WO2016065453A1 - Cassette d'expression pour la transformation de cellules eucaryotes, procédé pour la transformation de cellules eucaryotes, micro-organisme génétiquement modifié, procédé de production de biocombustibles et/ou d'agents biochimiques et biocombustible et/ou agent biochimique ainsi produits - Google Patents

Cassette d'expression pour la transformation de cellules eucaryotes, procédé pour la transformation de cellules eucaryotes, micro-organisme génétiquement modifié, procédé de production de biocombustibles et/ou d'agents biochimiques et biocombustible et/ou agent biochimique ainsi produits 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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Portuguese (pt)
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WO2016065453A9 (fr
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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/pt
Publication of WO2016065453A1 publication Critical patent/WO2016065453A1/fr
Publication of WO2016065453A9 publication Critical patent/WO2016065453A9/fr

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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

La présente invention concerne une cassette d'expression pour la transformation de cellules eucaryotes comprenant la séquence de nucléotides (SEQ ID NO: 2) codant pour un peptide à caractéristique xylose isomérase (SEQ ID NO:1), comprenant éventuellement aussi d'autres gènes de la voie des pentoses phosphates. L'invention concerne également un micro-organisme génétiquement modifié par insertion de la cassette d'expression décrite, capable de consomer le xylose et de convertir l'éthanol en comparaison avec son équivalent sans lesdites modifications. L'invention se rapporte en outre à un procédé de production de biocombustibles et d'agents biochimiques, de préférence de l'éthanol, à partir de sucres abondants dans la partie lignocellulosique de la biomasse végétale, notamment les pentoses et, parmi les pentoses, principalement le xylose. L'invention porte en outre sur des biocombustibles, de préférence de l'éthanol, et des agents biochimiques produits au moyen du procédé de l'invention.
PCT/BR2015/050198 2014-10-30 2015-10-30 Cassette d'expression pour la transformation de cellules eucaryotes, procédé pour la transformation de cellules eucaryotes, micro-organisme génétiquement modifié, procédé de production de biocombustibles et/ou d'agents biochimiques et biocombustible et/ou agent biochimique ainsi produits WO2016065453A1 (fr)

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