudesse ser retirada pela expressão da recombinase Cre (SEQ ID NO:15) e o marcador auxotrófico URA3 pudesse ser utilizado em todos os cassetes de expressão com os genes descritos. Adjacent to the terminator of each cassette, the URA3 gene flanked by two loxP regions in the same orientation was cloned, allowing that region to be removed by expression of Cre recombinase (SEQ ID NO: 15) and the URA3 auxotrophic marker could be deleted. used in all expression cassettes with the described genes.
[0088] Em relação à construção do cassete de expressão contendo gene que codifica xilose isomerase, adicionalmente à construção acima descrita, nas extremidades do cassete foram clonadas 126 pb de cada lado com homologia a uma região próxima ao cromossomo cinco de Saccharomyces cerevisiae, permitindo a integração via recombinação homóloga nessa região.  Regarding the expression cassette construction containing gene encoding xylose isomerase, in addition to the construction described above, 126 bp on each side was cloned on either side with homology to a region near Saccharomyces cerevisiae chromosome five, allowing integration via homologous recombination in this region.
[0089] Para a construção do cassete de expressão com gene que codifica xiluloquinase, por exemplo, amplificou-se esse gene por PCR do genoma de S. cerevisiae e ele foi clonado adjacente ao promotor e terminador do gene que codifica Álcool desidrogenase (ADH1 ) representado nas sequências SED ID NO:8 e SEQ ID NO:10, respectivamente. Após o terminador, foi inserido o gene URA3 flanqueado por duas regiões loxP (SEQ ID NO:4) na mesma orientação. Na extremidade do cassete foi clonado regiões de homologia próximas ao centrômero dois e oito de S. cerevisiae. Foram realizadas duas transformações para inserir os cassetes expressando o gene XKS1 a 288 pb do centrômero dois e a 228 pb do centrômero oito. Dessa maneira, além da cópia endógena, o transformante tem mais duas cópias do gene XKS1 sob a ação de um promotor constitutivo de alta expressão. For the construction of the xylulokinase-encoding gene expression cassette, for example, that gene was amplified by PCR of the S. cerevisiae genome and cloned adjacent to the promoter and terminator of the Alcohol dehydrogenase (ADH1) encoding gene. SED ID NO: 8 and SEQ ID NO: 10, respectively. After the terminator, the URA3 gene flanked by two loxP regions (SEQ ID NO: 4) was inserted in the same orientation. At the end of the cassette, homology regions were cloned near S. cerevisiae centromere two and eight. Two transformations were performed to insert the cassettes expressing the XKS1 gene at 288 bp from centromere two and 228 bp from centromere eight. Thus, in addition to the endogenous copy, the transformant has two more copies of the XKS1 gene under the action of a high-expressive constitutive promoter.
[0090] Em relação ao cassete de expressão que contém genes codificadores de Transaldolase {TAL 1), representado em SEQ ID NO:5 e Ribose 5-Fosfato Isomerase, {RKI1), representado em SEQ ID NO:7, por exemplo, esses genes foram clonados sob a ação dos promotores e terminadores dos genes Gliceraldeído 3-Fosfato Desidrogenase, isoenzima 1 , {TDH1), representado em SEQ ID NO:18 e 3-fosfoglicerato Quinase, {PGK1), representado em SEQ ID NO:3, respectivamente, separados pelo marcador URA3 flanqueado pelos sítios loxP, e devidamente inseridos no cromossomo da célula hospedeira.  With respect to the expression cassette containing Transaldolase (TAL 1) encoding genes represented by SEQ ID NO: 5 and Ribose 5-Phosphate Isomerase (RKI1) represented by SEQ ID NO: 7, for example, genes were cloned under the action of the Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1, (TDH1) gene promoters and terminators represented by SEQ ID NO: 18 and 3-phosphoglycerate kinase, {PGK1) represented by SEQ ID NO: 3, respectively, separated by the URA3 marker flanked by the loxP sites, and properly inserted into the host cell chromosome.
[0091] Em relação ao cassete de expressão que contém genes codificadores de Transcetolase (TKL1 ), representado em SEQ ID NO:11 e Ribose 5-Fosfato Epimerase (RPE1 ), representado em SEQ ID NO:12, por exemplo, esses genes foram clonados sob a ação dos promotores e terminadores dos genes Gliceraldeído 3-Fosfato Desidrogenase, isoenzima 1 { TDH1) e 3-fosfoglicerato Quinase {PGK1), representado em SEQ ID NO:13, respectivamente, separados pelo marcador URA3 flanqueado pelos sítios loxP, e devidamente inseridos no cromossomo da célula hospedeira. With respect to the expression cassette containing Transcetolase (TKL1) encoding genes represented by SEQ ID NO: 11 and Ribose 5-Phosphate Epimerase (RPE1) represented by SEQ ID NO: 12, for example, these genes were cloned under the action of the Glyceraldehyde 3-Phosphate Dehydrogenase, isoenzyme 1 (TDH1) and 3-phosphoglycerate kinase (PGK1) gene promoters and terminators, represented by SEQ ID NO: 13, respectively, separated by the URA3 marker flanked by the loxP sites, and properly inserted into the host cell chromosome.
[0092]A transformação da célula hospedeira com cada um dos cassetes contendo genes da fase não oxidativa da via das pentoses fosfato seguiu o protocolo de Gietz e Schiestl [Nature Protocols 2, 31 - 34; 2007], via acetato de lítio, e cada um dos genes, flanqueados por promotores e terminadores forte e constitutivos de Saccharomyces cerevisiae, foi estavelmente integrado ao cromossomo da célula hospedeira. A correta integração foi confirmada por PCR. Após a confirmação, a região URA3 foi excisada do genoma pela expressão transiente da recombinase Cre (SEQ ID NO:15), deixando apenas um sítio loxP no local, após o terminador do gene inserido.  Transformation of the host cell with each of the cassettes containing non-oxidative phase genes of the pentose phosphate pathway followed the protocol of Gietz and Schiestl [Nature Protocols 2, 31 - 34; 2007], via lithium acetate, and each of the genes, flanked by strong and constitutive Saccharomyces cerevisiae promoters and terminators, was stably integrated into the host cell chromosome. Correct integration was confirmed by PCR. Upon confirmation, the URA3 region was excised from the genome by transient expression of Cre recombinase (SEQ ID NO: 15), leaving only one loxP site in place after the inserted gene terminator.
[0093] Nas extremidades do cassete da Xilose Isomerase (SEQ ID NO:2), foram clonadas 126 pb de cada lado com homologia a uma região próxima ao cromossomo cinco de Saccharomyces cerevisiae, permitindo a integração via recombinação homóloga nessa região. At the ends of the Xylose Isomerase cassette (SEQ ID NO: 2), 126 bp was cloned from each side with homology to a region close to the Saccharomyces cerevisiae chromosome five, allowing integration via homologous recombination in this region.
Exemplo 3 - INSERÇÃO DO CASSETE DE XILOSE ISOMERASE NO GENOMA EM ALTO NÚMERO DE CÓPIAS  Example 3 - Inserting the Xylose Isomerase Cassette into the High Number of Copies Genome
[0094] Para garantir a integração estável e em alto número de cópias na célula hospedeira, o cassete que contém o gene SEQ ID NO:2, capaz de expressar o peptídeo com função Xilose Isomerase representada por SEQ ID NO:1 foi modificado com a inclusão, nas extremidades do cassete, de elementos delta do retrotransposon Ty1, (elemento presente em alto número de cópias no genoma de S. cerevisiae e representado por SEQ ID NO:16).  To ensure stable and high copy number integration into the host cell, the cassette containing the SEQ ID NO: 2 gene capable of expressing the Xylose Isomerase peptide represented by SEQ ID NO: 1 has been modified with the inclusion, at the ends of the cassette, delta elements of the retrotransposon Ty1, (element present in high copy number in the S. cerevisiae genome and represented by SEQ ID NO: 16).
[0095] O marcador URA3 flanqueado pelas regiões loxP é substituído nesse plasmídeo pelo marcador LEU2 (SEQ ID NO:17). Previamente, o gene LEU2 é deletado em uma etapa de manipulação genética. Nessa etapa, integra-se o gene URA3, flanqueado pelas regiões loxP adjacentes a regiões de homologia ao promotor e terminador de LEU2, resultando na deleção desse gene. Em seguida, é inserido o cassete da XI, flanqueado pelos elementos Ty1 e usando o marcador auxotrófico LEU2 para seleção dos transformantes. The URA3 marker flanked by the loxP regions is replaced in that plasmid by the marker LEU2 (SEQ ID NO: 17). Previously, the LEU2 gene is deleted in a step of genetic manipulation. In this step, the URA3 gene is integrated, flanked by loxP regions adjacent to LEU2 promoter and terminator homology regions, resulting in deletion of this gene. Then, the XI cassette flanked by the Ty1 elements is inserted and using the auxotrophic marker LEU2 to select the transformants.
ÇJt MTIJJiOj— Líc L.cl¾§? ¾JÍ LiX U Ci«C lni!cL«ã ÇJt MTIJJiOj— Líc L.cl¾ § ? ÍJ Í LiX U Ci «Cnni! CL« ã
[0096] A deleção do gene GRE3, o qual codifica uma aldose redutase e está representado em SEQ ID NO:14, foi realizado em duas etapas através de manipulação genética, visando a diminuição da produção de xilitol a partir de xilose. Na primeira etapa, foi integrado o gene URA3, flanqueado pelas regiões loxP adjacentes a regiões de homologia ao promotor e terminador do gene GRE3, resultando na deleção dessa região. Na segunda etapa, após confirmada a deleção, o marcador URA3 foi retirado pela expressão transiente da recombinase Cre.  The deletion of the GRE3 gene, which encodes an aldose reductase and is represented in SEQ ID NO: 14, was performed in two steps by genetic manipulation, aimed at decreasing xylitol production from xylose. In the first step, the URA3 gene was integrated, flanked by loxP regions adjacent to GRE3 gene promoter and terminator homology regions, resulting in deletion of this region. In the second step, after the deletion was confirmed, the URA3 marker was removed by transient expression of Cre recombinase.
Exemplo 5 - COMPROVAÇÃO DA INSERÇÃO DOS CASSETES NO MICROORGANISMO GENETICAMENTE MODIFICADO  Example 5 - PROOFING CASSETTE INSERTION IN GENETICALLY MODIFIED MICROORGANISM
[0097] O DNA do micro-organismo geneticamente modificado foi utilizado como modelo para a reação de polimerase em cadeia utilizando oligonucleotídeos que anelam em uma região externa ao local de inserção dos cassetes de expressão gênica. Para cada reação foi utilizado um par de oligos específicos para a região externa de cada cassete inserido. The genetically modified microorganism DNA was used as a template for the polymerase chain reaction using oligonucleotides that ring in a region outside the insertion site of gene expression cassettes. For each reaction, a pair of oligos specific for the external region of each inserted cassette was used.
[0098] Após experimento de reação em cadeira da polimerase (PCR), uma alíquota foi aplicada em gel de agarose 0,8%, corado com GelRed, e submetido a eletroforese para a separação dos fragmentos amplificados. After a polymerase chair reaction (PCR) experiment, an aliquot was applied to a 0.8% agarose gel, stained with GelRed, and electrophoresed to separate the amplified fragments.
[0099] A Figura 3 mostra o gel de eletroforese obtido a partir da amplificação das regiões externas aos cassetes inseridos, comprovando a integração às leveduras. Nessa figura, M representa o marcador 1 kb ladder; 1 a, o cassete do gene XKS1 inserido próximo ao centrômero 2; 1 b, o branco da reação 1 ; 2a é o cassete do gene XKS1 inserido próximo ao centrômero 8; 2b é o branco da reação 2; 3a é o cassete dos genes TAL1 e RKI1 inseridos próximo ao centrômero 12; 3b é o branco da reação 3; 4a é o cassete dos genes TKL1 e RKI1 inseridos próximo ao centrômero 13; 4b é o branco da reação 4; e 5b é o branco da reação 5. [0099] Figure 3 shows the electrophoresis gel obtained from the amplification of the external regions to the inserted cassettes, proving the integration with the yeasts. In this figure, M represents the marker 1 kb ladder; 1a, the XKS1 gene cassette inserted near centromere 2; 1 b, the blank of reaction 1; 2a is the XKS1 gene cassette inserted near centromere 8; 2b is the blank of reaction 2; 3a is the cassette of the TAL1 and RKI1 genes inserted near centromere 12; 3b is the blank of reaction 3; 4a is the cassette of the TKL1 and RKI1 genes inserted near centromere 13; 4b is the blank of reaction 4; and 5b is the blank of reaction 5.
[0100] Dessa maneira, nas Figuras 3 e 4 vemos que os cassetes foram inseridos corretamente no local pretendido. Esse resultado é observado devido à amplificação de bandas com tamanho similar ao cassete construído.  Thus, in Figures 3 and 4 we see that the cassettes were inserted correctly in the intended location. This result is observed due to the amplification of bands of similar size to the built cassette.
Exemplo 6 - ANÁLISE DE CRESCIMENTO EM XILOSE COMO ÚNICA FONTE DE CARBONO DA LINHAGEM GENETICAMENTE MODIFICADA EM RELAÇÃO À LINHAGEM SELVAGEM Example 6 - XYLOSIS GROWTH ANALYSIS AS THE ONLY SOURCE OF GENETICALLY MODIFIED CARBON LINES TO WILD LINES
[0101] Foi realizada comparação em relação ao consumo de xilose da linhagem de Saccharomyces cerevisiae sem as modificações genéticas descritas na presente invenção ( A) e a linhagem que contém o cassete de expressão que compreende SEQ ID NO:2 (■), capaz de codificar proteína com função xilose isomerase compreendida em SEQ ID NO:1.  Comparison was made with respect to xylose consumption of the Saccharomyces cerevisiae strain without the genetic modifications described in the present invention (A) and the strain containing the expression cassette comprising SEQ ID NO: 2 (■) capable of encode protein with xylose isomerase function within SEQ ID NO: 1.
[0102] Para o inoculo da fermentação, as linhagens, tanto a que contém as modificações genéticas descritas na presente invenção, bem como as que não continham, foram crescidas em meio mínimo com glicose, sem uracila, por aproximadamente 16 horas, a 30QC, 200 rpm. As células foram lavadas duas vezes com água destilada estéril e utilizadas para inoculo em meio mínimo, sem uracila, contendo apenas xilose como fonte de carbono. O inoculo foi iniciado com uma OD de aproximadamente 1 ,0 e a fermentações foram conduzidas em erlenmeyer de 250 mL, com volume de trabalho de 100 mL e incubadas em shaker a 30QC, 200 rpm. Esporadicamente, alíquotas eram retiradas para observar o crescimento das células. For the inoculation of fermentation, strains, both containing the genetic modifications described in the present invention as well as those not containing them, were grown in minimal medium with glucose without uracil for approximately 16 hours at 30 ° C. C, 200 rpm. The cells were washed two times. times with sterile distilled water and used for inoculation in minimal medium without uracil, containing only xylose as carbon source. The inoculum was started with an OD of approximately 1, and 0 Fermentations were conducted in 250 mL Erlenmeyer flask with a volume of 100 ml work and incubated in a shaker at 30 Q C, 200 rpm. From time to time, aliquots were taken to observe cell growth.
[0103] Apenas a linhagem geneticamente modificada (■) com inserção de cassete de expressão que compreende SEQ ID NO:2, capaz de codificar proteína com função xilose isomerase que compreende SEQ ID NO:1 apresentou crescimento em meio contendo xilose, comprovando a importância da expressão funcional da xilose isomerase de Melampsora pinitorqua em S. cerevisiae para conversão de xilose em xilulose (Figuras 5 e 6). Após essa etapa, a molécula é fosforilada e segue pela fase oxidativa da pentose fosfato, sendo convertida posteriormente em frutose-6-fosfato e gliceraldeído-3-fosfato, intermediários da via glicolítica convencional da levedura, utilizada para a produção de etanol (Figura 7). Only the genetically modified (■) expression cassette-insert strain comprising SEQ ID NO: 2 capable of encoding xylose isomerase protein comprising SEQ ID NO: 1 showed growth in xylose-containing medium, proving the importance of the functional expression of xylose isomerase of Melampsora pinitorqua in S. cerevisiae for xylose to xylulose conversion (Figures 5 and 6). After this step, the molecule is phosphorylated and follows the oxidative phase of pentose phosphate and is later converted to fructose-6-phosphate and glyceraldehyde-3-phosphate, intermediates of the conventional yeast glycolytic pathway used for ethanol production (Figure 7 ).
LISTAGEM DE SEQUÊNCIAS LIST OF SEQUENCES
A Requerente informa que a "Listagem de Sequências" apresentada em formato impresso é idêntica àquela contida no formato de arquivo eletronico, exceto quanto à numeração de suas respectivas páginas. The Applicant informs that the "Sequence Listing" presented in print format is identical to that contained in the electronic file format, except for the numbering of their respective pages.
<110> BioCelere <110> BioCelere
<120> CASSETE DE EXPRESSÃO PARA A TRANSFORMAÇÃO DE CÉLULA EUCARIÓTICA, <120> EXPRESSION CASSETTE FOR EUCHARIOTIC CELL TRANSFORMATION,
PROCESSO PARA A TRANSFORMAÇÃO DE CÉLULA EUCARIÓTICA,  PROCESS FOR EUCHARIOTIC CELL TRANSFORMATION,
MICRO-ORGANISMO GENETICAMENTE MODIFICADO, PROCESSO DE PRODUÇÃO DE BIOCOMBUSTÍVEIS E/OU BIOQUÍMICOS E BIOCOMBUSTÍVEL E/OU  GENETICALLY MODIFIED MICRO-ORGANISM, BIOFUEL AND / OR BIOCHEMICAL AND / OR BIOFUEL PRODUCTION PROCESS
BIOQUÍMICO ASSIM PRODUZIDOS  BIOCHEMICAL SO PRODUCED
<130> BioCelere Patente <130> BioCelere Patent
<160> 18 <160> 18
<170> Patentln version 3.5 <170> Patentln version 3.5
<210> 1 <210> 1
<211> 442  <211> 442
<212> Peptideo  <212> Peptide
<213> Sequência Artificial  <213> Artificial Sequence
<220> <220>
<223> Sequência de peptideo com função de xilose isomerase <400> 1  <223> Xylose Isomerase Function Peptide Sequence <400> 1
Met Thr Val Ala Leu Lys Gln Arg Thr Tyr Tyr Ala Asn lie Pro Gln Met Thr Val Wing Read Lys Gln Arg Thr Tyr Tyr Wing Asn lie Pro Gln
1 5 10 15 1 5 10 15
lie Lys Phe Glu Gly Gln Glu Thr Asp Asn Pro Leu Ala Tyr Arg Trp lie Lys Phe Glu Gly Gln Glu Thr Asp Asn Pro Read Wing Tyr Arg Trp
20 25 30  20 25 30
Tyr Asp Glu Asn Arg Val Val Ala Gly Lys Thr Met Lys Asp His Leu 35 40 45 Tyr Asp Glu Asn Arg Val Val Gly Lys Wing Thr Met Lys Asp His Leu 35 40 45
Arg Leu Ala Cys Ala Tyr Trp His Ser Phe Thr Ala Asn Gly Ser Asp 50 55 60 Arg Leu Wing Cys Wing Tyr Trp His Ser Phe Thr Wing Asn Gly Ser Asp 50 55 60
Pro Phe Gly Ala Pro Thr His lie Phe Pro Trp Asp Asp Tyr Ser AspPro Phe Gly Ala Pro Thr His lie Phe Pro Trp Asp Asp Tyr Ser Asp
65 70 75 80 65 70 75 80
Pro lie Asp Lys Ala Tyr Ser Lys Ala Asp Ala Ala Phe Glu Phe Leu Pro lie Asp Lys Wing Tyr Ser Lys Wing Asp Wing Wing Phe Glu Phe Leu
85 90 95  85 90 95
Thr Lys Leu Gly Leu Pro Tyr Tyr Cys Phe His Asp Val Asp Ala Val Thr Lys Leu Gly Leu Pro Tyr Cyr Phe His Asp Val Asp Val Wing
100 105 110  100 105 110
Glu Tyr Thr Asp Asp lie His Glu Asn Glu Arg Arg Leu Gln Ala lie Glu Tyr Thr Asp Asp lie His Glu Asn Glu Arg Arg Leu Gln Ala lie
115 120 125  115 120 125
Thr Gly Tyr Leu Lys Gln Lys Gln Gln Glu Ser Gly Val Lys Leu Leu 130 135 140 Thr Gly Tyr Leu Lys Gln Lys Gln Gln Glu Ser Gly Val Lys Leu Leu 130 135 140
Trp Gly Thr Ala Asn Leu Phe Ser His Arg Arg Tyr Met Asn Gly Ala 145 150 155 160 Trp Gly Thr Wing Asn Leu Phe Be His Arg Arg Tyr Met Asn Gly Wing 145 150 155 160
Ser Thr Asn Pro Asp Phe His Val Leu Ala His Ala Gly Ala Gln Val Ser Thr Asn Pro Asp Phe His Val Leu Wing His Wing Gly Wing Gln Val
165 170 175  165 170 175
Lys Ala Ala Leu Asp Ala Thr lie Glu Leu Gly Gly Glu Asn Tyr Val Lys Wing Wing Leu Asp Wing Thr lie Glu Leu Gly Gly Glu Asn Tyr Val
180 185 190  180 185 190
Phe Trp Gly Gly Arg Glu Gly Tyr Met Ser Leu Leu Asn Thr Asn Met 195 200 205 Lys Arg Glu Lys Glu His Phe Ala Arg Phe Leu His Ala Ala Lys Asp 210 215 220 Phe Trp Gly Gly Arg Glu Gly Tyr Met Being Read Leu Asn Thr Asn Met 195 200 205 Lys Arg Glu Lys Glu His Phe Wing Arg Phe Read His Wing Wing Lys Asp 210 215 220
Tyr Ala Arg Lys Asn Gly Phe Arg Gly Thr Phe Phe lie Glu Pro Lys 225 230 235 240 Tyr Ala Arg Lys Asn Gly Phe Arg Gly Thr Phe Phe lie Glu Pro Lys 225 230 235 240
Pro Ala Glu Pro Ser Lys His Gln Tyr Asp Tyr Asp Cys Glu Thr Val Pro Glu Wing Pro Ser Lys His Gln Tyr Asp Tyr Asp Cys Glu Thr Val
245 250 255  245 250 255
lie Gly Phe Leu Arg Gln Tyr Asp Leu Leu Asn Asp Phe Lys lie Asn lie Gly Phe Leu Arg Gln Tyr Asp Leu Leu Asn Asp Phe Lys lie Asn
260 265 270  260 265 270
lie Glu Val Asn His Ala Thr Leu Ala Gly His Thr Phe Ser His Glulie Glu Val Asn His Wing Thr Read Leu Wing Gly His Thr Phe Be His Glu
275 280 285 275 280 285
Leu Gln Val Ala Ala Asp Ala Gly Val Leu Gly Ser Met Asp Ala Asn 290 295 300 Leu Gln Val Wing Wing Asp Wing Gly Val Wing Leu Gly Wing Met Asp Wing Wing 290 295 300
Arg Gly Asp Tyr Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro Asn Asn 305 310 315 320 Arg Gly Asp Tyr Gln Asn Gly Trp Asp Thr Asp Gln Phe Pro Asn 305 310 315 320
lie Asn Glu Leu Thr Glu Ala Met Leu Val lie Leu Glu Ala Gly Gly lie Asn Glu Leu Thr Glu Ala Met Leu Val lie Leu Glu Ala Gly Gly
325 330 335  325 330 335
Phe Gln Gly Gly Gly lie Asn Phe Asp Ala Lys lie Arg Arg Asn Ser Phe Gln Gly Gly Gly lie Asn Phe Asp Wing Lys lie Arg Arg Asn Ser
340 345 350  340 345 350
Thr Asp Pro Glu Asp Leu Phe His Ala His lie Gly Gly lie Asp Ser Thr Asp Pro Glu Asp Leu Phe His Wing His Lie Gly Gly lie Asp Ser
355 360 365 Phe Ala Arg Ala Leu Ile Val Ala Asp Asn Val Leu Gln Lys Ser Glu 355 360 365 Phe Wing Arg Wing Leu Ile Val Wing Asp Asn Val Leu Gln Lys Ser Glu
370 375 380  370 375 380
Tyr Arg Lys Phe Arg Lys Glu Arg Tyr Ser Ser Phe Asp Ser Gly Ala Tyr Arg Lys Phe Arg Lys Glu Arg Tyr Be Ser Phe Asp Ser Gly Ala
385 390 395 400 385 390 395 400
Gly Arg Asp Phe Glu Gln Gly Lys Leu Ser Leu Glu Asp Leu Arg Gln Gly Arg Asp Phe Glu Gln Gly Lys Reads Being Read Glu Asp Reads Arg Gln
405 410 415  405 410 415
Tyr Ala Met Glu Asn Gly Glu Pro Glu Val Arg Ser Gly Lys Gln Glu Tyr Ala Met Glu Asn Gly Glu Pro Glu Val Arg Be Gly Lys Gln Glu
420 425 430  420 425 430
Trp Leu Glu Asn Ile Ile Asn Arg Tyr Ile Trp Leu Glu Asn Ile Ile Asn Arg Tyr Ile
435 440  435 440
<210> 2 <210> 2
<211> 1329 <211> 1329
<212> DNA <212> DNA
<213> Sequência Artificial <220>  <213> Artificial Sequence <220>
<223> Sequência codificadora de peptideo com função de xilose isomerase <400> 2  <223> Xylose isomerase function peptide coding sequence <400> 2
atgactgttg cgcttaagca acgtacctac tacgcgaaca tacctcagat caagttcgaa 60 ggtcaagaga ctgataaccc attggcatac agatggtatg acgaaaacag agtcgtcgca 120 ggcaagacaa tgaaggatca tttaagatta gcttgtgctt actggcattc ctttacagcc 180 aatggctctg atccattcgg agccccaact cacatattcc cttgggatga ttactcagac 240 cctattgata aagcatactc taaagctgat gctgcctttg aattcttgac taaactaggt 300 ttgccatact attgcttcca tgacgtagat gcagtggagt acacagacga tattcacgaa 360 aacgaacgta gactacaggc aattactggc tacttaaagc aaaagcaaca agagtcaggc 420 gtgaagttgt tgtggggaac agctaacctg ttttctcaca gaagatacat gaacggagct 480 agtacaaatc cagattttca cgtgctagca catgccgggg cacaagtaaa ggctgctctt 540 gatgcaacaa tcgagttggg tggagaaaac tacgttttct ggggtggaag agaaggttac 600 atgtcactgc ttaacaccaa tatgaaaaga gagaaagagc actttgcgag attcttacac 660 gctgcaaagg attatgccag aaaaaacggc ttcagaggta ctttcttcat cgaaccaaag 720 ccagcagaac catcaaagca tcagtacgat tatgattgtg aaacagttat agggttcttg 780 agacagtacg atctcttgaa cgacttcaag atcaatatag aggttaatca tgccacttta 840 gctggtcata ccttttccca tgaattacaa gttgccgctg acgccggtgt cttaggtagt 900 atggatgcaa atagaggaga ttaccaaaac ggttgggata ccgaccagtt cccaaacaac 960 attaacgagc tgacagaagc aatgcttgtc attttggaag cgggtggctt tcaaggtggc 1020 gggatcaatt tcgatgctaa aatcagacgt aattctacag atcctgaaga tcttttccat 1080 gcacatattg gtggtattga ttctttcgct agagctttga ttgttgcaga caacgtattg 1140 caaaaatctg aatacagaaa gtttagaaag gagagatact cctcatttga cagcggtgct 1200 gggcgtgact ttgaacaagg aaagttatca ctagaagatc taagacaata cgccatggaa 1260 aatggtgaac ctgaagttag atctggtaaa caagagtggc tcgaaaacat tatcaacaga 1320 tacatctga 1329 atgactgttg cgcttaagca acgtacctac tacgcgaaca tacctcagat caagttcgaa 60 ggtcaagaga ctgataaccc attggcatac agatggtatg acgaaaacag agtcgtcgca 120 ggcaagacaa tgaaggatca tttaagatta gcttgtgctt actggcattc ctttacagcc 180 aatggctctg atccattcgg agccccaact cacatattcc cttgggatga ttactcagac 240 cctattgata aagcatactc taaagctgat gctgcctttg aattcttgac taaactaggt 300 ttgccatact attgcttcca tgacgtagat gcagtggagt acacagacga 360 tattcacgaa aacgaacgta gactacaggc aattactggc tacttaaagc aaaagcaaca agagtcaggc 420 gtgaagttgt tgtggggaac agctaacctg ttttctcaca gaagatacat gaacggagct 480 agtacaaatc cagattttca cgtgctagca catgccgggg cacaagtaaa ggctgctctt 540 gatgcaacaa tcgagttggg tggagaaaac tacgttttct ggggtggaag agaaggttac 600 atgtcactgc ttaacaccaa tatgaaaaga gagaaagagc actttgcgag attcttacac 660 gctgcaaagg attatgccag aaaaaacggc ttcagaggta ctttcttcat cgaaccaaag 720 ccagcagaac catcaaagca tcagtacgat tatgattgtg aaacagttat agggttcttg 780 agacagtacg atctcttgaa atcaatatag aggttaatca tgccacttta cgacttcaag 840 gctggtcata ccttttccca tgaattacaa gttgccgctg acgccggtgt cttaggtagt 900 atggatgcaa atagaggaga ttaccaaaac ggttgggata ccgaccagtt cccaaacaac 960 attaacgagc tgacagaagc aatgcttgtc attttggaag cgggtggctt tcaaggtggc 1020 gggatcaatt tcgatgctaa aatcagacgt aattctacag atcctgaaga tcttttccat 1080 gcacatattg gtggtattga ttctttcgct agagctttga ttgttgcaga caacgtattg 1140 caaaaatctg aatacagaaa gtttagaaag gagagatact cctcatttga cagcggtgct 1200 gggcgtgact ttgaa caagg aaagttatca ctagaagatc taagacaata cgccatggaa 1260 aatggtgaac ctgaagttag atctggtaaa caagagtggc tcgaaaacat tatcaacaga 1320 tacatctga 1329
<210> 3 <210> 3
<211> 305  <211> 305
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220> <213> Nucleotide Sequence <220>
<223> Terminador Gliceraldeido 3-Fosfato Desidrogenase, isoenzima 1  <223> Glyceraldehyde 3-Phosphate Dehydrogenase Terminator, Isoenzyme 1
<400> 3 <400> 3
ataaagcaat cttgatgagg ataatgattt ttttttgaat atacataaat actaccgttt 60 ttctgctaga ttttgtaaag acgtaaataa gtacatatta ctttttaagc caagacaaga 120 ttaagcatta actttaccct tttctcttct aagttttaac actagttatc actgttaaaa 180 aattatggcg agaacgtcgg cggttaaaat atattaccct gaatgtggtg aattgaagtt 240 cttggatggt ttaaagattt ttcctttttg ggaaataagt aaacaatata ttgctgcctt 300 tgcaa 305 ataaagcaat cttgatgagg ataatgattt ttttttgaat atacataaat actaccgttt 60 ttctgctaga ttttgtaaag acgtaaataa gtacatatta ctttttaagc caagacaaga 120 ttaagcatta actttaccct tttctcttct aagttttaac actagttatc actgttaaaa 180 aattatggcg agaacgtcgg cggttaaaat atattaccct gaatgtggtg aattgaagtt 240 cttggatggt ttaaagattt ttcctttttg ggaaataagt aaacaatata ttgctgcctt 300 305 tgcaa
<210> 4 <210> 4
<211> 1349  <211> 1349
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Sequência codificadora dos genes URA3 e loxp  <223> URA3 and loxp coding sequence
<400> 4 <400> 4
cactataggg cgaattgggc ccgacgtcgc atgctcccgg ccgccatggc ggccgcggga 60 attcgatata acttcgtata gcatacatta tacgaagtta tggtccataa agcttttcaa 120 ttcatctttt ttttttttgt tctttttttt gattccggtt tctttgaaat ttttttgatt 180 cggtaatctc cgagcagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata 240 tacgcatatg tggtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca 300 gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg 360 ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa 420 caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag 480 cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca 540 tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg 600 aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat 660 acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg 720 ttagcggttt gaagcaggcg gcggaagaag taacaaagga acctagaggc cttttgatgt 780 tagcagaatt gtcatgcaag ggctccctag ctactggaga atatactaag ggtactgttg 840 acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg 900 gaagagatga aggttacgat tggttgatta tgacacccgg tgtgggttta gatgacaagg 960 gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca 1020 ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac 1080 gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa 1140 ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat 1200 atcagttatt acccgggaat ctcggtcgta atgatttcta taacttcgta tagcatacat 1260 tatacgaagt tatatcacta gtgaattcgc ggccgcctgc aggtcgacca tatgggagag 1320 ctcccaacgc gttggatgca tagcttgag 1349 cactataggg cgaattgggc ccgacgtcgc atgctcccgg ccgccatggc ggccgcggga 60 attcgatata acttcgtata gcatacatta tacgaagtta tggtccataa agcttttcaa 120 ttcatctttt ttttttttgt tctttttttt gattccggtt tctttgaaat ttttttgatt 180 cggtaatctc cgagcagaag gaagaacgaa ggaaggagca cagacttaga ttggtatata 240 tacgcatatg tggtgttgaa gaaacatgaa attgcccagt attcttaacc caactgcaca 300 gaacaaaaac ctgcaggaaa cgaagataaa tcatgtcgaa agctacatat aaggaacgtg 360 ctgctactca tcctagtcct gttgctgcca agctatttaa tatcatgcac gaaaagcaaa 420 caaacttgtg tgcttcattg gatgttcgta ccaccaagga attactggag ttagttgaag 480 cattaggtcc caaaatttgt ttactaaaaa cacatgtgga tatcttgact gatttttcca 540 tggagggcac agttaagccg ctaaaggcat tatccgccaa gtacaatttt ttactcttcg 600 aagacagaaa atttgctgac attggtaata cagtcaaatt gcagtactct gcgggtgtat 660 acagaatagc agaatgggca gacattacga atgcacacgg tgtggtgggc ccaggtattg 720 ttagcggttt gaagcaggcg gcggaagaag taacaaagga acctagaggc cttttgatgt 780 tagcagaatt gtcatgcaag ggctccctag ctactggaga atatactaag ggtactgttg 840 acattgcgaa gagcgacaaa gattttgtta tcggctttat tgctcaaaga gacatgggtg 900 gaagagatga aggttacgat tgacacccgg tgtgggttta gatgacaagg tggttgatta 960 gagacgcatt gggtcaacag tatagaaccg tggatgatgt ggtctctaca ggatctgaca 1020 ttattattgt tggaagagga ctatttgcaa agggaaggga tgctaaggta gagggtgaac 1080 gttacagaaa agcaggctgg gaagcatatt tgagaagatg cggccagcaa aactaaaaaa 1140 ctgtattata agtaaatgca tgtatactaa actcacaaat tagagcttca atttaattat 1200 atcagttatt acccgggaat ctcggtcgta atgatttcta taacttcgta tagcatacat 1260 tatacgaagt tatatcacta gtgaattcgc ggccgcctgc aggtcgacca tatgggagag 1320 ctcccaacgc gtt ggatgca tagcttgag 1349
<210> 5 <210> 5
<211> 1008  <211> 1008
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Transaldolase ( TAL1 )  <223> Transaldolase (TAL1)
<400> 5 atgtctgaac cagctcaaaa gaaacaaaag gttgctaaca actctctaga acaattgaaa 60 gcctccggca ctgtcgttgt tgccgacact ggtgatttcg gctctattgc caagtttcaa 120 cctcaagact ccacaactaa cccatcattg atcttggctg ctgccaagca accaacttac 180 gccaagttga tcgatgttgc cgtggaatac ggtaagaagc atggtaagac caccgaagag 240 caagtcgaaa atgctgtgga cagattgtta gtcgaattcg gtaaggagat cttaaagatt 300 gttccaggca gagtctccac cgaagttgat gctagattgt cttttgacac tcaagctacc 360 attgaaaagg ctagacatat cattaaattg tttgaacaag aaggtgtctc caaggaaaga 420 gtccttatta aaattgcttc cacttgggaa ggtattcaag ctgccaaaga attggaagaa 480 aaggacggta tccactgtaa tttgactcta ttattctcct tcgttcaagc agttgcctgt 540 gccgaggccc aagttacttt gatttcccca tttgttggta gaattctaga ctggtacaaa 600 tccagcactg gtaaagatta caagggtgaa gccgacccag gtgttatttc cgtcaagaaa 660 atctacaact actacaagaa gtacggttac aagactattg ttatgggtgc ttctttcaga 720 agcactgacg aaatcaaaaa cttggctggt gttgactatc taacaatttc tccagcttta 780 ttggacaagt tgatgaacag tactgaacct ttcccaagag ttttggaccc tgtctccgct 840 aagaaggaag ccggcgacaa gatttcttac atcagcgacg aatctaaatt cagattcgac 900 ttgaatgaag acgctatggc cactgaaaaa ttgtccgaag gtatcagaaa attctctgcc 960 gatattgtta ctctattcga cttgattgaa aagaaagtta ccgcttaa 1008 <400> 5 atgtctgaac cagctcaaaa gaaacaaaag gttgctaaca actctctaga acaattgaaa 60 gcctccggca ctgtcgttgt tgccgacact ggtgatttcg gctctattgc caagtttcaa 120 cctcaagact ccacaactaa cccatcattg atcttggctg ctgccaagca accaacttac 180 gccaagttga tcgatgttgc cgtggaatac ggtaagaagc atggtaagac caccgaagag 240 caagtcgaaa atgctgtgga cagattgtta gtcgaattcg gtaaggagat cttaaagatt 300 gttccaggca gagtctccac cgaagttgat gctagattgt cttttgacac tcaagctacc 360 attgaaaagg ctagacatat cattaaattg tttgaacaag aaggtgtctc caaggaaaga 420 gtccttatta aaattgcttc cacttgggaa ggtattcaag ctgccaaaga attggaagaa 480 aaggacggta tccactgtaa tttgactcta ttattctcct tcgttcaagc agttgcctgt 540 gccgaggccc aagttacttt gatttcccca tttgttggta gaattctaga ctggtacaaa 600 tccagcactg gtaaagatta caagggtgaa gccgacccag gtgttatttc cgtcaagaaa 660 atctacaact actacaagaa gtacggttac aagactattg ttatgggtgc ttctttcaga 720 agcactgacg aaatcaaaaa cttggctggt gttgactatc taacaatttc tccagcttta 780 ttggacaagt tgatgaacag tactgaacct ttcccaagag ttttggaccc tgtctccgct 840 aagaaggaag ccggcgacaa gatttcttac atcagcgacg aatctaaatt cagattcgac 900 ttgaatgaag acgctatggc cactgaaaaa ttgtccgaag gtatcagaaa attctctgcc 960 gatattgtta ctctattcga cttgattgaa aagaaagta ccgcta
<210> 6 <210> 6
<211> 873  <211> 873
<212> DNA  <212> DNA
<213> Sequência Nucleotidica  <213> Nucleotide Sequence
<220> <223> Promotor 3-Fosfato Quinase (PGK1) <220> <223> Promoter 3-Phosphate Kinase (PGK1)
<400> 6 <400> 6
tactgtaatt gcttttagtt gtgtattttt agtgtgcaag tttctgtaaa tcgattaatt 60 tttttttctt tcctcttttt attaacctta atttttattt tagattcctg acttcaactc 120 aagacgcaca gatattataa catctgcata ataggcattt gcaagaatta ctcgtgagta 180 aggaaagagt gaggaactat cgcatacctg catttaaaga tgccgatttg ggcgcgaatc 240 ctttattttg gcttcaccct catactatta tcagggccag aaaaaggaag tgtttccctc 300 cttcttgaat tgatgttacc ctcataaagc acgtggcctc ttatcgagaa agaaattacc 360 gtcgctcgtg atttgtttgc aaaaagaaca aaactgaaaa aacccagaca cgctcgactt 420 cctgtcttcc tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc 480 tcacaggttt tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca 540 catgctatga tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct 600 ctctctttca aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc 660 ttttcttcta accaaggggg tggtttagtt tagtagaacc tcgtgaaact tacatttaca 720 tatatataaa cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt 780 cttagttttt caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag 840 taattatcta ctttttacaa caaatataaa aca 873 tactgtaatt gcttttagtt gtgtattttt agtgtgcaag tttctgtaaa tcgattaatt 60 tttttttctt tcctcttttt attaacctta atttttattt tagattcctg acttcaactc 120 aagacgcaca gatattataa catctgcata ataggcattt gcaagaatta ctcgtgagta 180 aggaaagagt gaggaactat cgcatacctg catttaaaga tgccgatttg ggcgcgaatc 240 ctttattttg gcttcaccct catactatta tcagggccag aaaaaggaag tgtttccctc 300 cttcttgaat tgatgttacc ctcataaagc acgtggcctc ttatcgagaa agaaattacc 360 gtcgctcgtg atttgtttgc aaaaagaaca aaactgaaaa aacccagaca cgctcgactt 420 cctgtcttcc tattgattgc agcttccaat ttcgtcacac aacaaggtcc tagcgacggc 480 tcacaggttt tgtaacaagc aatcgaaggt tctggaatgg cgggaaaggg tttagtacca 540 catgctatga tgcccactgt gatctccaga gcaaagttcg ttcgatcgta ctgttactct 600 ctctctttca aacagaattg tccgaatcgt gtgacaacaa cagcctgttc tcacacactc 660 ttttcttcta accaaggggg tagtagaacc tcgtgaaact tacatttaca tggtttagtt 720 tatatataaa cttgcataaa ttggtcaatg caagaaatac atatttggtc ttttctaatt 780 cttagttttt caagttctta gatgctttct ttttctcttt tttacagatc atcaaggaag 840 taattatcta ctttttacaa caaatataaa aca 873
<210> 7 <210> 7
<211> 777  <211> 777
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Ribose 5-Fosfato Isomerase (RKI1) <400> 7 <223> Ribose 5-Phosphate Isomerase (RKI1) <400> 7
atggctgccg gtgtcccaaa aattgatgcg ttagaatctt tgggtaatcc tttggaggat 60 gccaagagag ctgcagcata cagagcagtt gatgaaaatt taaaatttga tgatcacaaa 120 ataattggaa ttggtagtgg tagcacagtg gtttatgttg ccgaaagaat tggacaatat 180 ttgcatgacc ctaaatttta tgaagtagcg tctaaattca tttgcattcc aacaggattc 240 caatcaagaa acttgatttt ggataacaag ttgcaattag gctccattga acagtatcct 300 cgcattgata tagcgtttga cggtgctgat gaagtggatg agaatttaca attgattaaa 360 ggtggtggtg cttgtctatt tcaagaaaaa ttggttagta ctagtgctaa aaccttcatt 420 gtcgttgctg attcaagaaa aaagtcacca aaacatttag gtaagaactg gaggcaaggt 480 gttcccattg aaattgtacc ttcctcatac gtgagggtca agaatgatct attagaacaa 540 ttgcatgctg aaaaagttga catcagacaa ggaggttctg ctaaagcagg tcctgttgta 600 actgacaata ataacttcat tatcgatgcg gatttcggtg aaatttccga tccaagaaaa 660 ttgcatagag aaatcaaact gttagtgggc gtggtggaaa caggtttatt catcgacaac 720 gcttcaaaag cctacttcgg taattctgac ggtagtgttg aagttacgga aaagtga 777 atggctgccg gtgtcccaaa aattgatgcg ttagaatctt tgggtaatcc tttggaggat 60 gccaagagag ctgcagcata cagagcagtt gatgaaaatt taaaatttga tgatcacaaa 120 ataattggaa ttggtagtgg tagcacagtg gtttatgttg ccgaaagaat tggacaatat 180 ttgcatgacc ctaaatttta tgaagtagcg tctaaattca tttgcattcc aacaggattc 240 caatcaagaa acttgatttt ggataacaag ttgcaattag gctccattga acagtatcct 300 cgcattgata tagcgtttga cggtgctgat gaagtggatg agaatttaca attgattaaa 360 ggtggtggtg cttgtctatt tcaagaaaaa ttggttagta ctagtgctaa aaccttcatt 420 gtcgttgctg attcaagaaa aaagtcacca aaacatttag gtaagaactg gaggcaaggt 480 gttcccattg aaattgtacc ttcctcatac gtgagggtca agaatgatct attagaacaa 540 ttgcatgctg aaaaagttga catcagacaa ggaggttctg ctaaagcagg tcctgttgta 600 actgacaata ataacttcat tatcgatgcg gatttcggtg aaatttccga tccaagaaaa 660 ttgcatagag aaatcaaact gttagtgggc gtggtggaaa caggtttatt catcgacaac 720 gcttcaaaag cctacttcgg taattctgac ggtagtgttg aagttacgga aaagtga 777
<210> 8 <210> 8
<211> 702 <211> 702
<212> DNA <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Promotor da enzima Álcool Desidrogenase 1 (ADH1) <400> 8  <223> Alcohol Dehydrogenase 1 (ADH1) enzyme promoter <400> 8
ttccgggtgt acaatatgga cttcctcttt tctggcaacc aaacccatac atcgggattc 60 ctataatacc ttcgttggtc tccctaacat gtaggtggcg gaggggagat atacaataga 120 acagatacca gacaagacat aatgggctaa acaagactac accatttaca ctgcctcatt 180 gatggtggta cataacgaac taatactgta gccctagact tgatagccat catcatatcg 240 aagtttcact accctttttc catttgccat ctattgaagt aataataggc gcatgcaact 300 tcttttcttt tttttttttt ctctctcccc cgttgttgtc tcaccatatc cgcaatgaca 360 aaaaaatgat ggaagacact aaaggaaaaa attaacgaca aagacagcac caacagatgt 420 cgttgttcca gagctgatga ggggtatctc gaagcacacg aaactttttc cttccttcat 480 tcacgcacac tactctctaa tgagcaacga tatacggcct tccttccagt tacttgaatt 540 tgaaataaaa aaagtttgct gtcttgctat caagtataaa tagacctgca attattaatc 600 ttttgtgttc tcgtcattgt tctcgttccc tttcttcctt gtttcttttt ctgcacaata 660 tttcaagcta taccaagcat acaatcaact atctcatata ca 702 ttccgggtgt acaatatgga cttcctcttt tctggcaacc aaacccatac atcgggattc 60 ctataatacc ttcgttggtc tccctaacat gtaggtggcg gaggggagat atacaataga 120 acagatacca gacaagacat aatgggctaa acaagactac accatttaca ctgcctcatt 180 gatggtggta cataacgaac taatactgta gccctagact tgatagccat catcatatcg 240 aagtttcact accctttttc catttgccat ctattgaagt aataataggc gcatgcaact 300 tcttttcttt tttttttttt ctctctcccc cgttgttgtc tcaccatatc cgcaatgaca 360 aaaaaatgat ggaagacact aaaggaaaaa attaacgaca aagacagcac caacagatgt 420 cgttgttcca gagctgatga ggggtatctc gaagcacacg aaactttttc cttccttcat 480 tcacgcacac tactctctaa tgagcaacga tatacggcct tccttccagt tacttgaatt 540 tgaaataaaa aaagtttgct gtcttgctat caagtataaa tagacctgca attattaatc 600 ttttgtgttc tcgtcattgt tctcgttccc tttcttcctt gtttcttttt ctgcacaata 660 tttcaagcta taccaagcat acaatcaact atctcat 70
<210> 9 <210> 9
<211> 1803 <211> 1803
<212> DNA <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Xiluloquinase (XKS1) <400> 9  <223> Xylulokinase (XKS1) <400> 9
atgttgtgtt cagtaattca gagacagaca agagaggttt ccaacacaat gtctttagac 60 tcatactatc ttgggtttga tctttcgacc caacaactga aatgtctcgc cattaaccag 120 gacctaaaaa ttgtccattc agaaacagtg gaatttgaaa aggatcttcc gcattataac 180 acaaagaagg gtgtctatat acacggcgac actatcgaat gtcccgtagc catgtggtta 240 gaggctctag atctggttct ctcgaaatat cgcgaggcta aatttccatt gaacaaagtt 300 atggccgtct cagggtcctg ccagcagcac gggtctgtct actggtcctc ccaagccgaa 360 tctctgttag agcaattgaa taagaaaccg gaaaaagatt tattgcacta cgtgagctct 420 gtagcatttg caaggcaaac cgcccccaat tggcaagacc acagtactgc aaagcaatgt 480 caagagtttg aagagtgcat aggtgggcct gaaaaaatgg ctcaattaac agggtccaga 540 gcccatttta gatttactgg tcctcaaatt ctgaaaattg cacaattaga accagaagct 600 tacgaaaaaa caaagaccat ttctttagtg tctaattttt tgacttctat cttagtgggc 660 catcttgttg aattagagga ggcagatgcc tgtggtatga acctttatga tatacgtgaa 720 agaaaattca gtgatgagct gctacatcta attgatagtt cttctaagga taaaactatc 780 agacaaaaat taatgagagc acccatgaaa aatttgatag cgggtaccat ctgtaaatat 840 tttattgaga agtacggttt caatacaaac tgcaaggtct ctcccatgac tggggataat 900 ttagccacta tatgttcttt acccctgcgg aagaatgacg ttctcgtttc cctaggaaca 960 agtactacag ttcttctggt caccgataag tatcacccct ctccgaacta tcatcttttc 1020 attcatccaa ctctgccaaa ccattatatg ggtatgattt gttattgtaa tggttctttg 1080 gcaagggaga ggataagaga cgagttaaac aaagaacggg aaaataatta tgagaagact 1140 aacgattgga ctctttttaa tcaagctgtg ctagatgact cagaaagtag tgaaaatgaa 1200 ttaggtgtat attttcctct gggggagatc gttcctagcg taaaagccat aaacaaaagg 1260 gttatcttca atccaaaaag gggtatgatt gaaagagagg tggccaagtt caaagacaag 1320 aggcacgatg ccaaaaatat tgtagaatca caggctttaa gttgcagggt aagaatatct 1380 ccactgcttt cggattcaaa cgcaagctca caacagagac tgaacgaaga tacaatcgtg 1440 aagtttgatt acgatgaatc tccgctgcgg gactacctaa ataaaaggcc agaaaggact 1500 ttttttgtag gtggggcttc taaaaacgat gctattgtga agaagtttgc tcaagtcatt 1560 ggtgctacaa agggtaattt taggctagaa acaccaaact catgtgccct tggtggttgt 1620 tataaggcca tgtggtcatt gttatacgac tctaataaaa ttgcagttcc ttttgataaa 1680 tttctgaatg acaattttcc atggcatgta atggaaagca tatccgatgt ggataatgaa 1740 aattgggatc gctataattc caaaattgtc cccttaagcg aactggaaaa gactctcatc 1800 taa 1803 atgttgtgtt cagtaattca gagacagaca agagaggttt ccaacacaat gtctttagac 60 tcatactatc ttgggtttga tctttcgacc caacaactga aatgtctcgc cattaaccag 120 gacctaaaaa ttgtccattc agaaacagtg gaatttgaaa aggatcttcc gcattataac 180 acaaagaagg gtgtctatat acacggcgac actatcgaat gtcccgtagc catgtggtta 240 gaggctctag atctggttct ctcgaaatat cgcgaggcta aatttccatt 300 gaacaaagtt atggccgtct cagggtcctg ccagcagcac gggtctgtct actggtcctc ccaagccgaa 360 tctctgttag agcaattgaa taagaaaccg gaaaaagatt tattgcacta cgtgagctct 420 gtagcatttg caaggcaaac cgcccccaat tggcaagacc acagtactgc aaagcaatgt 480 caagagtttg aagagtgcat aggtgggcct gaaaaaatgg ctcaattaac agggtccaga 540 gcccatttta gatttactgg tcctcaaatt ctgaaaattg cacaattaga accagaagct 600 tacgaaaaaa caaagaccat ttctttagtg tctaattttt tgacttctat cttagtgggc 660 catcttgttg aattagagga ggcagatgcc tgtggtatga acctttatga tatacgtgaa 720 agaaaattca gtgatgagct gctacatcta attgatagtt cttctaagga taaaactatc 780 agacaaaaat taatgagagc acccatgaaa aatttgatag cgggtaccat ctgtaaatat 840 tttattgaga agtacggttt caatacaaac tgcaaggtct ctcccatgac tggggataat 900 ttagccacta tatgttcttt acccctgcgg aagaatgacg ttctcgtttc cctaggaaca 960 agtactacag ttcttctggt caccgataag tatcacccct ctccgaacta tcatcttttc 1020 attcatccaa ctctgccaaa ccattatatg ggtatgattt gttattgtaa tggttctttg 1080 gcaagggaga ggataagaga cgagttaaac aaagaacggg aaaataatta tgagaagact 1140 aacgattgga ctcttt ttaa tcaagctgtg ctagatgact cagaaagtag tgaaaatgaa 1200 ttaggtgtat attttcctct gggggagatc gttcctagcg taaaagccat aaacaaaagg 1260 gttatcttca atccaaaaag gggtatgatt gaaagagagg tggccaagtt caaagacaag 1320 aggcacgatg ccaaaaatat tgtagaatca caggctttaa gttgcagggt aagaatatct 1380 ccactgcttt cggattcaaa cgcaagctca caacagagac tgaacgaaga tacaatcgtg 1440 aagtttgatt acgatgaatc tccgctgcgg gactacctaa ataaaaggcc agaaaggact 1500 ttttttgtag gtggggcttc taaaaacgat gctattgtga agaagtttgc 1560 tcaagtcatt ggtgctacaa agggtaattt taggctagaa acaccaaact catgtgccct tggtggttgt 1620 tataaggcca tgtggtcatt gttatacgac tctaataaaa ttgcagttcc ttttgataaa 1680 tttctgaatg acaattttcc atggcatgta atggaaagca tatccgatgt ggataatgaa 1740 aattgggatc gctataattc caaaattgtc cccttaagcg aactggaaaa gactctcatc 1800 cup 1803
<210> 10 <210> 10
<211> 236  <211> 236
<212> DNA  <212> DNA
<213> Sequência Nucleotidica  <213> Nucleotide Sequence
Terminador de Álcool Desidrogenase (ADH1) Alcohol Dehydrogenase Terminator (ADH1)
<400> 10 <400> 10
gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120 ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacatc 180 tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca attgta 236 gcgaatttct tatgatttat gatttttatt attaaataag ttataaaaaa aataagtgta 60 tacaaatttt aaagtgactc ttaggtttta aaacgaaaat tcttattctt gagtaactct 120 ttcctgtagg tcaggttgct ttctcaggta tagcatgagg tcgctcttat tgaccacatc 180 tctaccggca tgccgagcaa atgcctgcaa atcgctcccc atttcaccca 236 attgta
<210> 11 <210> 11
<211> 2043  <211> 2043
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Transcetolase (TKL1)  <223> Transcetolase (TKL1)
<400> 11 <400> 11
atgactcaat ttactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60 gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120 gcgcacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180 gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240 actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300 ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360 ggtatctcca acgctgttgg tatggccatg gctcaagcta accttgctgc cacttacaac 420 aagccaggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480 caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540 attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600 gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660 aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720 ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780 tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840 ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900 attttaaagc caggtgtcga agccaacaac aagtggaaca agttgttcag cgaataccaa 960 aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020 aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080 ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140 gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200 tcttccggtt caggtaacta ctctggtaga tacatcagat acggtattag agaacacgct 1260 atgggtgcca tcatgaacgg tatttcagct ttcggtgcca actacaaacc atacggtggt 1320 actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380 cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440 catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500 ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560 ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620 agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680 gctactggtt ccgaagtatc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740 atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800 tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860 acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920 aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980 caaaagacca ttgcattcta taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040 taa 2043 atgactcaat ttactgacat tgataagcta gccgtctcca ccataagaat tttggctgtg 60 gacaccgtat ccaaggccaa ctcaggtcac ccaggtgctc cattgggtat ggcaccagct 120 gcgcacgttc tatggagtca aatgcgcatg aacccaacca acccagactg gatcaacaga 180 gatagatttg tcttgtctaa cggtcacgcg gtcgctttgt tgtattctat gctacatttg 240 actggttacg atctgtctat tgaagacttg aaacagttca gacagttggg ttccagaaca 300 ccaggtcatc ctgaatttga gttgccaggt gttgaagtta ctaccggtcc attaggtcaa 360 ggtatctcca acgctgttgg tatggccatg gctcaagcta accttgctgc cacttacaac 420 aagccaggct ttaccttgtc tgacaactac acctatgttt tcttgggtga cggttgtttg 480 caagaaggta tttcttcaga agcttcctcc ttggctggtc atttgaaatt gggtaacttg 540 attgccatct acgatgacaa caagatcact atcgatggtg ctaccagtat ctcattcgat 600 gaagatgttg ctaagagata cgaagcctac ggttgggaag ttttgtacgt agaaaatggt 660 aacgaagatc tagccggtat tgccaaggct attgctcaag ctaagttatc caaggacaaa 720 ccaactttga tcaaaatgac cacaaccatt ggttacggtt ccttgcatgc cggctctcac 780 tctgtgcacg gtgccccatt gaaagcagat gatgttaaac aactaaagag caaattcggt 840 ttcaacccag acaagtcctt tgttgttcca caagaagttt acgaccacta ccaaaagaca 900 attttaaagc caggtgtcg agccaacaac the aagtggaaca agttgttcag cgaataccaa 960 aagaaattcc cagaattagg tgctgaattg gctagaagat tgagcggcca actacccgca 1020 aattgggaat ctaagttgcc aacttacacc gccaaggact ctgccgtggc cactagaaaa 1080 ttatcagaaa ctgttcttga ggatgtttac aatcaattgc cagagttgat tggtggttct 1140 gccgatttaa caccttctaa cttgaccaga tggaaggaag cccttgactt ccaacctcct 1200 tcttccggtt caggtaacta ctctggtaga tacatcagat acggtattag agaacacgct 1260 atgggtgcca tcatgaacgg tatttcagct ttcggtgcca actacaaacc 1320 atacggtggt actttcttga acttcgtttc ttatgctgct ggtgccgtta gattgtccgc tttgtctggc 1380 cacccagtta tttgggttgc tacacatgac tctatcggtg tcggtgaaga tggtccaaca 1440 catcaaccta ttgaaacttt agcacacttc agatccctac caaacattca agtttggaga 1500 ccagctgatg gtaacgaagt ttctgccgcc tacaagaact ctttagaatc caagcatact 1560 ccaagtatca ttgctttgtc cagacaaaac ttgccacaat tggaaggtag ctctattgaa 1620 agcgcttcta agggtggtta cgtactacaa gatgttgcta acccagatat tattttagtg 1680 gctactggtt ccgaagtatc tttgagtgtt gaagctgcta agactttggc cgcaaagaac 1740 atcaaggctc gtgttgtttc tctaccagat ttcttcactt ttgacaaaca acccctagaa 1800 tacagactat cagtcttacc agacaacgtt ccaatcatgt ctgttgaagt tttggctacc 1860 acatgttggg gcaaatacgc tcatcaatcc ttcggtattg acagatttgg tgcctccggt 1920 aaggcaccag aagtcttcaa gttcttcggt ttcaccccag aaggtgttgc tgaaagagct 1980 caaaagacca ttgcattcta taagggtgac aagctaattt ctcctttgaa aaaagctttc 2040 cup 2043
<210> 12 <210> 12
<211> 717  <211> 717
<212> DNA  <212> DNA
<213> Sequênci  <213> Sequence
<220> <220>
<223> Ribose 5  <223> Ribose 5
<400> 12 <400> 12
atggtcaaac caattatagc tcccagtatc cttgcttctg acttcgccaa cttgggttgc 60 gaatgtcata aggtcatcaa cgccggcgca gattggttac atatcgatgt catggacggc 120 cattttgttc caaacattac tctgggccaa ccaattgtta cctccctacg tcgttctgtg 180 ccacgccctg gcgatgctag caacacagaa aagaagccca ctgcgttctt cgattgtcac 240 atgatggttg aaaatcctga aaaatgggtc gacgattttg ctaaatgtgg tgctgaccaa 300 tttacgttcc actacgaggc cacacaagac cctttgcatt tagttaagtt gattaagtct 360 aagggcatca aagctgcatg cgccatcaaa cctggtactt ctgttgacgt tttatttgaa 420 ctagctcctc atttggatat ggctcttgtt atgactgtgg aacctgggtt tggaggccaa 480 aaattcatgg aagacatgat gccaaaagtg gaaactttga gagccaagtt tccccatttg 540 aatatccaag tcgatggtgg tttgggcaag gagaccatcc cgaaagccgc caaagccggt 600 gccaacgtta ttgtcgctgg taccagtgtt ttcactgcag ctgacccgca cgatgttatc 660 tccttcatga aagaagaagt ctcgaaggaa ttgcgttcta gagatttgct agattag 717 atggtcaaac caattatagc tcccagtatc cttgcttctg acttcgccaa cttgggttgc 60 gaatgtcata aggtcatcaa cgccggcgca gattggttac atatcgatgt catggacggc 120 cattttgttc caaacattac tctgggccaa ccaattgtta cctccctacg tcgttctgtg 180 ccacgccctg gcgatgctag caacacagaa aagaagccca ctgcgttctt cgattgtcac 240 atgatggttg aaaatcctga aaaatgggtc gacgattttg ctaaatgtgg tgctgaccaa 300 tttacgttcc actacgaggc cacacaagac cctttgcatt tagttaagtt gattaagtct 360 aagggcatca aagctgcatg cgccatcaaa cctggtactt ctgttgacgt tttatttgaa 420 ctagctcctc atttggatat ggctcttgtt atgactgtgg aacctgggtt tggaggccaa 480 aaattcatgg aagacatgat gccaaaagtg gaaactttga gagccaagtt tccccatttg 540 aatatccaag tcgatggtgg tttgggcaag gagaccatcc cgaaagccgc caaagccggt 600 gccaacgtta ttgtcgctgg taccagtgtt ttcactgcag ctatacccgca cgatgttatc 660 tccttcatga aagaagaagt cggagagga cgtagga
<210> 13 <210> 13
<211> 189  <211> 189
<212> DNA  <212> DNA
<213> Sequência Nucleotidica  <213> Nucleotide Sequence
Terminador 3-Fosfato Quinase (PGK1) 3-Phosphate Kinase Terminator (PGK1)
<400> 13 <400> 13
attgaattga attgaaatcg atagatcaat ttttttcttt tctctttccc catcctttac 60 gctaaaataa tagtttattt tattttttga atatttttta tttatatacg tatatataga 120 ctattattta tcttttaatg attattaaga tttttattaa aaaaaaattc gctcctcttt 180 taatgcctt 189 attgaattga attgaaatcg atagatcaat ttttttcttt tctctttccc catcctttac 60 gctaaaataa tagtttattt tattttttga atatttttta tttatatacg tatatataga 120 ctattattta tcttttaatg attattaaga ttttta
<210> 14 <210> 14
<211> 984 <212> DNA <211> 984 <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Aldose Redutase (GRE3)  <223> Aldose Reductase (GRE3)
<400> 14 <400> 14
atgtcttcac tggttactct taataacggt ctgaaaatgc ccctagtcgg cttagggtgc 60 tggaaaattg acaaaaaagt ctgtgcgagt caaatttatg aagctatcaa attaggctac 120 cgtctattcg atggtgcttg cgactacggc aacgaaaagg aagttggtga aggtatcagg 180 aaagccatct ccgaaggtct tgtttctaga aaggatatat ttgttgtttc aaagttatgg 240 aacaattttc accatcctga tcatgtaaaa ttagctttaa agaagacctt aagcgatatg 300 ggacttgatt atttagacct gtattatatt cacttcccaa tcgccttcaa atatgttcca 360 tttgaagaga aataccctcc aggattctat acgggcgcag atgacgagaa gaaaggtcac 420 atcaccgaag cacatgtacc aatcatagat acgtaccggg ctctggaaga atgtgttgat 480 gaaggcttga ttaagtctat tggtgtttcc aactttcagg gaagcttgat tcaagattta 540 ttacgtggtt gtagaatcaa gcccgtggct ttgcaaattg aacaccatcc ttatttgact 600 caagaacacc tagttgagtt ttgtaaatta cacgatatcc aagtagttgc ttactcctcc 660 ttcggtcctc aatcattcat tgagatggac ttacagttgg caaaaaccac gccaactctg 720 ttcgagaatg atgtaatcaa gaaggtctca caaaaccatc caggcagtac cacttcccaa 780 gtattgctta gatgggcaac tcagagaggc attgccgtca ttccaaaatc ttccaagaag 840 gaaaggttac ttggcaacct agaaatcgaa aaaaagttca ctttaacgga gcaagaattg 900 aaggatattt ctgcactaaa tgccaacatc agatttaatg atccatggac ctggttggat 960 ggtaaattcc ccacttttgc ctga 984 <210> 15 atgtcttcac tggttactct taataacggt ctgaaaatgc ccctagtcgg cttagggtgc 60 tggaaaattg acaaaaaagt ctgtgcgagt caaatttatg aagctatcaa attaggctac 120 cgtctattcg atggtgcttg cgactacggc aacgaaaagg aagttggtga aggtatcagg 180 aaagccatct ccgaaggtct tgtttctaga aaggatatat ttgttgtttc aaagttatgg 240 aacaattttc accatcctga tcatgtaaaa ttagctttaa agaagacctt aagcgatatg 300 ggacttgatt atttagacct gtattatatt cacttcccaa tcgccttcaa atatgttcca 360 tttgaagaga aataccctcc aggattctat acgggcgcag atgacgagaa gaaaggtcac 420 atcaccgaag cacatgtacc aatcatagat acgtaccggg ctctggaaga atgtgttgat 480 gaaggcttga ttaagtctat tggtgtttcc aactttcagg gaagcttgat tcaagattta 540 ttacgtggtt gtagaatcaa gcccgtggct ttgcaaattg aacaccatcc ttatttgact 600 caagaacacc tagttgagtt ttgtaaatta cacgatatcc aagtagttgc ttactcctcc 660 ttcggtcctc aatcattcat tgagatggac ttacagttgg caaaaaccac gccaactctg 720 ttcgagaatg atgtaatcaa gaaggtctca caaaaccatc caggcagtac cacttcccaa 780 gtattgctta gatgggcaac tcagagaggc attgccgtca ttccaaaatc ttccaagaag 840 gaaaggttac ttggcaacct agaaatcgaa aaaaagttca ctttaacgga gcaagaattg 900 aaggatattt ctgcactaaa tgccaacatc agatttaatg atccatggac ctggttggat 960 ggtaaattcc ccacttttgc ctga 984 <210> 15
<211> 1032  <211> 1032
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> Recombinase CRE  <223> Recombinase CRE
<400> 15 <400> 15
ctaatcgcca tcttccagca ggcgcaccat tgcccctgtt tcactatcca gggtacggat 60 atagttcatg acaatattta cattggtcca gccaccagct tgcatgatct ccggtattga 120 aactccagcg cgggccatat ctcgcgcggc tccgacacgg gcactgtgtc cagaccaggc 180 caggtatctc tgaccagagt catccttagc gccgtaaatc aatcgatgag ttgcttcaaa 240 aatcccttcc agggcgcgag ttgatagctg gctggtggca gatggcgcgg caacaccatt 300 ttttctgacc cggcaaaaca ggtagttatt cggatcatca gctacaccag agacggaaat 360 ccatcgctcg accagtttag ttacccccag gctaagtgcc ttctctacac ctgcggtgct 420 aaccagcgtt ttcgttctgc caatatggat taacattctc ccaccgtcag tacgtgagat 480 atctttaacc ctgatcctgg caatttcggc tatacgtaac agggtgttat aagcaatccc 540 cagaaatgcc agattacgta tatcctggca gcgatcgcta ttttccatga gtgaacgaac 600 ctggtcgaaa tcagtgcgtt cgaacgctag agcctgtttt gcacgttcac cggcatcaac 660 gttttctttt cggatccgcc gcataaccag tgaaacagca ttgctgtcac ttggtcgtgg 720 cagcccggac cgacgatgaa gcatgtttag ctggcccaaa tgttgctgga tagtttttac 780 tgccagaccg cgcgcctgaa gatatagaag ataatcgcga acatcttcag gttctgcggg 840 aaaccatttc cggttattca acttgcacca tgccgcccac gaccggcaaa cggacagaag 900 cattttccag gtatgctcag aaaacgcctg gcgatccctg aacatgtcca tcaggttctt 960 gcgaacctca tcactcgttg catcgaccgg taatgcaggc aaattttggt gtacggtcag 1020 taaattggac at 1032 ctaatcgcca tcttccagca ggcgcaccat tgcccctgtt tcactatcca gggtacggat 60 atagttcatg acaatattta cattggtcca gccaccagct tgcatgatct ccggtattga 120 aactccagcg cgggccatat ctcgcgcggc tccgacacgg gcactgtgtc cagaccaggc 180 caggtatctc tgaccagagt catccttagc gccgtaaatc aatcgatgag ttgcttcaaa 240 aatcccttcc agggcgcgag ttgatagctg gctggtggca gatggcgcgg caacaccatt 300 ttttctgacc cggcaaaaca ggtagttatt cggatcatca gctacaccag agacggaaat 360 ccatcgctcg accagtttag ttacccccag gctaagtgcc ttctctacac ctgcggtgct 420 aaccagcgtt ttcgttctgc caatatggat taacattctc ccaccgtcag tacgtgagat 480 atctttaacc ctgatcctgg caatttcggc tatacgtaac agggtgttat aagcaatccc 540 cagaaatgcc agattacgta tatcctggca gcgatcgcta ttttccatga gtgaacgaac 600 ctggtcgaaa tcagtgcgtt cgaacgctag agcctgtttt gcacgttcac cggcatcaac 660 gttttctttt cggatccgcc gcataaccag tgaaacagca ttgctgtcac ttggtcgtgg 720 cagcccggac cgacgatgaa gcatgtttag ctggcccaaa tgttgctgga tagtttttac 780 tgccagaccg cgcgcctgaa gatatagaag ataatcgcga acatcttcag gttctgcggg 840 aaaccatttc cggttattca acttgcacca tgccgcccac gaccggcaaa cggacagaag 900 cattttccag gtatgctcag aaaacgcctg gcgatccctg aacatgtcca tcaggttctt 960 gcgaacctca tcactcgttg catcgaccgg taatgcaggc aaattttggt gtacggtcag 1020 taaattggac at 1032
<210> 16 <210> 16
<211> 327 <211> 327
<212> DNA <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> LTR do retrotransposon Tyl <400> 16  <223> Tyl retrotransposon LTR <400> 16
ctgagagatt ggtgaatttt gagatgattg taggcattcc attgttgatc aaggctacaa 60 tattatgtat acagaatata ctaaaagttc tcctcgaggg tagaggaatc ctcaaagggg 120 aagcgatatt tctacataat attattacga ttattcctca ttccgtttta tatgtttcat 180 tatcctatta cattatcaat ccttgcactt cagcttcctc taacttcgat gacagtttct 240 cataccttat gtcatcgtct aacaccgtat atgataatat actggtagtg tgactactag 300 tttatagacg atagttgatt tttattc 327 ctgagagatt ggtgaatttt gagatgattg taggcattcc attgttgatc aaggctacaa 60 tattatgtat acagaatata ctaaaagttc tcctcgaggg tagaggaatc ctcaaagggg 120 aagcgatatt tctacataat attattacga ttattcctca ttccgtttta tatgtttcat 180 tatcctatta cattatcaat ccttgcactt cagcttcctc taacttcgat gacagtttct 240 cataccttat gtcatcgtct aacaccgtat atgataatat actggtagtg tgactactag 300 327 tttatagacg atagttgatt tttattc
<210> 17 <210> 17
<211> 2216  <211> 2216
<212> DNA  <212> DNA
<213> Sequência Nucleotidica <220>  <213> Nucleotide Sequence <220>
<223> LEU2 (ORF + promotor e terminador)  <223> LEU2 (ORF + promoter and terminator)
<400> 17 <400> 17
tcgaggagaa cttctagtat atccacatac ctaatattat tgccttatta aaaatggaat 60 cccaacaatt acatcaaaat ccacattctc ttcaaaatca attgtcctgt acttccttgt 120 tcatgtgtgt tcaaaaacgt tatatttata ggataattat actctatttc tcaacaagta 180 attggttgtt tggccgagcg gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt 240 aactgtggga atactcaggt atcgtaagat gcaagagttc gaatctctta gcaaccatta 300 tttttttcct caacataacg agaacacaca ggggcgctat cgcacagaat caaattcgat 360 gactggaaat tttttgttaa tttcagaggt cgcctgacgc atataccttt ttcaactgaa 420 aaattgggag aaaaaggaaa ggtgagaggc cggaaccggc ttttcatata gaatagagaa 480 gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc 540 tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt 600 acatttcagc aatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctaag 660 aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa tcacagccga agccattaag 720 gttcttaaag ctatttctga tgttcgttcc aatgtcaagt tcgatttcga aaatcattta 780 attggtggtg ctgctatcga tgctacaggt gtcccacttc cagatgaggc gctggaagcc 840 tccaagaagg ttgatgccgt tttgttaggt gctgtgggtg gtcctaaatg gggtaccggt 900 agtgttagac ctgaacaagg tttactaaaa atccgtaaag aacttcaatt gtacgccaac 960 ttaagaccat gtaactttgc atccgactct cttttagact tatctccaat caagccacaa 1020 tttgctaaag gtactgactt cgttgttgtc agagaattag tgggaggtat ttactttggt 1080 aagagaaagg aagacgatgg tgatggtgtc gcttgggata gtgaacaata caccgttcca 1140 gaagtgcaaa gaatcacaag aatggccgct ttcatggccc tacaacatga gccaccattg 1200 cctatttggt ccttggataa agctaatgtt ttggcctctt caagattatg gagaaaaact 1260 gtggaggaaa ccatcaagaa cgaattccct acattgaagg ttcaacatca attgattgat 1320 tctgccgcca tgatcctagt taagaaccca acccacctaa atggtattat aatcaccagc 1380 aacatgtttg gtgatatcat ctccgatgaa gcctccgtta tcccaggttc cttgggtttg 1440 ttgccatctg cgtccttggc ctctttgcca gacaagaaca ccgcatttgg tttgtacgaa 1500 ccatgccacg gttctgctcc agatttgcca aagaataagg ttgaccctat cgccactatc 1560 ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc ctgaagaagg taaggccatt 1620 gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa ctggtgattt aggtggttcc 1680 aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag ttaagaaaat ccttgcttaa 1740 aaagattctc tttttttatg atatttgtac ataaacttta taaatgaaat tcataataga 1800 aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta tatacgcaat 1860 ctacatacat ttatcaagaa ggagaaaaag gaggatagta aaggaataca ggtaagcaaa 1920 ttgatactaa tggctcaacg tgataaggaa aaagaattgc actttaacat taatattgac 1980 aaggaggagg gcaccacaca aaaagttagg tgtaacagaa aatcatgaaa ctacgattcc 2040 taatttgata ttggaggatt ttctctaaaa aaaaaaaaat acaacaaata aaaaacactc 2100 aatgacctga ccatttgatg gagtttaagt caataccttc ttgaaccatt tcccataatg 2160 gtgaaagttc cctcaagaat tttactctgt cagaaacggc cttacgacgt agtcga 2216 tcgaggagaa cttctagtat atccacatac ctaatattat tgccttatta aaaatggaat 60 cccaacaatt acatcaaaat ccacattctc ttcaaaatca attgtcctgt acttccttgt 120 tcatgtgtgt tcaaaaacgt tatatttata ggataattat actctatttc tcaacaagta 180 attggttgtt tggccgagcg gtctaaggcg cctgattcaa gaaatatctt gaccgcagtt 240 aactgtggga atactcaggt atcgtaagat gcaagagttc gaatctctta gcaaccatta 300 tttttttcct caacataacg agaacacaca ggggcgctat cgcacagaat caaattcgat 360 gactggaaat tttttgttaa tttcagaggt cgcctgacgc atataccttt ttcaactgaa 420 aaattgggag aaaaaggaaa ggtgagaggc cggaaccggc ttttcatata gaatagagaa 480 gcgttcatga ctaaatgctt gcatcacaat acttgaagtt gacaatatta tttaaggacc 540 tattgttttt tccaataggt ggttagcaat cgtcttactt tctaactttt cttacctttt 600 acatttcagc aatatatata tatatttcaa ggatatacca ttctaatgtc tgcccctaag 660 aagatcgtcg ttttgccagg tgaccacgtt ggtcaagaaa tcacagccga agccattaag 720 gttcttaaag ctatttctga tgttcgttcc aatgtcaagt tcgatttcga aaatcattta 780 attggtggtg ctgctatcga tgctacaggt gtcccacttc cagatgaggc gctggaagcc 840 tccaagaagg ttgatgccgt tttgttaggt gctgtgggtg gtcctaaatg gggtaccggt 900 agtgttagac ctgaacaagg tttactaaaa atccgtaaag aacttcaatt gtacgccaac 960 ttaagaccat gtaactttg c atccgactct cttttagact tatctccaat caagccacaa 1020 tttgctaaag gtactgactt cgttgttgtc agagaattag tgggaggtat ttactttggt 1080 aagagaaagg aagacgatgg tgatggtgtc gcttgggata gtgaacaata caccgttcca 1140 gaagtgcaaa gaatcacaag aatggccgct ttcatggccc tacaacatga gccaccattg 1200 cctatttggt ccttggataa agctaatgtt ttggcctctt caagattatg gagaaaaact 1260 gtggaggaaa ccatcaagaa cgaattccct acattgaagg ttcaacatca attgattgat 1320 tctgccgcca tgatcctagt taagaaccca acccacctaa atggtattat 1380 aatcaccagc aacatgtttg gtgatatcat ctccgatgaa gcctccgtta tcccaggttc cttgggtttg 1440 ttgccatctg cgtccttggc ctctttgcca gacaagaaca ccgcatttgg tttgtacgaa 1500 ccatgccacg gttctgctcc agatttgcca aagaataagg ttgaccctat cgccactatc 1560 ttgtctgctg caatgatgtt gaaattgtca ttgaacttgc ctgaagaagg taaggccatt 1620 gaagatgcag ttaaaaaggt tttggatgca ggtatcagaa ctggtgattt aggtggttcc 1680 aacagtacca ccgaagtcgg tgatgctgtc gccgaagaag ttaagaaaat ccttgcttaa 1740 aaagattctc tttttttatg atatttgtac ataaacttta taaatgaaat tcataataga 1800 aacgacacga aattacaaaa tggaatatgt tcatagggta gacgaaacta tatacgcaat 1860 ctacatacat ttatcaagaa ggagaaaaag gaggatagta aaggaataca ggtaagcaaa 1920 ttgatactaa tggctcaacg tgataaggaa aaagaattgc actttaacat taatattgac 1980 aaggaggagg gcaccacaca aaaagttagg tgtaacagaa aatcatgaaa ctacgattcc 2040 taatttgata ttggaggatt ttctctaaaa aaaaaaaaat acaacaaata aaaaacactc 2100 aatgacctga ccatttgatg gagtttaagt caataccttc ttgaaccatt tcccataatg 2160 gtgaaagttc cctcaagaat tttactctgt cagaaacggc cttacgacgt agtcga 2216
<210> 18 <210> 18
<211> 742  <211> 742
<212> DNA  <212> DNA
<213> Sequência Nucleotidica  <213> Nucleotide Sequence
<220> <220>
<223> Promotor Gliceraldeido 3-Fosfato Desidrogensase, isoenzima <400> 18  <223> Glyceraldehyde 3-Phosphate Dehydrogensase Promoter, isoenzyme <400> 18
aatgtatatg ctcatttaca ctccatatca ccatatggag gataagttgg gttgagcttc tgatccaatt tattctatcc attagttgct gatatgtccc accagccaac acttgatagt 120 atctactcgc cattcacttc cagcagcgcc agtagggttg ttgagcttag taaaaatgtg 180 cgcaccacaa gcctacatgt ctccacgtca catgaaacca caccgtgggg ccttgttgcg 240 ctaggaatag gatatgcgac gaagacgctt ctgcttagta accacaccac attttcaggg 300 ggtcgatctg cttgcttcct ttactgtcac gagcggccca taatcgcgct ttttttttaa 360 aagacgcgag acagcaaaca ggaagctcgg gtttcaacct tcggagtggt cgcagatctg 420 gagactggat ctttacaata cagtaaggca agccaccatc tgcttcttag gtgcatgcga 480 cggtatccac gtgcagaaca acatagtctg aagaaggggg gaggagcatg ttcattctct 540 gtagcagtaa gagcttggtg ataatgacca aaactggagt ctcgaaatca tataaataga 600 caatatattt tcacacaatg agatttgtag tacagttcta ttctctctct tgcataaata 660 agaaattcat caagaacttg gtttgatatt tcaccaacac acacaaaaaa cagtacttca 720 ctaaatttac acacaaaaca aa 742 aatgtatatg ctcatttaca ctccatatca ccatatggag gataagttgg gttgagcttc tgatccaatt tattctatcc attagttgct gatatgtccc accagccaac acttgatagt 120 atctactcgc cattcacttc cagcagcgcc agtagggttg ttgagcttag taaaaatgtg 180 cgcaccacaa gcctacatgt ctccacgtca catgaaacca caccgtgggg ccttgttgcg 240 ctaggaatag gatatgcgac gaagacgctt ctgcttagta accacaccac attttcaggg 300 ggtcgatctg cttgcttcct ttactgtcac gagcggccca taatcgcgct ttttttttaa 360 aagacgcgag acagcaaaca ggaagctcgg gtttcaacct tcggagtggt cgcagatctg 420 gagactggat ctttacaata cagtaaggca agccaccatc tgcttcttag gtgcatgcga 480 cggtatccac gtgcagaaca acatagtctg aagaaggggg gaggagcatg ttcattctct 540 gtagcagtaa gagcttggtg ataatgacca aaactggagt ctcgaaatca tataaataga 600 caatatattt tcacacaatg agatttgtag tacagttcta ttctctctct tgcataaata 660 agaaattcat caagaacttg gtttgatatt tcaccaacac acacaaaaaa cagtacttca 720 aa 742 ctaaatttac acacaaaaca

Claims

REIVINDICAÇÕES
1 . Cassete de expressão para a transformação de célula eucariótica caracterizado por compreender: 1 . Expression cassette for eukaryotic cell transformation comprising:
a) pelo menos uma sequência nucleotídica selecionada do grupo consistindo de: xilose isomerase (SEQ ID NO:2), ou sequência com pelo 80% de identidade com SEQ ID NO:2, transaldolase (SEQ ID NO:5), ribose 5- fosfato isomerase (SEQ ID NO:7), xiluloquinase (SEQ ID NO:9), transcetolase (SEQ ID NO:11 ) e ribose 5-fosfato epimerase (SEQ ID NO:12);  (a) at least one nucleotide sequence selected from the group consisting of: xylose isomerase (SEQ ID NO: 2), or sequence with at least 80% identity with SEQ ID NO: 2, transaldolase (SEQ ID NO: 5), ribose 5- phosphate isomerase (SEQ ID NO: 7), xylulokinase (SEQ ID NO: 9), transcetolase (SEQ ID NO: 11) and ribose 5-phosphate epimerase (SEQ ID NO: 12);
b) pelo menos uma sequência nucleotídica promotora selecionada do grupo consistindo de: promotor gliceraldeído 3-fosfato desidrogenase (SEQ ID NO:18), promotor 3-fosfato quinase (SEQ ID NO:6), promotor da enzima álcool desidrogenase 1 (SEQ ID NO:8);  b) at least one promoter nucleotide sequence selected from the group consisting of: glyceraldehyde 3-phosphate dehydrogenase promoter (SEQ ID NO: 18), 3-phosphate kinase promoter (SEQ ID NO: 6), alcohol dehydrogenase 1 enzyme promoter (SEQ ID NO: 8);
c) pelo menos uma sequência nucleotídica terminadora selecionada do grupo consistindo de: terminador gliceraldeído 3-fosfato desidrogenase (SEQ ID NO:3), terminador de álcool desidrogenase (SEQ ID NO:10), terminador 3- fosfato quinase (SEQ ID NO:13);  c) at least one terminator nucleotide sequence selected from the group consisting of: glyceraldehyde 3-phosphate dehydrogenase terminator (SEQ ID NO: 3), alcohol dehydrogenase terminator (SEQ ID NO: 10), 3-phosphate kinase terminator (SEQ ID NO: 13);
e em que a sequência nucleotídica definida em a) está funcionalmente ligada à sequência nucleotídica promotora definida em b) e à sequência nucleotídica terminadora definida em c), sendo heteróloga qualquer das referidas sequências.  and wherein the nucleotide sequence defined in a) is operably linked to the promoter nucleotide sequence defined in b) and the terminator nucleotide sequence defined in c), any of said sequences being heterologous.
2. Cassete, de acordo com a reivindicação 1 , caracterizado por ser selecionado do grupo consistindo de:  Cassette according to claim 1, characterized in that it is selected from the group consisting of:
a) cassete de expressão que compreende gene que codifica xilose isomerase descrita em SEQ ID NO:2, ou gene que codifica xilose isomerase com pelo menos 80% de identidade com SEQ ID NO:2, promotor TDH1 de sequência nucleotídica SEQ ID NO:18, e terminador TDH1 de sequência nucleotídica SEQ ID NO:3; b) cassete de expressão que compreende promotor ADH1 representado pela sequência SEQ ID NO:8, gene XKS1 representado pela sequência SEQ ID NO:9 e terminador ADH1 representado pela sequência SEQ ID NO:10; a) Expression cassette comprising gene encoding xylose isomerase described in SEQ ID NO: 2, or gene encoding xylose isomerase with at least 80% identity to SEQ ID NO: 2, TDH1 nucleotide sequence promoter SEQ ID NO: 18 , and TDH1 nucleotide sequence terminator SEQ ID NO: 3; b) expression cassette comprising ADH1 promoter represented by the sequence SEQ ID NO: 8, XKS1 gene represented by the sequence SEQ ID NO: 9 and ADH1 terminator represented by the sequence SEQ ID NO: 10;
c) cassete de expressão que compreende promotor TDH1 de sequência nucleotídica SEQ ID NO:18, gene TAL1 de sequência SEQ ID NO:5, gene terminador TDH1 de sequência SEQ ID NO:3, seguido de promotor PGK1 de sequência SEQ ID NO:6, de gene RKI1 (SEQ ID NO:7) e de terminador de sequência nucleotídica SEQ ID NO:13;  c) expression cassette comprising TDH1 nucleotide sequence promoter SEQ ID NO: 18, sequence TAL1 gene SEQ ID NO: 5, sequence TDH1 terminator gene SEQ ID NO: 3, followed by sequence promoter PGK1 SEQ ID NO: 6 , RKI1 gene (SEQ ID NO: 7), and nucleotide sequence terminator SEQ ID NO: 13;
d) cassete de expressão que compreende promotor TDH1 de sequência SEQ ID NO:18, gene TKL1 de sequência SEQ ID NO:11 , gene codificador de Ribose 5-Fosfato Epimerase SEQ ID NO:7, terminador TDH1 de sequência SEQ ID NO:3, seguido de promotor PGK1 de sequência SEQ ID NO:6, gene RPE1 de sequência SEQ ID NO:12 e terminador PGK1 de sequência SEQ ID NO:13; e combinações de pelo menos dois cassetes de expressão conforme descritos acima.  d) expression cassette comprising sequence TDH1 promoter SEQ ID NO: 18, sequence TKL1 gene SEQ ID NO: 11, Ribose 5-Phosphate Epimerase encoding gene SEQ ID NO: 7, sequence TDH1 terminator SEQ ID NO: 3 , followed by SEQ ID NO: 6 sequence PGK1 promoter, SEQ ID NO: 12 sequence RPE1 gene and SEQ ID NO: 13 sequence PGK1 terminator; and combinations of at least two expression cassettes as described above.
e em que o(s) dito(s) cassete(s) de expressão é(são) funcional(is) na(s) célula(s) eucariótica(s).  and wherein said expression cassette (s) is functional in the eukaryotic cell (s).
3. Cassete de expressão de acordo com a reivindicação 1 ou 2 caracterizado pelo fato de que o(s) referido(s) promotor(es) é(são) constitutivo(s) ou naturalmente induzível(is).  Expression cassette according to claim 1 or 2, characterized in that said promoter (s) is constitutive or naturally inducible.
4. Processo para a transformação de célula eucariótica caracterizado por compreender a introdução, na célula a ser transformada, de pelo menos um cassete de expressão conforme definido em qualquer uma das reivindicações 1 a 3.  Process for the transformation of eukaryotic cell comprising introducing into the cell to be transformed at least one expression cassette as defined in any one of claims 1 to 3.
5. Processo, de acordo com a reivindicação 4, caracterizado por adicionalmente compreender a inativação ou deleção do gene GRE3 (SEQ ID NO:14) no genoma da referida célula eucariótica.  A process according to claim 4, further comprising inactivating or deleting the GRE3 gene (SEQ ID NO: 14) in the genome of said eukaryotic cell.
6. Micro-organismo geneticamente modificado caracterizado por compreender pelo menos um cassete de expressão conforme definido em qualquer uma das reivindicações 1 a 3. Genetically modified microorganism comprising at least one expression cassette as defined in any one of claims 1 to 3.
7. Micro-organismo geneticamente modificado caracterizado por compreender cassete de expressão que compreende SEQ ID NO:2 ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:2. 7. Genetically modified microorganism comprising expression cassette comprising SEQ ID NO: 2 or any sequence having at least 80% identity to SEQ ID NO: 2.
8. Micro-organismo geneticamente modificado caracterizado por expressar o peptídeo compreendido em SEQ ID NO:1 , ou qualquer sequência com pelo menos 80% de identidade com SEQ ID NO:1 , em sua forma ativa de xilose isomerase.  8. Genetically modified micro-organism characterized by expressing the peptide comprised in SEQ ID NO: 1, or any sequence having at least 80% identity to SEQ ID NO: 1, in its active form of xylose isomerase.
9. Micro-organismo, de acordo com a reivindicação 6, caracterizado pelo fato de que um ou mais dos referidos cassetes de expressão é (são) inserido na região entre o centromero e seu primeiro gene diretamente adjacente, na direção upstream ou downatream.  Microorganism according to claim 6, characterized in that one or more of said expression cassettes is inserted into the region between the centromere and its first directly adjacent gene in the upstream or downatream direction.
10. Micro-organismo, de acordo com qualquer uma das reivindicações 6 a 9, caracterizado pelo fato de que as sequências promotoras, sequências codificantes e sequências terminadoras dos cassetes de expressão são estáveis no genoma do micro-organismo e/ou estão presentes em no mínimo 5 cópias no genoma do micro-organismo.  Microorganism according to any one of claims 6 to 9, characterized in that the promoter sequences, coding sequences, and terminator sequences of the expression cassettes are stable in the microorganism genome and / or present in the minimum 5 copies in the genome of the microorganism.
1 1 . Micro-organismo, de acordo com qualquer uma das reivindicações 6 a 10, caracterizado pelo fato de que o gene GRE3 (SEQ ID NO:14) é inativado ou deletado em/de seu genoma.  1 1. Microorganism according to any one of claims 6 to 10, characterized in that the GRE3 gene (SEQ ID NO: 14) is inactivated or deleted in / from its genome.
12. Micro-organismo, de acordo com qualquer uma das reivindicações 6 a 1 1 , caracterizado pelo fato de que é uma levedura do género selecionado õmdo grupo consistindo de: Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Cândida, Kluyveromyces, Schizosaccharomyces, Brettanomyces, Hansenula e Yarrowia.  Microorganism according to any one of claims 6 to 11, characterized in that it is a yeast of the genus selected from the group consisting of: Saccharomyces, Scheffersomyces, Spathaspora, Pichia, Candida, Kluyveromyces, Schizosaccharomyces, Brettanomyces, Hansenula and Yarrowia.
13. Processo de produção de biocombustíveis e/ou bioquímicos caracterizado por compreender uma etapa de cultivo do micro-organismo conforme definido em qualquer uma das reivindicações 6 a 12, em meio contendo xilose como uma das fontes de carbono.  Biofuel and / or biochemical production process comprising a step of cultivating the microorganism as defined in any one of claims 6 to 12 in xylose-containing medium as one of the carbon sources.
14. Processo de produção de biocombustíveis e/ou bioquímicos, de acordo com a reivindicação 13, caracterizado por compreender: a) colocar o micro-organismo, como descrito nas reivindicações de 6 a 12 em contato com material que compreende açúcares como aqueles provenientes de biomassa vegetal lignocelulósica, os quais, por sua vez, compreendem pentoses, como a xilose, sendo que o material lignocelulosico pode, opcionalmente ser submetido a prévio pré-tratamento e hidrólise. Essa etapa pode ocorrer, preferencialmente em condições anaeróbias, não sendo esta, entretanto, uma condição restritiva para o processo; e Biofuel and / or biochemical production process according to claim 13, characterized in that it comprises: a) bringing the microorganism as described in claims 6 to 12 in contact with material comprising sugars such as those from lignocellulosic plant biomass, which in turn comprise pentoses such as xylose, the lignocellulosic material being may optionally undergo pre-treatment and hydrolysis. This stage may occur, preferably under anaerobic conditions, but this is not a restrictive condition for the process; and
b) opcionalmente, fazer o posterior recolhimento do composto gerado. b) optionally retracting the generated compound.
15. Biocombustível caracterizado por ser obtido pelo processo conforme definido em qualquer uma das reivindicações 13 e 14. Biofuel characterized in that it is obtained by the process as defined in any one of claims 13 and 14.
16. Biocombustível de acordo com a reivindicação 15, caracterizado por ser etanol.  Biofuel according to Claim 15, characterized in that it is ethanol.
17. Bioquímico caracterizado por ser obtido pelo processo conforme definido em qualquer uma das reivindicações 13 e 14.  Biochemical characterized in that it is obtained by the process as defined in any one of claims 13 and 14.
PCT/BR2015/050198 2014-10-30 2015-10-30 Expression cassette for eukaryotic cell transformation, method for transforming eukaryotic cells, genetically modified microorganism, method for producing biofuels and/or biochemicals, and biofuel and/or biochemical thereby produced WO2016065453A1 (en)

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