EP2167533A2 - SÉQUENCE D'ADN CODANT POUR UN TRANSPORTEUR DU L-ARABINOSE SPÉCIFIQUE, MOLÉCULE D'ADNc, PLASMIDE COMPRENANT LADITE SÉQUENCE D'ADN, CELLULE HÔTE TRANSFORMÉE AVEC CE PLASMIDE ET LEUR APPLICATION - Google Patents

SÉQUENCE D'ADN CODANT POUR UN TRANSPORTEUR DU L-ARABINOSE SPÉCIFIQUE, MOLÉCULE D'ADNc, PLASMIDE COMPRENANT LADITE SÉQUENCE D'ADN, CELLULE HÔTE TRANSFORMÉE AVEC CE PLASMIDE ET LEUR APPLICATION

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Publication number
EP2167533A2
EP2167533A2 EP08766990A EP08766990A EP2167533A2 EP 2167533 A2 EP2167533 A2 EP 2167533A2 EP 08766990 A EP08766990 A EP 08766990A EP 08766990 A EP08766990 A EP 08766990A EP 2167533 A2 EP2167533 A2 EP 2167533A2
Authority
EP
European Patent Office
Prior art keywords
acid
arabinose
host cell
xylitol
plasmid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP08766990A
Other languages
German (de)
English (en)
Inventor
César SIMÕES DA FONSECA
Rita BÄRBEL HAHN-HÄGERDAL
Isabel Maria Spencer Vieira Martins
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universidade Nova de Lisboa
Original Assignee
Universidade Nova de Lisboa
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universidade Nova de Lisboa filed Critical Universidade Nova de Lisboa
Publication of EP2167533A2 publication Critical patent/EP2167533A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • 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

  • a CDNA MOLECULE A PLASMID COMPRISING THE SAID DNA SEQUENCE, HOST CELL TRANSFORMED WITH SUCH PLASMID AND
  • the present invention refers to a DNA sequence containing the codifying region of a new gene encoding a specific L- arabinose transporter with high uptake capacity, which once inserted in a yeast, preferably Saccharomyces cerevisiae, modifies it in terms of its capacity to make a highly- effective use of L-arabinose.
  • the objective of the present invention is to provide the biorefinary, biofuels and bioethanol production industry with a genetically-modified yeast capable of consuming L- arabinose more rapidly and with larger specificity in D- glucose and D-xylose mixtures as well as of fermenting L- arabinose with higher productivity.
  • biofuels bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L- ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.
  • the cellulose component of lignocellulose materials is exclusively composed of glucose polymers, whereas the hemicellulose fraction is composed of polymers containing a mixture of hexoses (D-glucose, D-galactose, D-manose) and pentoses (D-xylose, L-arabinose) .
  • D-Xylose is the main pentose present in hemicelluloses, accounting up to .40% of the carbohydrate fraction.
  • L-Arabinose may also be present in significant amounts, mainly in agricultural residues, accounting up to 20% of the carbohydrate fraction.
  • the majority of these pentoses (D-xylose and L-arabinose) can be recovered as fermentable sugars in the hemicellulose hydrolysate.
  • lignocellulose materials for a cost-effective production of biofuels and platform chemicals by S. cerevisiae requires the complete fermentation of pentoses.
  • this yeast does not have natural ability to use D-xylose and L-arabinose.
  • yeasts and different groups of microorganisms comprising that capacity, but which are not necessarily adequate for industrial application.
  • S. cerevisiae is clearly the most adequate yeast to this aggressive environment, in many cases maintaining good fermentation capacity.
  • D-xylose and L-arabinose Different strategies have been applied to produce recombinant strains of S. cerevisiae able to use D-xylose and L-arabinose.
  • XR cerevisiae already modified with XR and XDH: a Trichoderma reesei gene, encoding a L-arabitol-4-dehydrogenase (LAD) , which oxidizes L-arabitol into L-xylulose, and the expression of the L-xylulose reductase gene (LXR) of T. reesei or Ambrosiozyma monospora, which reduces L-xylulose into xylitol (WO2002066616 and WO2005026339) .
  • LXR L-xylulose reductase gene
  • the enzyme XR is unspecific and might convert L-arabinose into L-arabitol
  • the XDH enzyme converts xylitol, produced by LAD and LXR, into D-xylulose.
  • a S. cerevisiae strain able to utilize D-xylose and L- arabinose was obtained by combining the heterologous expression of XR/XDH and AI/RK/RPE (WO2006096130) .
  • the sugar transporter from the environment into the cell can be ' an obstacle to the efficient fermentation of pentoses by S. cerevisiae.
  • the L-arabinose is transported by GAL2, an unspecific transporter capable to carry D-galactose, D-glucose, D-xylose and L-arabinose.
  • GAL2 an unspecific transporter capable to carry D-galactose, D-glucose, D-xylose and L-arabinose.
  • the overexpression of the gene GAL2 improved L- arabinose fermentation capacity in the S. cerevisiae strain modified with AI/RK/RPE.
  • this strain shows relatively low yields of ethanol yield and productivity.
  • the ethanol yield and productivity will be improved when S. cerevisiae strains are obtained with an efficient pentose fermentation capacity.
  • Specific pentose transporters are necessary to increase the metabolic flux and the consequent ethanol production, by means of fermentation, or other compounds obtained from the sugar which is present in the hemicellulose hydrolysates from plant biomass handling.
  • Candida arabinofermentans PYCC 5603 T was considered the best yeast to isolate the gene, encoding the high-capacity and specific L-arabinose transporter (ARTl), to be expressed in S. cerevisiae.
  • the present invention provides a means for allowing the process for the use of lignocellulose materials to become more efficient and highly cost effective, namely in production of biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic) , glycerol, sorbitol, mannitol, xylitol/arabinitol, L-ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone, fatty acids, fatty-derived molecules, isoprenoids, isoprenoid-derived molecules, alkanes, isopentanol, isoamylacetate.
  • the solution to this problem is based on the fact that the present inventors have identified and isolated a gene encoding a C. arabinofermentans transporter with a surprisingly uptake capacity and specificity for L- arabinose, when compared to sugar transporters that occur naturally in fermenting yeasts.
  • the amino acid sequence and the corresponding coding genes are not known for a specific and high capacity L-arabinose transporter. One of these genes is now disclosed.
  • this transporter is specific for L-arabinose, not using D-xylose or D-glucose as substrates, when the corresponding gene is inserted into a host cell, this gene turns the cell into a potentially more efficient cell to consume and ferment the L-arabinose which is present in the hexose and pentose mixture resulting from lignocellulose plant biomass material, which is one of the raw materials of industrial interest, namely for production of biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L- ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3-hydroxybutyrolactone,
  • a first aspect of the invention refers to an isolated DNA fragment encoding a transporter with high uptake capacity and specificity for L-arabinose, comprising:
  • nucleotide sequence SEQ ID No. 1 / or b a nucleotide sequence SEQ ID No. 1, or complementary strings thereof.
  • the invention refers to a ⁇ DNA molecule comprising: a) a nucleotide sequence SEQ ID No. 1; or b) a functionally-equivalent variant of the nucleotide sequence SEQ ID No. 1, or complementary strings thereof.
  • the invention refers to plasmids comprising a DNA fragment according to claim 1.
  • the invention refers to a host cell characterized in that it is transformed with the DNA fragment according to claim 1, in order to allow the host cell to express the said L-arabinose transporter.
  • the invention refers to a process for using plant biomass or other lignocellulose materials in the production of biofuels and platform chemicals, comprising the use of L-arabinose from an environment including a L-arabinose source with a host cell transformed according to claims 4 to 6, wherein the host cell uses L- arabinose, generating value-added compounds, such as biofuels, bulk and platform chemicals including ethanol, butanol, lactate, 1,4-diacids (succinate, fumaric, malic), glycerol, sorbitol, mannitol, xylitol/arabinitol, L- ascorbic acid, xylitol, hydrogen gas, 2,5-furan dicarboxylic acid, 3-hydroxy propionic acid, aspartic acid, glutaric acid, glutamic acid, itaconic acid, levulinic acid, and 3 ⁇ hydroxybutyrolactone, fatty acids, fatty- derived molecules, isoprenoids, is
  • Figure J. Gel electrophoresis of denaturant polyacrylamide (10% T) with 20 ⁇ g total protein from plasma or mitochondrial membranes isolated from C. arabinofermentans cells cultivated in 0.5% L-arabinose (Ara) or 0.5% D- glucose (GIu) . The gel was stained with Coomassie Blue. M - Sigma Marker (Wide Range), MW - molecular weight; pm - plasma membranes; mm - mitochondrial membranes.
  • Figure 2 Amino acid sequence of the N-terminus region of the ARTl protein and degenerate primers designed from this region.
  • FIG. 3 Analysis by Northern blot of the expression of ARTl gene.
  • the total RNA was isolated from C. arabinofermentans PYCC 5603 T cultures in mineral medium containing 0.5% L-arabinose (L-Ara) or 0.5% D-glucose (D- GIu) as the sole carbon and energy source. Each sample contains 10 ⁇ g total RNA, separated in 1.2% denaturing agarose gel and subsequently transferred to a nylon membrane (Hybond-N) .
  • Figure 4 a) Nucleotide sequence of ARTl gene (SEQ ID No. 1), from the first (ATG) to the last codon (TAA); b) Amino acid sequence of the ARTl protein.
  • a process was developed to express a specific L- arabinose transporter in Saccharomyces cerevisiae.
  • This process includes the introduction of heterologous DNA in yeasts which become integral within a gene for the transport of L-arabinose, with high specificity and uptake capacity.
  • the identified membrane protein was isolated from a preparative gel loaded with 250 ⁇ g of total membrane protein of C. arabinofermentans cultivated in 0.5% L-arabinose. After electrophoresis, the proteins were transferred to a PVDF membrane (sequi-blot of BIO-RAD) . The electrophoresis and the transfer were carried out according to the manufacturer's instructions. The gel and the membrane containing the prote i n were used for sequencing of the N-terminus of the protein ⁇ Protein Core Facility, Columbia University, USA) . The 15-amino acid sequence obtained is shown in Figure 2. From this sequence degenerate primers were drawn (Figure 2) .
  • the primers design was based on the amino acid sequence of the protein N-terminus.
  • the amplified fragments were cloned into the pMOSBlue vector (Amersham Biosciences) and sequenced using an ALFexpressTM II DNA Analyzer (Amersham Biosciences) , and 5' -Cy5-labelled vector-specific primers (Thermo Sequenase Primer Cycle Sequencing kit) .
  • the protein encoded by this molecule presented the characteristic properties of a sugar transporter.
  • An analysis by Northern blot was followed, which demonstrated that the respective mRNA was very abundant in cells cultivated in 0.5% L-arabinose, but it was not detectable in cells cultivated in 0.5% glucose ( Figure 3) .
  • the cDNA end 5 1 was obtained by 5'-RACE, using the ATCAO3_REV (5' CTGAACCAATAATCCAAAATCCAC-3 ' ) primer. The obtained fragment was cloned and sequenced as described in the previous paragraph, being demonstrated that tb ⁇ cDNA encodes a further amino acid (the initiation methionine) and a not-translated sequence '5' of 29 amino acids.
  • the new gene was designated ARTl (ARabinose Transporter 1 ) .
  • the respective nucleotide sequence (SEQ ID No. 1) is shown in Figure 4.
  • a new vector was built, using the YEplacl95 plasmid (multi ⁇ copy) (Gietz et al., 1988), the HXT7 truncated promoter of S. cerevisiae, the ARTl gene and the PGKl terminator of 5. cerevisiae.
  • a DNA fragment comprising the nucleotides -392 to -1 of the HXT7 promoter was amplified by PCR using the HXT7prom_F0R (S'-AACCTGCAGCTCGTAGGAACAATTTCGG-S') and HXT7prom_REV ( 5 ' -GGACGGGACATATGCTGATTAAAATTAAAAAAACTT-S ' ) primers and the YEpkHXT7 plasmid (Krampe et al., 1998) as template. These primers contain, in the 5' end, Pstl and Ndel restriction sites, respectively.
  • the codifying region of the ARTl gene was amplified using the ART1__FOR2 (5' ATAGCAGATCTCATATGGTTTTCGGTAACAGGCAAAT-S ' ) and ART1_REV2 ( 5 ' -ATAGCAGATCTTCTAGATTAACTATCTAAAGACCGAACG-S ' ) primers and genomic DNA from C. arabinofermentans PYCC 5603 T as template. In the 5' end, these primers contain Ndel and Xbal restriction sites, respectively.
  • the fragment containing the HXT7 promotor was digested with Pstl and Ndel, the fragment containing the ARTl gene was digested with Ndel and Xbal and the YEplacl95 plasmid was digested with Pstl and Xbal. These three DNA elements were linked, forming the pHXT7p-ARTl plasmid. Subsequently, a 0,3 kb fragment containing the terminator region of the PGKl gene was amplified from the pMA91 plasmid, with the PGKT_FOR2
  • PGKT_REV 5 ' -TAATTAGAGCTCTCGAAAGCTTTAACGAACGCAGAA- 3 ' primers that, in the 5' end, contain Xbal and Sad restriction sites, respectively.
  • the fragment containing the terminator region of the PGKl gene was subsequently- digested with these enzymes and linked among the Xbal and Sad restriction sites of the pHXT7p-ART2 plasmid, thus resulting in the pHXT7p-ARTl-PGKlt plasmid.
  • the ARTl gene is translated into a protein of 521 amino acids (Artlp; Fig. 4).
  • This protein has homology with sugar transporters from other yeasts and filamentous fungi.
  • the protein further a homology region with a bacterial L-arabinose transporter family, AraJ (a gene encoding a Escherichia coli L-arabinose permease) .

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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Mycology (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

La présente invention concerne des microorganismes génétiquement modifiés par des cellules hôtes par l'introduction d'une séquence d'acide nucléique codant pour un transporteur du L-arabinose spécifique d'une levure. Les cellules hôtes sont de préférence celles d'un microorganisme eucaryote telles que les levures ou les champignons filamenteux. L'invention est utile pour l'utilisation complète d'un sucre obtenu à partir de la biomasse d'une plante et d'autres matières ligno-cellulosiques d'importance biotechnologique dans la production de biocarburants, produits chimiques complexes et de base incluant l'éthanol, le butanol, le lactate, les 1,4-diacides (succinate, fumarique, malique), le glycérol, le sorbitol, le mannitol, le xylitol/arabinitol, l'acide L-ascorbique, le xylitol, l'hydrogène gazeux, l'acide 2,5-furan-dicarboxylique, l'acide 3-hydroxypropionique, l'acide aspartique, l'acide glutarique, l'acide glutamique, l'acide itaconique, l'acide lévulinique, et la 3-hydroxybutyrolactone, les acides gras, les molécules dérivées d'acides gras, les isoprénoïdes, les molécules dérivées d'isoprénoïde, les alcanes, l'isopentanol, l'acétate d'isoamyle, en utilisant des microorganismes génétiquement modifiés pour consumer et fermenter le L-arabinose dans des mélanges d'hexose et de pentose provenant de matières premières renouvelables.
EP08766990A 2007-07-06 2008-07-04 SÉQUENCE D'ADN CODANT POUR UN TRANSPORTEUR DU L-ARABINOSE SPÉCIFIQUE, MOLÉCULE D'ADNc, PLASMIDE COMPRENANT LADITE SÉQUENCE D'ADN, CELLULE HÔTE TRANSFORMÉE AVEC CE PLASMIDE ET LEUR APPLICATION Withdrawn EP2167533A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
PT103780A PT103780A (pt) 2007-07-06 2007-07-06 Sequência de dna que codifica um transportador específico para l- arabinose, molécula de cdna, plasmideo compreendendo a referida sequência de dna, célula hospedeira transformada com esse plamídeo e sua aplicação.
PCT/PT2008/000026 WO2009008756A2 (fr) 2007-07-06 2008-07-04 SÉQUENCE D'ADN CODANT POUR UN TRANSPORTEUR DU L-ARABINOSE SPÉCIFIQUE, MOLÉCULE D'ADNc, PLASMIDE COMPRENANT LADITE SÉQUENCE D'ADN, CELLULE HÔTE TRANSFORMÉE AVEC CE PLASMIDE ET LEUR APPLICATION

Publications (1)

Publication Number Publication Date
EP2167533A2 true EP2167533A2 (fr) 2010-03-31

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EP08766990A Withdrawn EP2167533A2 (fr) 2007-07-06 2008-07-04 SÉQUENCE D'ADN CODANT POUR UN TRANSPORTEUR DU L-ARABINOSE SPÉCIFIQUE, MOLÉCULE D'ADNc, PLASMIDE COMPRENANT LADITE SÉQUENCE D'ADN, CELLULE HÔTE TRANSFORMÉE AVEC CE PLASMIDE ET LEUR APPLICATION

Country Status (3)

Country Link
EP (1) EP2167533A2 (fr)
PT (1) PT103780A (fr)
WO (1) WO2009008756A2 (fr)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8237035B1 (en) 2008-04-15 2012-08-07 Pioneer Hi Bred International Inc Maize variety PHVNV
WO2011059314A1 (fr) * 2009-11-12 2011-05-19 Stichting Voor De Technische Wetenschappen Transporteurs de pentoses et leurs utilisations
DE102011081649A1 (de) 2011-08-26 2013-02-28 Evonik Röhm Gmbh Längerkettige Methacrylate aus nachwachsenden Rohstoffen
WO2013049711A1 (fr) * 2011-09-29 2013-04-04 Bio Architecture Lab, Inc. Procédés pour préparer l'acide 2,5-furanedicarboxylique
US9598689B2 (en) 2011-11-10 2017-03-21 Lallemand Hungary Liquidity Management Llc Genetically modified strain of S. cerevisiae engineered to ferment xylose and arabinose

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
SE0202090D0 (sv) * 2002-05-08 2002-07-04 Forskarpatent I Syd Ab A modifierd yeast consuming L-arabinose
WO2006096130A1 (fr) * 2005-03-11 2006-09-14 Forskarpatent I Syd Ab Souches de saccharomyces cerevisiae capables de faire fermenter l'arabinose et le xylose
WO2007143247A2 (fr) * 2006-06-02 2007-12-13 Midwest Research Institute Clonage et caractérisation de transporteurs de l-arabinose issus d'une levure non conventionnelle
DE102006060381B4 (de) * 2006-12-20 2010-04-08 Johann Wolfgang Goethe-Universität Frankfurt am Main Neuer spezifischer Arabinose-Transporter aus der Hefe Pichia stipitis und dessen Verwendungen

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2009008756A2 *

Also Published As

Publication number Publication date
WO2009008756A2 (fr) 2009-01-15
PT103780A (pt) 2009-01-06
WO2009008756A4 (fr) 2009-04-23
WO2009008756A3 (fr) 2009-03-05

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