US20120165569A1 - Succinic acid production in a eukaryotic cell - Google Patents

Succinic acid production in a eukaryotic cell Download PDF

Info

Publication number
US20120165569A1
US20120165569A1 US12/743,106 US74310608A US2012165569A1 US 20120165569 A1 US20120165569 A1 US 20120165569A1 US 74310608 A US74310608 A US 74310608A US 2012165569 A1 US2012165569 A1 US 2012165569A1
Authority
US
United States
Prior art keywords
succinic acid
seq
nucleotide sequence
cell according
acid
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.)
Abandoned
Application number
US12/743,106
Other languages
English (en)
Inventor
René Verwaal
Liang Wu
Robbertus Antonius Damveld
Cornelis Maria Jacobus Sagt
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.)
DSM IP Assets BV
Original Assignee
DSM IP Assets BV
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 DSM IP Assets BV filed Critical DSM IP Assets BV
Assigned to DSM IP ASSETS B.V. reassignment DSM IP ASSETS B.V. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: DAMVELD, ROBBERTUS ANTONIUS, SAGT, CORNELIS MARIA JACOBUS, VERWAAL, RENE, WU, LIANG
Publication of US20120165569A1 publication Critical patent/US20120165569A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • 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
    • 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
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/001—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid
    • 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
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

Definitions

  • the present invention relates to a recombinant eukaryotic cell comprising a nucleotide sequence encoding a fumarate reductase and a process for the production of succinic acid wherein the recombinant eukaryotic cell is used.
  • Succinic acid is a potential precursor for numerous chemicals.
  • succinic acid can be converted into 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone.
  • BDO 1,4-butanediol
  • Another product derived from succinic acid is a polyester polymer which is made by linking succinic acid and BDO.
  • Succinic acid is predominantly produced through petrochemical processes by hydrogenation of butane. These processes are considered harmful for the environment and costly.
  • the fermentative production of succinic acid may be an attractive alternative process for the production of succinic acid, wherein renewable feedstock as a carbon source may be used.
  • a number of different bacteria such as Escherichia coli , and the rumen bacteria Actinobacillus, Anaerobiospirillum, Bacteroides, Mannheimia, or Succinimonas , sp. are known to produce succinic acid. Metabolic engineering of these bacterial strains have improved the succinic acid yield and/or productivity, or reduced the by-product formation.
  • WO2007/061590 discloses a pyruvate decarboxylase negative yeast for the production of malic acid and/or succinic acid which is transformed with a pyruvate carboxylase enzyme or a phosphoenolpyruvate carboxylase, a malate dehydrogenase enzyme, and a malic acid transporter protein (MAE).
  • a pyruvate carboxylase enzyme or a phosphoenolpyruvate carboxylase a malate dehydrogenase enzyme
  • MAE malic acid transporter protein
  • the aim of the present invention is an alternative microorganism for the production of succinic acid.
  • the aim is achieved according to the invention with a recombinant eukaryotic cell selected from the group consisting of a yeast and a filamentous fungus comprising a nucleotide sequence encoding NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid.
  • the recombinant eukaryotic cell according to the present invention produces an increased amount of succinic acid compared to the amount of succinic acid produced by a wild-type eukaryotic cell.
  • a eukaryotic cell according to the present invention produces at least 1.2, preferably at least 1.5, preferably at least 2 times more succinic acid than a wild-type eukaryotic cell which does not comprise the nucleotide sequence encoding NAD(H)-dependent fumarate reductase.
  • a recombinant eukaryotic cell is defined as a cell which contains, or is transformed or genetically modified with a nucleotide sequence or polypeptide that does not naturally occur in the eukaryotic cell, or it contains additional copy or copies of an endogenous nucleic acid sequence.
  • a wild-type eukaryotic cell is herein defined as the parental cell of the recombinant cell.
  • the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid may be a heterologous or homologous nucleotide sequence, or encodes a heterologous or homologous NAD(H)-dependent fumarate reductase, which may have been further genetically modified by mutation, disruption or deletion.
  • Recombinant DNA techniques are well known in the art such as in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory Press.
  • homologous when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain.
  • heterologous when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature.
  • Heterologous nucleic acids or proteins are not endogenous to the cell into which it is introduced, but have been obtained from another cell or synthetically or recombinantly produced.
  • a NAD(H)-dependent fumarate reductase according to the present invention uses NAD(H) as a cofactor, whereas most eukaryotic cells comprise a FADH 2 -dependent fumarate reductase, wherein FADH 2 is the cofactor. It was found advantageous that the eukaryotic cell comprises a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase, since the NAD(H)-dependent fumarate reductase provides the cell with further options to oxidise NAD(H) to NAD + and influence the redox balance in the cell.
  • the cell expresses a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid, wherein the nucleotide sequence preferably encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 40%, preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 1, and/or SEQ ID NO: 3, and/or SEQ ID NO: 4, and/or SEQ ID NO: 6.
  • a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid
  • the nucleotide sequence preferably encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 40%, preferably at least 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 97, 98, 99% sequence
  • the nucleotide sequence encodes a NAD(H)-dependent fumarate reductase comprising the amino acid sequence of SEQ ID NO: 1, and/or SEQ ID NO: 3, and/or SEQ ID NO: 4, and/or SEQ ID NO: 6.
  • Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. Usually, sequence identities or similarities are compared over the whole length of the sequences compared. In the art, “identity” also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences.
  • Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include BLASTP and BLASTN, publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894). Preferred parameters for amino acid sequences comparison using BLASTP are gap open 11.0, gap extend 1, Blosum 62 matrix.
  • Nucleotide sequences encoding the enzymes expressed in the cell of the invention may also be defined by their capability to hybridise with the nucleotide sequences encoding a NAD(H) dependent fumarate reductase of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and/or SEQ ID NO: 6, under moderate, or preferably under stringent hybridisation conditions.
  • Stringent hybridisation conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridise at a temperature of about 65° C.
  • the hybridisation is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution.
  • Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridise at a temperature of about 45° C. in a solution comprising about 1 M salt, preferably 6 ⁇ SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 ⁇ SSC or any other solution having a comparable ionic strength.
  • the hybridisation is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution.
  • These conditions will usually allow the specific hybridisation of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridisation conditions in order to specifically identify sequences varying in identity between 50% and 90%.
  • the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen eukaryote host cell.
  • codon optimisation are known in the art.
  • a preferred method to optimise codon usage of the nucleotide sequences to that of the eukaryotic cell is a codon pair optimization technology as disclosed in WO2008/000632.
  • Codon-pair optimization is a method for producing a polypeptide in a host cell, wherein the nucleotide sequences encoding the polypeptide have been modified with respect to their codon-usage, in particular the codon-pairs that are used, to obtain improved expression of the nucleotide sequence encoding the polypeptide and/or improved production of the polypeptide.
  • Codon pairs are defined as a set of two subsequent triplets (codons) in a coding sequence.
  • gene refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein.
  • nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e. a polynucleotide, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).
  • a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
  • polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • the essential nature of such analogues of naturally occurring amino acids is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited to the same protein but consisting entirely of naturally occurring amino acids.
  • polypeptide “peptide” and “protein” are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.
  • enzyme as used herein is defined as a protein which catalyses a (bio)chemical reaction in a cell.
  • nucleotide sequence encoding an enzyme is operably linked to a promoter that causes sufficient expression of the corresponding nucleotide sequence in the eukaryotic cell according to the present invention to confer to the cell the ability to produce succinic acid.
  • operably linked refers to a linkage of polynucleotide elements (or coding sequences or nucleic acid sequence) in a functional relationship.
  • a nucleic acid sequence is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • promoter refers to a nucleic acid fragment that functions to control the transcription of one or more genes, located upstream with respect to the direction of transcription of the transcription initiation site of the gene, and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA sequences known to one of skilled in the art.
  • a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
  • a promoter that could be used to achieve the expression of a nucleotide sequence coding for an enzyme such as NAD(H)-dependent fumarate reductase or any other enzyme introduced in the eukaryotic cell of the invention may be not native to a nucleotide sequence coding for the enzyme to be expressed, i.e. a promoter that is heterologous to the nucleotide sequence (coding sequence) to which it is operably linked.
  • the promoter is homologous, i.e. endogenous to the host cell.
  • Suitable promoters in this context include both constitutive and inducible natural promoters as well as engineered promoters, which are well known to the person skilled in the art.
  • Suitable promoters in eukaryotic host cells may be GAL7, GAL10, or GAL 1, CYC1, HIS3, ADH1, PGL, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, and AOX1.
  • Other suitable promoters include PDC, GPD1, PGK1, TEF1, and TDH.
  • nucleotide sequence encoding an enzyme comprises a terminator.
  • Any terminator which is functional in the eukaryotic cell, may be used in the present invention.
  • Preferred terminators are obtained from natural genes of the host cell. Suitable terminator sequences are well known in the art. Preferably, such terminators are combined with mutations that prevent nonsense mediated mRNA decay in the host cell of the invention (see for example: Shirley et al., 2002, Genetics 161:1465-1482).
  • a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase may be overexpressed to achieve a sufficient production of succinic acid by the cell.
  • nucleotide sequences encoding enzymes in a eukaryotic cell of the invention there are various means available in the art for overexpression of nucleotide sequences encoding enzymes in a eukaryotic cell of the invention.
  • a nucleotide sequence encoding an enzyme may be overexpressed by increasing the copy number of the gene coding for the enzyme in the cell, e.g. by integrating additional copies of the gene in the cell's genome, by expressing the gene from a centromeric vector, from an episomal multicopy expression vector or by introducing an (episomal) expression vector that comprises multiple copies of the gene.
  • overexpression of the enzyme according to the invention is achieved with a (strong) constitutive promoter.
  • the invention also relates to a nucleotide construct comprising one or more nucleotide sequence(s) selected from the group consisting of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10.
  • the nucleic acid construct may be a plasmid, for instance a low copy plasmid or a high copy plasmid.
  • the eukaryotic cell according to the present invention may comprise a single, but preferably comprises multiple copies of the nucleotide sequence encoding a NAD(H) dependent fumarate reductase, for instance by multiple copies of a nucleotide construct.
  • the nucleic acid construct may be maintained episomally and thus comprise a sequence for autonomous replication, such as an autosomal replication sequence.
  • a suitable episomal nucleic acid construct may e.g. be based on the yeast 2 ⁇ or pKD1 plasmids (Gleer et al., 1991, Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489).
  • each nucleic acid construct may be integrated in one or more copies into the genome of the eukaryotic cell. Integration into the cell's genome may occur at random by non-homologous recombination but preferably, the nucleic acid construct may be integrated into the cell's genome by homologous recombination as is well known in the art.
  • the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase may be a heterologous or a homologous nucleotide sequence.
  • the NADH-dependent fumarate reductase is a heterologous enzyme, which may be derived from any suitable origin, for instance bacteria, fungi, protozoa or plants.
  • the cell according to the invention comprises hetereologous a NAD(H)-dependent fumarate reductase, preferably derived from a Trypanosoma sp, for instance a Trypanosoma brucei.
  • nucleotide sequence encoding a NAD(H)-dependent fumarate reductase is expressed in the cytosol.
  • cytosolic activity of the enzyme resulted in an increased productivity of succinic acid by the eukaryotic cell.
  • nucleotide sequence encoding a NAD(H)-dependent fumarate reductase comprises a peroxisomal or mitochondrial targeting signal
  • it may be essential to modify or delete a number of amino acids (and corresponding nucleotide sequences in the encoding nucleotide sequence) in order to prevent peroxisomal or mitochondrial targeting of the enzyme.
  • the presence of a peroxisomal targeting signal may for instance be determined by the method disclosed by Schluter et al, Nucleic acid Research 2007, 35, D815-D822.
  • the NAD(H)-dependent fumarate reductase lacks a peroxisomal or mitochondrial targeting signal for cytosolic activity of the enzyme upon expression of the encoding nucleotide sequence.
  • the cell expresses a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid, wherein the nucleotide sequence preferably encodes a NAD(H)-dependent fumarate reductase, preferably a fumarate reductase comprising an amino acid sequence that has at least 40%, preferably at least 45, 50, 55, 60, 65 70, 75, 80, 85, 90, 95, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 3, and/or SEQ ID NO: 6.
  • the nucleotide sequence encodes a NAD(H)-dependent fumarate reductase comprising the amino acid sequence of SEQ ID NO: 3, and/or SEQ ID NO: 6.
  • the eukaryotic cell selected from the group consisting of a yeast and a filamentous fungus preferably belongs to one of the genera Saccharomyces, Aspergillus, Penicillium, Pichia, Kluyveromyces, Yarrowia, Candida, Hansenula, Humicola, Rhizopus, Torulaspora, Trichosporon, Brettanomyces, Zygosaccharomyces, Pachysolen or Yamadazyma .
  • the eukaryotic cell is a Saccharomyces cervisiae, Saccharomyces uvarum, Saccharomyces bayanus, Aspergillus niger, Penicillium chrysogenum, Pichia stipidis, Kluyveromyces marxianus, K. lactis, K. thermotolerans, Yarrowia lipolytica, Candida sonorensis, C. glabrata, Hansenula polymorpha, Torulaspora delbrueckii, Brettanomyces bruxellensis, Rhizopus orizae or Zygosaccharomyces bailiff.
  • recombinant eukaryotic cell may comprise further genetic modifications, for instance mutations, deletions or disruptions, in homologous nucleotide sequences and/or transformation with nucleotide sequences that encode homologous or heterologous enzymes that catalyse a reaction in the cell resulting in an increased flux towards succinic acid.
  • heterologous and/or homologous nucleotide sequences encoding i) an enzyme that catalyses the conversion of phosphoenolpyruvate or pyruvate to oxaloacetate; ii) a malate dehydrogenase which catalyses the conversion from OAA to malic acid; or iii) a fumarase, which catalyses the conversion of malic acid to fumaric acid.
  • a eukaryotic cell may be transformed or genetically modified with any suitable nucleotide sequence catalyzing the reaction from a C3 to C4 carbon molecule, such as phosphoenolpyruvate (PEP, C3) to oxaloacetate (OAA, C4) and pyruvate (C3) to OAA or malic acid (C3).
  • PEP phosphoenolpyruvate
  • OAA oxaloacetate
  • C3 pyruvate
  • C3 pyruvate
  • malic acid C3
  • Suitable enzymes are PEP carboxykinase (EC 4.1.1.49, EC 4.1.1.38) and PEP carboxylase (EC 4.1.1.31) which catalyse the conversion of PEP to OAA; pyruvate carboxylase (EC 6.4.1.1.), that catalyses the reaction from pyruvate to OAA; or malic enzyme (EC 1.1.1.38), that catalyses the reaction from pyruvate to malic acid.
  • a eukaryotic cell according to the present invention overexpresses a nucleotide sequence encoding a pyruvate carboxylase (PYC), preferably a pyruvate carboxylase that is active in the cytosol upon expression of the nucleotide sequence encoding a PYC, for instance a PYC comprising an amino acid sequence according to SEQ ID NO: 41.
  • PYC pyruvate carboxylase
  • an endogenous or homologous pyruvate carboxylase is overexpressed. Surprisingly, it was found that overexpressing an endogenous pyruvate carboxylase resulted in increased succinic acid production levels by the eukaryotic cell according to the present invention.
  • a eukaryotic cell according to the present invention further comprises a nucleotide sequence encoding a heterologous PEP carboxykinase (EC 4.1.1.49) catalysing the reaction from phosphoenolpyruvate to oxaloacetate.
  • a eukaryotic cell according to the present invention which further comprises a heterologous PEP carboxykinase produced an increased amount of succinic acid as compared to a eukaryotic cell that does not comprise the heterologous PEP carboxykinase.
  • a PEP carboxykinase that is derived from bacteria, more preferably the enzyme having PEP carboxykinase activity is derived from Escherichia coli, Mannheimia sp., Actinobacillus sp., or Anaerobiospirillum sp., more preferably Mannheimia succiniciproducens, Actinobacillus succinogenes, or Anaerobiospirillum succiniciproducens.
  • the PEP carboxykinase is active in the cytosol upon expression of the nucleotide sequence encoding PEP carboxykinase since it was found that this resulted in an increase succinic acid production.
  • a eukaryotic cell comprises a PEP carboxykinase which has at least 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 17, preferably a PEP carboxykinase comprising SEQ ID NO: 14 or SEQ ID NO: 17.
  • a PEP carboxykinase which has at least 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 14 or SEQ ID NO: 17, preferably a PEP carboxykinase comprising SEQ ID NO: 14 or SEQ ID NO: 17.
  • a cell according to the present invention further comprises a nucleotide sequence encoding a malate dehydrogenase (MDH) which is active in the cytosol upon expression of the nucleotide sequence.
  • MDH malate dehydrogenase
  • a cytosolic MDH may be any suitable homologous or heterologous malate dehydrogenase.
  • the MDH may be a S. cerevisiae MDH3 or S. cerevisiae MDH1.
  • the MDH lacks a peroxisomal or mitochondrial targeting signal in order to localize the enzyme in the cytosol.
  • the MDH is S. cerevisiae MDH2 which has been modified such that it is not inactivated in the presence of glucose and is active in the cytosol.
  • Mdh2p deleted for the first 12 amino-terminal amino acids is less-susceptible for glucose-induced degradation (Minard and McAlister-Henn, J. Biol Chem. 1992 Aug. 25; 267(24):17458-64).
  • a eukaryotic cell comprises a nucleotide sequence encoding a malate dehydrogenase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, 99% sequence identity with the amino acid sequence of SEQ ID NO: 19 or SEQ ID NO: 21.
  • the malate dehydrogenase comprises SEQ ID NO: 19 or SEQ ID NO: 21.
  • the activity of malate dehydrogenase is increased by overexpressing the encoding nucleotide sequence by known methods in the art.
  • a eukaryotic cell according to the present invention further comprises a nucleotide sequence encoding an enzyme that catalyses the conversion of malic acid to fumaric acid, which may be a heterologous or homologous enzyme, for instance a fumarase (FUM).
  • a nucleotide sequence encoding an heterologous enzyme that catalyses the conversion of malic acid to fumaric acid may be derived from any suitable origin, preferably from microbial origin, preferably from a yeast, for instance Saccharomyces cerevisiae or a filamentous fungus, for instance Rhizopus oryzae.
  • a eukaryotic cell according to the present invention comprises a nucleotide sequence encoding a fumarase that has at least 70%, preferably at least 75, 80, 85, 90, 92, 94, 95, 96, 97, 98, or 99% sequence identity with the amino acid sequence of SEQ ID NO: 23.
  • the fumarase comprises SEQ ID NO: 23.
  • the enzyme having fumarase activity is active in the cytosol upon expression of the nucleotide sequence encoding the enzyme having fumarase activity. Surprisingly, it was found that a eukaryotic cell further comprising an enzyme having fumarase activity as described herein produced an increased amount of succinic acid.
  • a eukaryotic cell comprises a nucleotide sequence encoding a dicarboxylic acid transporter protein, preferably a malic acid transporter protein (MAE).
  • a dicarboxylic acid transporter protein may be a homologous or heterologous protein.
  • the dicarboxylic acid transporter protein is a heterologous protein.
  • a dicarboxylic acid transporter protein may be derived from any suitable organism, preferably from Schizosaccharomyces pombe.
  • a dicarboxylic acid transporter protein is a malic acid transporter protein (MAE) which has at least 80, 85, 90, 95 or 99% sequence identity with SEQ ID NO: 36.
  • the MAE comprises SEQ ID NO: 36.
  • a eukaryotic cell according to the present invention further comprising a dicarboxylic acid transporter, such as a malic acid transporter as described herein produced an increased amount of succinic acid as compared to a eukaryote cell not comprising a dicarboxylic acid transporter protein.
  • the present invention also relates to the use of a dicarboxylic acid transporter, preferably a malic acid transporter protein, in a eukaryotic cell to increase succinic acid production.
  • a dicarboxylic acid transporter preferably a malic acid transporter protein
  • the malic acid transporter is derived from Schizosaccharomyces pombe.
  • a eukaryotic cell is a yeast comprising nucleotide sequences encoding a NAD(H)-dependent fumarate reductase, a malate dehydrogenase, a heterologous fumarase, a heterologous PEP carboxykinase and a heterologous dicarboxylic acid transporter and overexpresses a pyruvate carboxylase (PYC), as described, including the preferred embodiments, herein above.
  • PYC pyruvate carboxylase
  • a yeast of the invention comprising the nucleotide sequences encoding the enzymes as described herein produced an increased amount of succinic acid as compared to a yeast comprising either of the nucleotide sequences alone.
  • a eukaryotic cell according to the present invention comprises reduced activity of enzymes that convert NAD(H) to NAD + compared to the activity of these enzymes in a wild-type cell.
  • the cell according to the present invention is a cell wherein at least one gene encoding alcohol dehydrogenase is not functional.
  • An alcohol dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell which comprises a reduced alcohol dehydrogenase activity compared to a cell wherein all genes encoding an alcohol dehydrogenase are functional.
  • a gene may become not functional by known methods in the art, for instance by mutation, disruption, or deletion, for instance by the method disclosed by Gueldener et. al. 2002, Nucleic Acids Research, Vol. 30, No. 6, e23.
  • a eukaryotic cell is a yeast cell such as Saccharomyces cerevisiae, wherein one or more genes adh1 and/or adh2, encoding alcohol dehydrogenase are inactivated.
  • the cell according to the present invention further comprises at least one gene encoding glycerol-3-phosphate dehydrogenase which is not functional.
  • a glycerol-3-phosphate dehydrogenase gene that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced glycerol-3-phosphate dehydrogenase activity, for instance by mutation, disruption, or deletion of the gene encoding glycerol-3-phosphate dehydrogenase, resulting in a decreased formation of glycerol as compared to the wild-type cell.
  • the eukaryotic cell comprising reduced alcohol dehydrogenase activity and/or glycerol-3-phosphate dehydrogenase activity and a NAD(H)-dependent fumarase resulted in an increased production of succinic acid as compared to a cell wherein one or more gene(s) encoding alcohol dehydrogenase and/or glycerol-3-phosphate dehydrogenase are not inactivated.
  • the present invention also relates to a process for the production of succinic acid comprising fermenting a eukaryotic cell comprising at least one gene encoding alcohol dehydrogenase is not functional and / or at least one gene encoding glycerol-3-phosphate dehydrogenase which is not functional.
  • the recombinant eukaryotic cell according to the present invention comprises at least one gene encoding succinate dehydrogenase that is not functional.
  • a succinate dehydrogenase that is not functional is used herein to describe a eukaryotic cell, which comprises a reduced succinate dehydrogenase activity by mutation, disruption, or deletion, of at least one gene encoding succinate dehydrogenase resulting in a increased formation of succinic acid as compared to the wild-type cell.
  • a eukaryotic cell comprising a gene encoding succinate dehydrogenase that is not functional may for instance be Aspergillus niger , preferably an Aspergillus niger , wherein one or more genes encoding succinate dehydrogenase, such as sdhA and sdhB is/are not functional, for instance by deletion of these genes.
  • a eukaryotic cell according to the invention is a yeast, preferably Saccharomyces cerevisiae , preferably a Saccharomyces cerevisiae comprising one or more of the nucleotide sequences selected from SEQ ID NO: 9 and SEQ ID NO: 10.
  • a eukaryotic cell according to the present invention may also be a filamentous fungus, preferably A. niger , preferably A. niger comprising one or more nucleotide sequences selected from SEQ ID NO: 7 and SEQ ID NO: 8.
  • a eukaryotic cell according to the present invention comprising any one of the genetic modifications described herein is capable of producing at least 0.3, 0.5, 0.7, g/L succinic acid, preferably at least 1 g/L succinic acid, preferably at least 1.5 preferably at least 2, or 2.5, 4.5 preferably at least 8, 10, 15, or 20 g / L succinic acid but usually below 200 or below 150 g / L.
  • a preferred eukaryotic cell according to the present invention may be able to grow on any suitable carbon source known in the art and convert it to succinic acid.
  • the eukaryotic cell may be able to convert directly plant biomass, celluloses, hemicelluloses, pectines, rhamnose, galactose, fucose, maltose, maltodextrines, ribose, ribulose, or starch, starch derivatives, sucrose, lactose and glycerol.
  • a preferred host organism expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (e.g.
  • the cell is able to convert a carbon source selected from the group consisting of glucose, fructose, galactose, xylose, arabinose, sucrose, raffinose, lactose and glycerol.
  • the present invention relates to a process for the preparation of succinic acid, comprising fermenting the eukaryotic cell according to the present invention, wherein succinic acid is prepared.
  • the succinic acid that is prepared in the process according to the present invention is further converted into a desirable product.
  • a desirable product may for instance be a polymer, such as polybutylene succinic acid (PBS), a deicing agent, or a surfactant.
  • the process according to the present invention may be run under aerobic and anaerobic conditions.
  • the process is carried out under anaerobic conditions or under micro-aerophilic or oxygen limited conditions.
  • An anaerobic fermentation process is herein defined as a fermentation process run in the absence of oxygen or in which substantially no oxygen is consumed, preferably less than 5, 2.5 or 1 mmol/L/h, and wherein organic molecules serve as both electron donor and electron acceptors.
  • An oxygen-limited fermentation process is a process in which the oxygen consumption is limited by the oxygen transfer from the gas to the liquid.
  • the degree of oxygen limitation is determined by the amount and composition of the ingoing gasflow as well as the actual mixing/mass transfer properties of the fermentation equipment used.
  • the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.
  • the process for the production of succinic acid according to the present invention may be carried out at any suitable pH between 1 and 9.
  • the pH in the fermentation broth is between 2 and 7, preferably between 3 and 5. It was found advantageous to be able to carry out the process according to the present invention at a low pH, since this prevents bacterial contamination. In addition, since the pH drops during succinic acid production, a lower amount of titrant may be needed to keep the pH at a desired level.
  • a suitable temperature at which the process according to the present invention may be carried out is between 5 and 60° C., preferably between 10 and 50° C., more preferably between 15 and 35° C., more preferably between 18° C. and 30° C.
  • the skilled man in the art knows which optimal temperatures are suitable for fermenting a specific eukaryotic cell.
  • succinic acid is recovered from the fermentation broth by a suitable method known in the art, for instance by crystallisation and ammonium precipitation.
  • the succinic acid that is prepared in the process according to the present invention is further converted into a pharmaceutical, cosmetic, food, feed, or chemical product.
  • Succinic acid may be further converted into a polymer, such as polybutylene succinate (PBS) or other suitable polymers derived therefrom.
  • PBS polybutylene succinate
  • the present invention also relates to a fermentation broth comprising a succinic acid obtainable by a process according to the present invention.
  • the invention relates to a process for the production of succinic acid by a yeast or a filamentous fungus as succinic acid producer, whereby fumarate reductase from Trypanosoma brucei is used to increase succinic acid production, wherein preferably the fumarate reductase is active in the cytosol.
  • Standard genetic techniques such as overexpression of enzymes in the host cells, genetic modification of host cells, or hybridisation techniques, are known methods in the art, such as described in Sambrook and Russel (2001) “Molecular Cloning: A Laboratory Manual (3 rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press , or F. Ausubel et al, eds., “Current protocols in molecular biology”, Green Publishing and Wiley Interscience, New York (1987). Methods for transformation, genetic modification etc of fungal host cells are known from e.g.
  • EP-A-0 635 574 WO 98/46772, WO 99/60102 and WO 00/37671, W090/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No. 6,265,186.
  • FIG. 1 Map of the pGBTOP-11 vector used for expression of fumarate reductase in A. niger
  • FIG. 2 Plasmid map of pGBS414SUS-07, encoding mitochondrial fumarate reductase m1 (FRDm1) from Trypanosoma brucei for expression in Saccharomyces cerevisiae.
  • CPO denotes codon pair optimized.
  • FIG. 3 Plasmid map of pGBS414SUS-08, encoding glycosomal fumarate reductase (FRDg) from Trypanosoma brucei for expression in Saccharomyces cerevisiae .
  • CPO denotes codon pair optimized.
  • FIG. 4 Plasmid map of pDEL-SDHA
  • FIG. 5 Map of plasmid pGBTPAn1, for overexpression FRDm1 in A. niger.
  • FIG. 6 Replacement scheme of sdhA
  • FIG. 7 Plasmid map of pGBS416FRD-1, encoding mitochondrial fumarate reductase m1 (FRDm1) from Trypanosoma brucei for expression in Saccharomyces cerevisiae. CPO denotes codon pair optimized.
  • FIG. 8 Plasmid map of pGBS416FRE-1, encoding glycosomal fumarate reductase (FRDg) from Trypanosoma brucei for expression in Saccharomyces cerevisiae.
  • FPDg glycosomal fumarate reductase
  • FIG. 9 Plasmid map of pGBS414PPK-1, containing PEP carboxykinase from Actinobacillus succinogenes (PCKa) for expression in Saccharomyces cerevisiae.
  • the synthetic gene construct TDH1 promoter-PCKa-TDH1 terminator was cloned into expression vector pRS414.
  • CPO denotes codon pair optimized.
  • FIG. 10 Plasmid map of pGBS414PPK-2, containing PEP carboxykinase from Actinobacillus succinogenes (PCKa) and mitochondrial fumarate reductase m1 from Trypanosoma brucei (FRDm1) for expression in Saccharomyces cerevisiae .
  • the synthetic gene constructs TDH1 promoter-PCKa-TDH1 terminator and TDH3 promoter-FRDm1-TDH3 terminator were cloned into expression vector pRS414.
  • CPO denotes codon pair optimized.
  • FIG. 11 Plasmid map of pGBS414PPK-3, containing PEP carboxykinase from Actinobacillus succinogenes (PCKa) and glycosomal fumarate reductase from Trypanosoma brucei (FRDg) for expression in Saccharomyces cerevisiae .
  • the synthetic gene constructs TDH1 promoter-PCKa-TDH1 terminator and TDH3 promoter-FRDg-TDH3 terminator were cloned into expression vector pRS414.
  • CPO denotes codon pair optimized.
  • FIG. 12 Plasmid map of pGBS414PEK-1, containing PEP carboxykinase from Mannheimia succiniciproducens (PCKm) for expression in Saccharomyces cerevisiae .
  • the synthetic gene construct TDH1 promoter-PCKm-TDH1 terminator was cloned into expression vector pRS414.
  • CPO denotes codon pair optimized.
  • FIG. 13 Plasmid map of pGBS414PEK-2, containing PEP carboxykinase from Mannheimia succiniciproducens (PCKm) and mitochondrial fumarate reductase m1 from Trypanosoma brucei (FRDm1) for expression in Saccharomyces cerevisiae .
  • the synthetic gene constructs TDH1 promoter-PCKm-TDH1 terminator and TDH3 promoter-FRDm1-TDH3 terminator were cloned into expression vector pRS414.
  • CPO denotes codon pair optimized.
  • FIG. 14 Plasmid map of pGBS414PEK-3, containing PEP carboxykinase from Mannheimia succiniciproducens (PCKm) and glycosomal fumarate reductase from Trypanosoma brucei (FRDg) for expression in Saccharomyces cerevisiae.
  • PCKm Mannheimia succiniciproducens
  • FPDg glycosomal fumarate reductase from Trypanosoma brucei
  • the synthetic gene constructs TDH1 promoter-PCKm-TDH1 terminator and TDH3 promoter-FRDg-TDH3 terminator were cloned into expression vector pRS414.
  • CPO denotes codon pair optimized.
  • FIG. 15 Plasmid map of pGBS415FUM-2, containing fumarase from Rhizopus oryzae (FUMR) and cytoplasmic malate dehydrogenase from Saccharomyces cerevisiae truncated for the first 12 amino acids (delta12N MDH2) for expression in Saccharomyces cerevisiae.
  • the synthetic gene constructs TDH1 promoter-FUMR-TDH1 terminator and DH3 promoter-MDH3-TDH3 terminator were cloned into expression vector pRS415.
  • CPO denotes codon pair optimized.
  • FIG. 16 Plasmid map of pGBS415FUM-3, containing fumarase from Rhizopus oryzae (FUMR) and peroxisomal malate dehydrogenase from Saccharomyces cerevisiae (MDH3) for expression in Saccharomyces cerevisiae .
  • the synthetic gene constructs TDH1 promoter-FUMR-TDH1 terminator and TDH3 promoter-MDH3-TDH3 terminator were cloned into expression vector pRS415.
  • CPO denotes codon pair optimized.
  • FIG. 17 Succinic acid levels in strains SUC-101 ( ⁇ , empty vectors control), SUC-148 ( ⁇ , overexpression of PCKa, MDH3, FUMR, FRDm1), SUC-149 ( ⁇ , PCKa, MDH3, FUMR, FRDg), SUC-150 ( ⁇ , PCKm, MDH3, FUMR, FRDm1), SUC-151 ( ⁇ , PCKm, MDH3, FUMR, FRDg), SUC-152 ( ⁇ , PCKa, MDH3, FUMR), SUC-154 ( ⁇ , PCKm, MDH3, FUMR) and SUC-169 ( ⁇ , PCKm, delta12NMDH2, FUMR, FRDm1).
  • FIG. 18 Plasmid map of pGBS416MAE-1, containing malate permease from Schizosaccharomyces pombe (SpMAE1) for expression in Saccharomyces cerevisiae .
  • the synthetic gene construct EnoI promoter-MAE1-Eno1 terminator was cloned into expression vector pRS416.
  • CPO denotes codon pair optimized.
  • FIG. 19 Succinic acid levels in strains SUC-101 ( ⁇ , empty vectors control), SUC-169 ( ⁇ , PCKm, delta12NMDH2, FUMR, FRDm1) and SUC-194 ( ⁇ , PCKm, delta12NMDH2, FUMR, FRDm1, SpMAE1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae. All data represent averages of 3 independent growth experiments of SUC-169 and SUC-194 and averages of 6 independent growth experiments of SUC-101.
  • FIG. 20 Succinic acid levels in strains SUC-103 ( ⁇ , adh1/2 and gpd1 deletion mutant; empty vectors control), SUC-201 ( ⁇ , adh1/2 and gpd1 deletion mutant; PCKa, MDH3, FUMR, FRDg) and SUC-200 ( ⁇ , adh1/2 and gpd1 deletion mutant; PCKa, MDH3, FUMR, FRDg, SpMAE1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae.
  • FIG. 21 Plasmid map of pGBS426PYC-2, containing pyruvate carboxylase from Saccharomyces cerevisiae for expression in Saccharomyces cerevisiae.
  • the PYC2 coding nucleotide sequence was obtained by PCR using genomic DNA from strain CEN.PK113-5D as template and the PCR product was cloned into expression vector p426GPD.
  • FIG. 22 Plasmid map of pGBS414FRE-1, encoding glycosomal fumarate reductase (FRDg) from Trypanosoma brucei for expression in Saccharomyces cerevisiae.
  • the synthetic gene construct TDH3 promoter-FRDg-TDH3 terminator was cloned into expression vector pRS414.
  • FIG. 23 Succinic acid levels in strains SUC-226 ( ⁇ , PCKa, MDH3, FUMR, FRDg), -227 ( ⁇ , PYC2, PCKa, MDH3, FUMR, FRDg), SUC-228 ( ⁇ , PYC2, MDH3, FUMR, FRDg) and SUC-230 ( ⁇ , MDH3, FUMR, FRDg). Data represents the average of 3 independent growth experiments.
  • Mitochondrial fumarate reductase ml [E.C. 1.3.1.6], GenBank accession number 60460035, from Trypanosoma brucei was analysed for the presence of signal sequences using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971. A putative mitochondrial targeting sequence in the N-terminal half of the protein was identified, including a possible cleavage site between pos. 25 and 26 (D-S).
  • SEQ ID NO: 3 was subjected to the codon-pair method as disclosed in WO2008/000632 for A. niger .
  • the resulting sequence SEQ ID NO: 7 was put behind the constitutive GPDA promoter sequence SEQ ID NO: 11, wherein the last 10 nucleotide sequences were replaced with optimal Kozak sequence CACCGTAAA. Convenient restriction sites were added.
  • the stop codon TAA in SEQ: ID NO: 7 was modified to TAAA.
  • the resulting sequence was synthesised at Sloning (Puchheim, Germany).
  • the fragment was SnaBI, SfiI cloned in the A. niger expression vector pGBTOP11 ( FIG. 1 ) using appropriate restriction sites.
  • the resulting plasmid comprising FRDm1 was named pGBTOPAn1 ( FIG. 5 ).
  • glycosomal fumarate reductase [E.C. 1.3.1.6], GenBank accession number 23928422, from Trypanosoma brucei was analysed for peroxisomal targeting in filamentous fungi using the PTS1 predictor http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp with the fungi-specific prediction function.
  • the C-terminal amino acids at position 1140-1142 (SKI) were removed from the protein SEQ ID NO: 4 (corresponding to nucleotide sequence SEQ ID NO: 5), resulting in SEQ ID NO: 6.
  • SEQ ID NO: 6 was subjected to the codon-pair method as disclosed in PCT/EP2007/05594 for A. niger .
  • the stop codon TAA in SEQ ID NO: 8 was modified to TAAA.
  • the resulting sequence SEQ ID NO: 8 was put behind the constitutive GPDA promoter sequence SEQ ID NO: 11, and convenient restriction sites were added.
  • the resulting sequence was synthesised at Sloning (Puchheim, Germany).
  • the fragment was SnaBI, SfiI cloned in the A. niger expression vector pGBTOP11 ( FIG. 1 ) using appropriate restriction sites.
  • A. niger WT-1 This A. niger strain is CBS513.88 comprising deletions of the genes encoding glucoamylase (glaA), fungal amylase and acid amylase.
  • A. niger WT 1 was constructed by using the “MARKER-GENE FREE” approach as described in EP 0 635 574 B1.
  • Aspergillus minimal medium contains per litre: 6 g NaNO 3 , 0.52 g KCl, 1.52 g KH 2 PO 4 , 1.12 ml 4 M KOH, 0.52 g MgSO 4 .7H 2 O, 10 g glucose, 1 g casaminoacids, 22 mg ZnSO 4 .7H 2 O, 11 mg H 3 BO 3 , 5 mg FeSO 4 .7H 2 O, 1.7 mg CoCl 2 .6H 2 O, 1.6 mg CuSO 4 .5H 2 O, 5 mg MnCl 2 .2H 2 O, 1.5 mg Na 2 MoO 4 .2H 2 O, 50 mg EDTA, 2 mg riboflavin, 2 mg thiamine-HCl, 2 mg nicotinamide, 1 mg pyri
  • Novozym 234TM Novo Industries
  • helicase instead of helicase is used for the preparation of protoplasts
  • KC buffer (0.8 M KCl, 9.5 mM citric acid, pH 6.2) is added to a final volume of 45 ml, the protoplast suspension is centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swinging-bucket rotor. The protoplasts are resuspended in 20 ml KC buffer and subsequently 25 ml of STC buffer (1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2 ) is added.
  • STC buffer 1.2 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2
  • the protoplast suspension is centrifuged for 10 minutes at 3000 rpm at 4 degrees Celsius in a swinging-bucket rotor, washed in STC-buffer and resuspended in STC-buffer at a concentration of 10E8 protoplasts/ml;
  • the DNA fragment dissolved in 10 microliter TE buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA) and 100 microliter of PEG solution (20% PEG 4000 (Merck), 0.8 M sorbitol, 10 mM Tris-HCl pH 7.5, 50 mM CaCl 2 ) is added;
  • the protoplasts are resuspended gently in 1 ml 1.2 M sorbitol and plated onto solid selective regeneration medium consisting of either Aspergillus minimal medium without riboflavin, thiamine.HCL, nicotinamide, pyridoxine, panthotenic acid, biotin, casaminoacids and glucose.
  • solid selective regeneration medium consisting of either Aspergillus minimal medium without riboflavin, thiamine.HCL, nicotinamide, pyridoxine, panthotenic acid, biotin, casaminoacids and glucose.
  • acetamide selection the medium contains 10 mM acetamide as the sole nitrogen source and 1 M sucrose as osmoticum and C-source.
  • protoplasts are plated onto PDA (Potato Dextrose Agar, Oxoid) supplemented with 1-50 microgram/ml phleomycin and 1M sucrose as osmosticum.
  • Regeneration plates are solidified using 2% agar (agar No. 1, Oxoid L11). After incubation for 6-10 days at 30 degrees Celsius, conidiospores of transformants are transferred to plates consisting of Aspergillus selective medium (minimal medium containing acetamide as sole nitrogen source in the case of acetamide selection or PDA supplemented with 1-50 microgram/ml phleomycin in the case of phleomycin selection) with 2% glucose and 1.5% agarose (Invitrogen) and incubated for 5-10 days at 30 degrees Celsius. Single transformants are isolated and this selective purification step is repeated once upon which purified transformants are stored.
  • Aspergillus selective medium minimal medium containing acetamide as sole nitrogen source in the case of acetamide selection or PDA supplemented with 1-50 microgram/ml phleomycin in the case of phleomycin selection
  • 2% glucose and 1.5% agarose Invitrogen
  • HPLC is performed for the determination of organic acids and sugars in different kinds of samples.
  • the principle of the separation on a Phenomenex Rezex-RHM-Monosaccharide column is based on size exclusion, ion-exclusion and ion-exchange using reversed phase mechanisms. Detection takes place by differential refractive index and ultra violet detectors.
  • Mitochondrial fumarate reductase m1 [E.C. 1.3.1.6], GenBank accession number 60460035, from Trypanosoma brucei was analysed for the presence of signal sequences and codon optimized as described in section 1.1 for expression in S. cerevisiae .
  • the resulting sequence SEQ ID NO: 9 was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, and convenient restriction sites were added.
  • the stop codon TGA in SEQ ID NO: 9 was modified to TAAG.
  • the resulting sequence was synthesised at Sloning (Puchheim, Germany).
  • the expression construct pGBS414SUS-07 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarate reductase synthetic gene construct ( FIG. 2 ).
  • the ligation mix is used for transformation of E. coli DH10B (Invitrogen) resulting in the yeast expression construct pGBS414SUS-07 ( FIG. 2 ).
  • glycosomal fumarate reductase (FRDg) [E.C. 1.3.1.6], GenBank accession number 23928422, from Trypanosoma brucei was analysed for peroxisomal targeting and codon optimisation was applied as described in section 1.1 for expression in S. cerevisiae .
  • the resulting sequence SEQ ID NO: 10 was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, and convenient restriction sites were added.
  • the stop codon TGA in SEQ ID NO: 10 was modified to TAAG. The resulting sequence was synthesised at Sloning (Puchheim, Germany).
  • the expression construct pGBS414SUS-08 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarate reductase synthetic gene construct ( FIG. 3 ).
  • the ligation mix is used for transformation of E. coli DH10B (Invitrogen) resulting in the yeast expression construct pGBS414SUS-08 ( FIG. 3 ).
  • the constructs pGBS414SUS-07 and pGBS414SUS-08 are independently transformed into S. cerevisiae strains CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), RWB066 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::Kanlox) and RWB064 (MATA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox). Transformation mixtures are plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose supplemented with appropriate amino acids.
  • YNB Yeast Nitrogen Base
  • Transformants are inoculated in Verduyn medium comprising glucose supplemented with appropriate amino acids (Verduyn et al., 1992, Yeast. July; 8(7):501-17) and grown under aerobic, anaerobic and oxygen-limited conditions in shake flasks.
  • the medium for anaerobic cultivation is supplemented with 0.01 g/l ergosterol and 0.42 g/l Tween 80 dissolved in ethanol (Andreasen and Stier, 1953, J. cell. Physiol, 41, 23-36; Andreasen and Stier, 1954, J. Cell. Physiol, 43: 271-281). All yeast cultures are grown at 30° C. in a shaking incubator at 250-280 rpm. At different incubation times, aliquots of the cultures are removed, centrifuged and the medium is analysed by HPLC for formation of oxalic acid, malic acid, fumaric acid and succinic acid as described in section 1.4.
  • Example 2A.1 mitochondrial fumarate reductase from Trypanosoma brucei (FRDm, SEQ ID NO: 9) was ligated in a S. cerevisiae expression vector pRS416 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27). The ligation mix was used for transformation of E. coli TOP10 cells (Invitrogen) resulting in the yeast expression constructs and pGBS416FRD-1 ( FIG. 7 ).
  • glycosomal fumarate reductase (FRDg, SEQ ID NO: 10) from Trypanosoma brucei was ligated in an S. cerevisiae expression vector pRS416.
  • the ligation mix was used for transformation of E. coli TOP10 cells (Invitrogen) resulting in the yeast expression construct pGBS416FRE-1 ( FIG. 8 ).
  • the constructs pGBS416FRD-1 and pGBS416FRE-1 were independently transformed into S. cerevisiae strain CEN.PK113-5D (MATA ura3-52). As negative control, empty vector pRS416 was transformed into strain CEN.PK 113-5D. Transformation mixtures were plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose.
  • YNB Yeast Nitrogen Base
  • Phosphoenolpyruvate carboxykinase [E.C. 4.1.1.49], Gen Bank accession number 152977907, from Actinobacillus succinogenes was analysed for the presence of signal sequences using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971.
  • This SEQ ID NO: 16 containing stop codon TAAG was put behind the constitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ ID NO: 26, and convenient restriction sites were added.
  • the resulting sequence SEQ ID NO: 29 was synthesised at Sloning (Puchheim, Germany).
  • SEQ ID NO: 18 containing stop codon TAAG was put behind the constitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ ID NO: 26. Convenient restriction sites were added. The resulting synthetic construct (SEQ ID NO: 30) was synthesised at Sloning (Puchheim, Germany).
  • Cytoplasmic malate dehydrogenase [E.C. 1.1.1.37], GenBank accession number 171915, is regulated by carbon catabolite repression: transcription of MDH2 is repressed and Mdh2p is degraded upon addition of glucose to glucose-starved cells. Mdh2p deleted for the 12 amino-terminal amino acids is less-susceptible for glucose-induced degradation (Minard and McAlister-Henn, J Biol Chem. 1992 Aug. 25; 267(24):17458-64).
  • SEQ ID NO: 19 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae .
  • SEQ ID NO: 20 containing a modified stop codon TAAG, encoding delta12NMDH2, was put behind the constitutive TDH3 promoter sequence SEQ ID NO: 12 and before the TDH3 terminator sequence SEQ ID NO: 13, and convenient restriction sites were added.
  • the resulting synthetic construct (SEQ ID NO: 31) was synthesised at Sloning (Puchheim, Germany).
  • Peroxisomal malate dehydrogenase [E.C. 1.1.1.37], GenBank accession number 1431095, was analysed for peroxisomal targeting in filamentous fungi using the PTS1 predictor http://mendel.imp.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp with the fungi-specific prediction function.
  • the C-terminal amino acids at position 341-343 (SKL) were removed from protein MDH3 resulting in SEQ ID NO: 21.
  • SEQ ID NO: 21 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae .
  • the stop codon TGA in the resulting sequence SEQ ID NO: 22 was modified to TAAG.
  • SEQ ID NO: 22 containing TAAG as stop codon was synthesized behind the constitutive TDH3 promoter sequence SEQ ID NO: 27 (600 by upstream of start codon) and before the TDH3 terminator sequence SEQ ID NO: 28 (300 by downstram of stop codon), and convenient restriction sites were added.
  • the resulting sequence SEQ ID NO: 32 was synthesised at Sloning (Puchheim, Germany).
  • Fumarase [E.C. 4.2.1.2], GenBank accession number 469103, from Rhizopus oryzae (FumR) was analysed for the presence of signal sequences using SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971. A putative mitochondrial targeting sequence in the first 23 amino acid of the protein was identified. To avoid potential targeting to mitochondria in S.
  • SEQ ID NO: 23 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae resulting in SEQ ID NO: 24.
  • the stop codon TAA in SEQ ID NO: 24 was modified to TAAG.
  • SEQ ID NO: 24 containing TAAG as stop codon was synthesized behind the constitutive TDH1 promoter sequence SEQ ID NO: 25 and before the TDH1 terminator sequence SEQ ID NO: 26 and convenient restriction sites were added.
  • the resulting synthetic construct SEQ ID NO: 33 was synthesised at Sloning (Puchheim, Germany).
  • the expression constructs pGBS414PPK-1 ( FIG. 9 ), pGBS414PPK-2 ( FIG. 10 ) and pGBS414PPK-3 ( FIG. 11 ) were created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the phosphoenolpyruvate carboxykinase (origin Actinobacillus succinogenes ) synthetic gene construct (SEQ ID NO: 29). The ligation mix was used for transformation of E.
  • an AscI/NotI restriction fragment consisting of glycosomal fumarate reductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 35) was ligated into the restricted pGBS414PPK-1 vector.
  • the ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-3 ( FIG. 11 ).
  • the expression constructs pGBS414PEK-1 ( FIG. 12 ), pGBS414PEK-2 ( FIG. 13 ) and pGBS414PEK-3 ( FIG. 14 ) were created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the phosphoenolpyruvate carboxykinase (origin Mannheimia succiniciproducens ) synthetic gene construct (SEQ ID NO: 30).
  • the ligation mix was used for transformation of E.
  • coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PEK-1.
  • pGBK414PEK-1 was restricted with AscI and NotI.
  • an AscI/NotI restriction fragment consisting of mitochondrial fumarate reductase from T. brucei (FRDm1) synthetic gene construct (SEQ ID NO: 34) was ligated into the restricted pGBS414PEK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PEK-2 ( FIG. 13 ).
  • an AscI/NotI restriction fragment consisting of glycosomal fumarate reductase from T. brucei (FRDg) synthetic gene construct (SEQ ID NO: 35) was ligated into the restricted pGBS414PEK-1 vector.
  • the ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PEK-3 ( FIG. 14 ).
  • the expression constructs pGBS415FUM-2 ( FIG. 15 ) and pGBS415FUM-3 ( FIG. 16 ) were created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS415 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the fumarase (origin Rhizopus oryzae ) synthetic gene construct (SEQ ID NO: 33).
  • the ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-1.
  • pGBK415FUM-1 was restricted with AscI and NotI.
  • an AscI/NotI restriction fragment consisting of cytoplasmic malate dehydrogenase from S. cerevisiae (delta12N MDH2) synthetic gene construct (SEQ ID NO: 31) was ligated into the restricted pGBS415FUM-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-2 ( FIG. 15 ).
  • an AscI/NotI restriction fragment consisting of peroxisomal malate dehydrogenase from S.
  • MDH3 cerevisiae (MDH3) synthetic gene construct (SEQ ID NO: 32) was ligated into the restricted pGBS415FUM-1 vector.
  • the ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS415FUM-3 ( FIG. 16 ).
  • Transformants were inoculated in 20 ml pre-culture medium consisting of Verduyn medium (Verduyn et al., 1992, Yeast. July; 8(7):501-17) comprising 2% galactose (w/v) and grown under aerobic conditions in 100 ml shake flasks in a shaking incubator at 30° C. at 250 rpm. After 72 hours, the culture was centrifuged for 5 minutes at 4750 rpm. 1 ml supernatant was used to measure succinic acid levels by HPLC as described in section 1.4. The remaining supernatant was decanted and the pellet (cells) was resuspended in 1 ml production medium.
  • the production medium consisted of Verduyn medium with 10% galactose (w/v) and 1% CaCO3 (w/v).
  • the resuspended cells were inoculated in 50 ml production medium in 100 ml shake flasks and grown in a shaking incubator at 30° C. at 100 rpm. At various time points, 1 ml sample was taken from the culture succinic acid levels were measured by HPLC as described in section 1.4 ( FIG. 17 ).
  • glycosomal fumarate reductase from T. brucei resulted in an even higher increase in succinic acid production levels; overexpression of PCKa, MDH3, FUMR and FRDg resulted in production of 3.9 g/L succinic acid, whereas overexexpression of PCKm, MDH3, FUMR and FRDg resulted in slightly lower production of 3.6 g/L succinic acid.
  • FRDg glycosomal fumarate reductase
  • SEQ ID NO: 37 Malate permease, GenBank accession number 119368831, from Schizosaccharomyces pombe (SEQ ID NO: 36) was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae resulting in SEQ ID NO: 37.
  • the stop codon TAA in SEQ ID NO: 37 was modified to TAAG.
  • SEQ ID NO: 37 containing TAAG as stop codon was put behind the constitutive ENO1 promoter sequence SEQ ID NO: 38 and before the ENO1 terminator sequence SEQ ID NO: 39, and convenient restriction sites were added.
  • T at position 596 ( ⁇ 5) was changed to A in order to obtain a better Kozak sequence.
  • the resulting sequence SEQ ID NO: 40 was synthesised at Sloning (Puchheim, Germany).
  • the expression constructs pGBS416MAE-1 ( FIG. 18 ) was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS416 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the Schizosaccharomyces pombe malate transporter synthetic gene construct (SEQ ID NO: 40). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS416MAE-1.
  • Plasmids pGBS414PEK-2, pGBS415FUM-2 and pGBS416MAE-1 (described under 2C.2.) were transformed into S. cerevisiae strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289) to create strain SUC-194, overexpressing PCKm, delta12NMDH2, FUMR, FRDm1 and SpMAE1. All genes were codon pair optimized for expression in S. cerevisiae.
  • the expression vectors were transformed into yeast by electroporation.
  • the transformation mixtures were plated on Yeast Nitrogen Base (YNB) w/o AA (Difco)+2% glucose.
  • YNB Yeast Nitrogen Base
  • AA Difco
  • strains SUC-101 is described in Table 2.
  • strains transformed with empty vectors produced up to 0.3 g/L succinic acid. Additional overexpression of SpMAE1 in strain SUC-194, overexpressing PCKm, delta12NMDH2, FUMR and FRDm1 resulted in increased succinic acid production levels to 4.6 g/L, whereas strain SUC-132, overexpressing PCKm, delta12NMDH2, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid.
  • Plasmids pGBS414PPK-3, pGBS415FUM-3 and pGBS416MAE-1 (described under 2C.2.) were transformed into S. cerevisiae strain RWB064 (MA TA ura3-52 leu2-112 trp1-289 adh1::lox adh2::lox gpd1::Kanlox) to create strain SUC-201, overexpressing PCKa, MDH3, FUMR, FRDg and SpMAE1. All genes were codon pair optimized for expression in S. cerevisiae.
  • Strain SUC-103 transformed with empty vectors (control strain) produced 0.9 g/L succinic acid after growth for 10 days in production medium ( FIG. 20 ).
  • Overexpression of PCKa, MDH3, FUMR and FRDg in strain RWB064 resulted in increased succinic acid production levels to 2.5 g/L (strain SUC-201, FIG. 20 ).
  • Additional overexpression of SpMAE1 besides PCKa, MDH3, FUMR and FRDg in strain RWB064 resulted in a further increase of succinic acid production levels to 11.9 g/L (strain SUC-200, FIG. 20 ).
  • cerevisiae strain CEN.PK113-5D (MATA ura3-52) was used as template to amplify the PYC2 coding sequence (SEQ ID NO: 42), using primers P1 (SEQ ID NO: 43) and P2 (SEQ ID NO: 44), and the Phusion DNA polymerase (Finnzymes, Finland) according to manufacturer's instructions. Convenient restriction sites were included in the primers for further cloning purposes.
  • the expression construct pGBS426PYC-2 ( FIG. 21 ) was created after a SpeI/XhoI restriction of the S. cerevisiae expression vector p426GPD (Mumberg et al., Gene. 1995 Apr. 14; 156(1):119-22) and subsequently ligating in this vector a SpeI/XhoI restriction fragment consisting of the amplified PYC2 nucleotide sequence (SEQ ID NO: 42). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS426PYC-2 ( FIG. 21 ).
  • Expression construct pGBS414FRE-1 was created after a BamHI/NotI restriction of the S. cerevisiae expression vector pRS414 (Sirkoski R. S. and Hieter P, Genetics, 1989, 122(1):19-27) and subsequently ligating in this vector a BamHI/NotI restriction fragment consisting of the glycosomal fumarate reductase (origin Trypanosoma brucei ) synthetic gene construct (SEQ ID NO: 35). The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414FRE-1 ( FIG. 22 ).
  • Strains SUC-226, SUC-227, SUC-228 and SUC-230 were obtained by transformation of different combinations of the plasmids pGBS414FRE-1, pGBS414PPK-3, pGBS415FUM-1, pGBS426PYC-2 and p426GPD into strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), as depicted in Table 5.
  • strain SUC-230 overexpressing MDH3, FUMR and FRDg, produced up to 3.0 g/L succinic acid.
  • Additional overexpression of PCKa increased succinic acid production up to 3.4 g/L (strain SUC-226)
  • additional overexpression of PYC2 increased succinic acid production up to 3.7 g/L (strain SUC-228).
  • overexpression of both PCKa and PYC2 resultsed in 1.5 increase of succinic acid production levels up to 5.0 g/L, as compared to the effect of PCK and PYC alone.
  • Genomic DNA of Aspergillus niger strain CBS513.88 was sequenced and analyzed. Two genes with translated proteins annotated as homologues to succinate dehydrogenase proteins were identified and named sdhA and sdhB respectively. Sequences of the sdhA (An16g07150) and sdhB (An02g12770) loci are available on genbank with accession numbers 145253004 and 145234071 respectively. Gene replacement vectors for sdhA and sdhB were designed according to known principles and constructed according to routine cloning procedures (see FIG. 6 ).
  • the vectors comprise approximately 1000 by flanking regions of the sdh ORFs for homologous recombination at the predestined genomic loci. In addition, they contain the A. nidulans bi-directional amdS selection marker driven by the gpdA promoter, in-between direct repeats.
  • the general design of these deletion vectors were previously described in EP635574B and WO 98/46772.
  • Linear DNA of deletion vector pDEL-SDHA ( FIG. 4 ) was isolated and used to transform Aspergillus niger CBS513.88 as described in: Biotechnology of Filamentous fungi: Technology and Products. (1992) Reed Publishing (USA); Chapter 6: Transformation p. 113 to 156.
  • This linear DNA can integrate into the genome at the sdhA locus, thus substituting the sdhA gene by the amdS gene as depicted in FIG. 6 .
  • Transformants were selected on acetamide media and colony purified according to standard procedures as described in EP635574B. Spores were plated on fluoro-acetamide media to select strains, which lost the amdS marker.
  • Strain dSDHA was selected as a representative strain with the sdhA gene inactivated.
  • the succinic acid production of dSDHA was determined in microtiter plates as described in Example 4.
  • A. niger strain dSDHA of example 3.2. was transformed with the expression construct pGBTOPAn1 ( FIG. 5 ) comprising truncated mitochondrial fumarate reductase m1 (FRDm1, SEQ ID NO:7) as described in Example 1.1.
  • E. coli DNA was removed by NotI digestion.
  • A. niger transformants were picked using Qpix and transferred onto MTP's containing Aspergillus selective media. After 7 days incubation at 30 degrees Celsius the biomass was transferred to microtiter plates (MTP's) containing PDA by hand or colony picker. After 7 days incubation at 30 degrees Celsius, the biomass was sporulated.
  • MTP's microtiter plates
  • Table 6 clearly shows an increased production of succinic acid by A. niger that comprises mitochondrial fumarate reductase from T. brucei

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Mycology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Medicinal Preparation (AREA)
  • Cosmetics (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Fuel Cell (AREA)
US12/743,106 2007-11-20 2008-11-14 Succinic acid production in a eukaryotic cell Abandoned US20120165569A1 (en)

Applications Claiming Priority (13)

Application Number Priority Date Filing Date Title
EP07121117.1 2007-11-20
EP07121113.0 2007-11-20
EP07121120 2007-11-20
EP07121117 2007-11-20
EP07121113 2007-11-20
EP07121120.5 2007-11-20
EP08156961 2008-05-27
EP08156960 2008-05-27
EP08156959.2 2008-05-27
EP08156960.0 2008-05-27
EP08156959 2008-05-27
EP08156961.8 2008-05-27
PCT/EP2008/065583 WO2009065778A1 (en) 2007-11-20 2008-11-14 Succinic acid production in a eukaryotic cell

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2008/065583 A-371-Of-International WO2009065778A1 (en) 2007-11-20 2008-11-14 Succinic acid production in a eukaryotic cell

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/044,722 Continuation US9689005B2 (en) 2007-11-20 2013-10-02 Succinic acid production in a eukaryotic cell

Publications (1)

Publication Number Publication Date
US20120165569A1 true US20120165569A1 (en) 2012-06-28

Family

ID=40268299

Family Applications (3)

Application Number Title Priority Date Filing Date
US12/743,927 Active 2030-05-05 US9340804B2 (en) 2007-11-20 2008-11-14 Dicarboxylic acid production in eukaryotes
US12/743,106 Abandoned US20120165569A1 (en) 2007-11-20 2008-11-14 Succinic acid production in a eukaryotic cell
US14/044,722 Active US9689005B2 (en) 2007-11-20 2013-10-02 Succinic acid production in a eukaryotic cell

Family Applications Before (1)

Application Number Title Priority Date Filing Date
US12/743,927 Active 2030-05-05 US9340804B2 (en) 2007-11-20 2008-11-14 Dicarboxylic acid production in eukaryotes

Family Applications After (1)

Application Number Title Priority Date Filing Date
US14/044,722 Active US9689005B2 (en) 2007-11-20 2013-10-02 Succinic acid production in a eukaryotic cell

Country Status (10)

Country Link
US (3) US9340804B2 (ja)
EP (3) EP2220232B1 (ja)
JP (2) JP5641938B2 (ja)
CN (3) CN101903522B (ja)
AT (1) ATE555203T1 (ja)
BR (2) BRPI0819273B8 (ja)
CA (2) CA2875319A1 (ja)
EA (1) EA032726B1 (ja)
ES (2) ES2383473T3 (ja)
WO (2) WO2009065780A1 (ja)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120040422A1 (en) * 2009-04-15 2012-02-16 Dsm Ip Asset B.V. Dicarboxylic acid production process
US20140363862A1 (en) * 2012-01-25 2014-12-11 BioAmber International S.à.r.l. Methods for Succinate Production
US9624514B2 (en) 2011-07-01 2017-04-18 Dsm Ip Assets B.V. Process for preparing dicarboxylic acids employing fungal cells
DE102016115425A1 (de) 2016-08-19 2018-02-22 Jacobs University Bremen Ggmbh Gentechnisch veränderte Hefe zur Fermentation von Glycerol
US10787649B2 (en) 2016-07-13 2020-09-29 Dsm Ip Assets B.V. Malate dehyrogenases
US11041176B2 (en) 2012-07-25 2021-06-22 Cargill, Incorporated Yeast cells having reductive TCA pathway from pyruvate to succinate and overexpressing an exogenous NAD(P)+ transhydrogenase enzyme
US11390873B2 (en) 2011-01-25 2022-07-19 Cargill, Incorporated Compositions and methods for succinate production

Families Citing this family (49)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009011974A1 (en) * 2007-05-18 2009-01-22 Microbia Precision Engineering, Inc. Organic acid production by fungal cells
CN101903522B (zh) * 2007-11-20 2015-08-05 帝斯曼知识产权资产管理有限公司 在真核生物中生产二羧酸
CA2714088A1 (en) * 2008-02-15 2009-08-20 Dsm Ip Assets B.V. Process for the production of a dicarboxylic acid
CN107267561A (zh) 2008-07-08 2017-10-20 帝斯曼知识产权资产管理有限公司 通过在低pH下发酵生产二羧酸
RU2422526C2 (ru) * 2009-01-30 2011-06-27 Федеральное государственное унитарное предприятие "Государственный научно-исследовательский институт генетики и селекции промышленных микроорганизмов (ФГУП ГосНИИгенетика) СПОСОБ ПОЛУЧЕНИЯ ЯНТАРНОЙ КИСЛОТЫ С ИСПОЛЬЗОВАНИЕМ ДРОЖЖЕЙ, ПРИНАДЛЕЖАЩИХ К РОДУ Yarrowia
UA108853C2 (uk) 2009-07-10 2015-06-25 Спосіб ферментації галактози
ES2655308T3 (es) 2009-08-27 2018-02-19 Dsm Ip Assets B.V. Procedimiento de fermentación de ácido dicarboxílico
BR112012004594A2 (pt) 2009-09-01 2016-06-21 Novozymes Inc célula hospedeira fúngica filamentosa, e, método para produzir um ácido dicarboxílico
US20120238722A1 (en) * 2009-11-24 2012-09-20 Roquette Freres Sa Process for the crystallization of succinic acid
JP2011120508A (ja) * 2009-12-09 2011-06-23 Oji Paper Co Ltd コハク酸を製造する方法
EP2519491A4 (en) 2009-12-31 2014-10-08 Groupe Novasep Sas PURIFICATION OF BERNSTEINIC ACID FROM THE FERMENTATION OF SPROUTS WITH AMMONIUM UCCINATE
CN102812127B (zh) 2010-03-09 2014-12-03 三菱化学株式会社 琥珀酸的制造方法
EP2371802A1 (en) 2010-03-30 2011-10-05 DSM IP Assets B.V. Process for the crystallization of succinic acid
CN102869766B (zh) 2010-04-21 2015-11-25 帝斯曼知识产权资产管理有限公司 适于发酵混合的糖组合物的细胞
US8617859B2 (en) 2010-06-04 2013-12-31 Novozymes, Inc. C4 dicarboxylic acid production in filamentous fungi
KR20130113937A (ko) 2010-06-21 2013-10-16 노보자임스 인코포레이티드 Ｃ4―디카르복실산 수송체 활성을 가진 아스페르질루스 아쿨레아투스 유래한 폴리펩티드 및 그것을 암호화하는 폴리뉴클레오티드
EP2582829B1 (en) 2010-06-21 2016-11-30 Novozymes, Inc. Methods for improved c4-dicarboxylic acid production in filamentous fungi
US9187772B2 (en) * 2010-09-01 2015-11-17 University Of Florida Research Foundation, Inc. L-malate production by metabolically engineered escherichia coli
EP2619314A1 (en) 2010-09-24 2013-07-31 DSM IP Assets B.V. Dicarboxylic acid production process
AR083613A1 (es) * 2010-10-28 2013-03-06 Total Sa Proceso para la produccion de acido polilactico usando monascus
EP2495304A1 (en) 2010-12-03 2012-09-05 DSM IP Assets B.V. Dicarboxylic acid production in a yeast cell
CN103492551A (zh) 2011-02-28 2014-01-01 诺维信股份有限公司 用于生产c4-二羧酸的微生物
ES2579706T3 (es) 2011-08-19 2016-08-16 Novozymes, Inc. Microorganismos recombinantes para la producción de ácidos C4-dicarboxílicos
CN102559518B (zh) * 2011-12-20 2013-10-16 江南大学 一种高产延胡索酸米根霉及其应用
FR2987678B1 (fr) 2012-03-02 2016-04-15 Roquette Freres Methode de mesure de la stabilite thermique d'un acide succinique cristallin destine a la fabrication de polymeres
FR2988733B1 (fr) 2012-03-27 2016-02-05 Carbios Microorganisme recombinant
KR101464656B1 (ko) 2012-06-15 2014-12-02 한국생명공학연구원 발효산물 고생성능을 가지는 변이 미생물 및 이를 이용한 발효산물의 제조방법
US10066246B2 (en) 2012-07-25 2018-09-04 Cargill, Incorporated Yeast cells having NADP(H)-dependent reductive TCA pathway from pyruvate to succinate
BR112015004488A2 (pt) * 2012-08-28 2018-11-21 Braskem Sa método de coprodução de um terpeno e pelo menos um coproduto a partir de uma fonte de carbono fermentável
KR102253532B1 (ko) 2012-09-14 2021-05-18 피티티 글로벌 케미컬 퍼블릭 컴퍼니 리미티드 낮은 ph에서 발효에 의한 유기산의 제조
EP2898082B1 (en) 2012-09-20 2019-09-04 LCY Bioscience Inc. Pathways to adipate semialdehyde and other organic products
WO2014135712A2 (en) 2013-03-08 2014-09-12 Dsm Ip Assets B.V. Polyester
WO2015007902A1 (en) * 2013-07-18 2015-01-22 Dsm Ip Assets B.V. Fermentation process
CA2931591C (en) * 2013-12-12 2023-09-19 Dsm Ip Assets B.V. Fumarate reductases
WO2016002680A1 (ja) * 2014-06-30 2016-01-07 旭硝子株式会社 形質転換体およびその製造方法、ならびに炭素数４のジカルボン酸の製造方法
US9882151B2 (en) 2014-11-14 2018-01-30 Universal Display Corporation Organic electroluminescent materials and devices
EP3227256B1 (en) 2014-12-02 2019-03-13 Roquette Freres Process for manufacturing succinic acid from a fermentation broth using nanofiltration to purify recycled mother liquor
EP3067378A1 (en) 2015-03-11 2016-09-14 DSM IP Assets B.V. Polyester
US20180179499A1 (en) 2015-06-04 2018-06-28 Bioamber Inc. Biobased production of functionalized alpha-substituted acrylates and c4-dicarboxylates
US11078504B2 (en) * 2015-12-23 2021-08-03 Dsm Ip Assets B.V. Yeast host cells for dicarboxylic acid production
JP2017121184A (ja) * 2016-01-04 2017-07-13 花王株式会社 変異リゾプス属菌
WO2018211132A1 (en) 2017-05-18 2018-11-22 Avantium Knowledge Centre B.V. Polyester copolymer
US11306179B2 (en) 2017-05-18 2022-04-19 Avantium Knowledge Centre B.V. Polyester copolymer
US11608488B2 (en) * 2017-07-11 2023-03-21 Adisseo France S.A.S. Enhanced metabolite-producing yeast
US20210207076A1 (en) * 2018-05-25 2021-07-08 Danisco Us Inc. Overexpression of fumarate reductase results in an increased fermentation rate in yeast
KR102129379B1 (ko) * 2018-10-10 2020-07-02 한국과학기술원 고활성의 말산 탈수소효소가 도입된 숙신산 생성용 변이 미생물 및 이를 이용한 숙신산 제조방법
JP7284262B2 (ja) 2018-11-22 2023-05-30 アバンティウム・ナレッジ・センター・ベー・フェー １種又は複数種のポリエステルコポリマーを製造するための方法、１種又は複数種のオリゴマーを調製するための方法、オリゴマー組成物、及びポリエステルコポリマー
CN113249238B (zh) * 2021-05-07 2022-08-23 江南大学 一株耐酸酿酒酵母及其在有机酸制备中的应用
CN114806913B (zh) * 2022-04-15 2023-11-28 盛虹控股集团有限公司 具有线粒体定位还原tca途径的高产琥珀酸酵母工程菌株及其构建方法和应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643758A (en) * 1987-03-10 1997-07-01 New England Biolabs, Inc. Production and purification of a protein fused to a binding protein

Family Cites Families (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3542861A1 (de) 1985-12-04 1987-06-11 Huels Chemische Werke Ag Verfahren zur gewinnung von l-aepfelsaeure
WO1990014423A1 (en) 1989-05-18 1990-11-29 The Infergene Company Microorganism transformation
DK0778348T3 (da) 1989-07-07 2000-12-04 Unilever Nv Fremgangsmåde til fremstilling af et protein ved anvendelse af en svamp transformeret ved multikopi-integration af en ekspr
EP1321523A3 (en) 1993-07-23 2004-03-03 DSM IP Assets B.V. Selection marker gene free recombinant strains; a method for obtaining them and the use of these strains
US6265186B1 (en) 1997-04-11 2001-07-24 Dsm N.V. Yeast cells comprising at least two copies of a desired gene integrated into the chromosomal genome at more than one non-ribosomal RNA encoding domain, particularly with Kluyveromyces
EP0979294B1 (en) 1997-04-11 2015-05-27 DSM IP Assets B.V. Gene conversion as a tool for the construction of recombinant industrial filamentous fungi
AU4144999A (en) 1998-05-19 1999-12-06 Dsm N.V. Improved (in vivo) production of cephalosporins
WO2000037671A2 (en) 1998-12-22 2000-06-29 Dsm N.V. Improved in vivo production of cephalosporins
BRPI0413403A (pt) * 2003-08-28 2006-10-17 Mitsubishi Chem Corp método para produzir ácido succìnico
DE102004011248A1 (de) * 2004-03-09 2005-09-22 Degussa Ag Verfahren zur Herstellung von L-Aminosäuren unter Verwendung coryneformer Bakterien
EP2460872A1 (en) * 2004-12-22 2012-06-06 Michigan Biotechnology Institute Recombinant microorganisms for increased production of organic acids
US7435168B2 (en) * 2005-01-14 2008-10-14 Archer-Daniels-Midland Company Compositions and methods for manipulating carbon flux in cells
DE102005031801B3 (de) * 2005-07-07 2006-08-24 Carsten Paulsen Reinigungsanlage für Türme von Windkraftanlagen
KR20080031504A (ko) 2005-08-05 2008-04-08 미시간 스테이트 유니버시티 악티노바실러스 숙시노제네스의 ｃ4-경로로부터 화학물질을생산하기 위해 악티노바실러스 숙시노제네스130ｚ(ａｔｃｃ 55618)로부터 얻은 유전자
KR100727054B1 (ko) 2005-08-19 2007-06-12 한국과학기술원 푸마레이트 하이드라타제 ｃ를 코딩하는 유전자로 형질전환된 재조합 미생물 및 이를 이용한 숙신산의 제조방법
AU2006287257A1 (en) * 2005-09-09 2007-03-15 Genomatica, Inc. Methods and organisms for the growth-coupled production of succinate
US20080090273A1 (en) * 2005-11-21 2008-04-17 Aaron Adriaan Winkler Malic Acid Production in Recombinant Yeast
EP1867727A1 (en) * 2006-06-15 2007-12-19 Danmarks Tekniske Universitet Enhanced citrate production
WO2008000632A1 (en) 2006-06-29 2008-01-03 Dsm Ip Assets B.V. A method for achieving improved polypeptide expression
CN101688176B (zh) * 2007-04-17 2013-11-06 味之素株式会社 具有羧基的酸性物质的生产方法
EP2155885A4 (en) 2007-05-18 2010-12-01 Microbia Prec Engineering Inc ACIDIC ACID PRODUCTION IN RECOMBINANT YEAST
WO2009011974A1 (en) 2007-05-18 2009-01-22 Microbia Precision Engineering, Inc. Organic acid production by fungal cells
CN101903522B (zh) * 2007-11-20 2015-08-05 帝斯曼知识产权资产管理有限公司 在真核生物中生产二羧酸
EP2807262A4 (en) 2012-01-25 2015-12-30 Bioamber Internat S A R L PROCESS FOR SUCCINATE MANUFACTURE
KR102253532B1 (ko) 2012-09-14 2021-05-18 피티티 글로벌 케미컬 퍼블릭 컴퍼니 리미티드 낮은 ph에서 발효에 의한 유기산의 제조

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5643758A (en) * 1987-03-10 1997-07-01 New England Biolabs, Inc. Production and purification of a protein fused to a binding protein

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
De Jongh et al. [Metabolic Engineering, Vol. 10, No. 2, pages 89-96 (17 November 2007). *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120040422A1 (en) * 2009-04-15 2012-02-16 Dsm Ip Asset B.V. Dicarboxylic acid production process
US9353387B2 (en) * 2009-04-15 2016-05-31 Dsm Ip Assets B.V. Dicarboxylic acid production process
US11390873B2 (en) 2011-01-25 2022-07-19 Cargill, Incorporated Compositions and methods for succinate production
US9624514B2 (en) 2011-07-01 2017-04-18 Dsm Ip Assets B.V. Process for preparing dicarboxylic acids employing fungal cells
US20140363862A1 (en) * 2012-01-25 2014-12-11 BioAmber International S.à.r.l. Methods for Succinate Production
US9885065B2 (en) * 2012-01-25 2018-02-06 Bioamber Inc. Methods for succinate production
US11041176B2 (en) 2012-07-25 2021-06-22 Cargill, Incorporated Yeast cells having reductive TCA pathway from pyruvate to succinate and overexpressing an exogenous NAD(P)+ transhydrogenase enzyme
US11821021B2 (en) 2012-07-25 2023-11-21 Cargill, Incorporated Yeast cells having reductive TCA pathway from pyruvate to succinate and overexpressing an exogenous NAD(P+) transhydrogenase enzyme
US10787649B2 (en) 2016-07-13 2020-09-29 Dsm Ip Assets B.V. Malate dehyrogenases
US11339379B2 (en) 2016-07-13 2022-05-24 Dsm Ip Assets B.V. Malate dehyrogenases
DE102016115425A1 (de) 2016-08-19 2018-02-22 Jacobs University Bremen Ggmbh Gentechnisch veränderte Hefe zur Fermentation von Glycerol
WO2018033176A1 (de) 2016-08-19 2018-02-22 Jacobs University Bremen Ggmbh Gentechnisch veränderte hefe zur fermentation von glycerol

Also Published As

Publication number Publication date
BRPI0819275B1 (pt) 2022-05-17
CA2704654C (en) 2015-02-10
EP2220233B1 (en) 2012-04-25
US20140031587A1 (en) 2014-01-30
CN101978063B (zh) 2018-10-16
WO2009065780A1 (en) 2009-05-28
EA201000837A1 (ru) 2010-12-30
US9689005B2 (en) 2017-06-27
JP2011502524A (ja) 2011-01-27
CN104818224A (zh) 2015-08-05
EP2687602A1 (en) 2014-01-22
US20110081694A1 (en) 2011-04-07
BRPI0819273B1 (pt) 2022-05-17
WO2009065778A1 (en) 2009-05-28
BRPI0819275A2 (pt) 2014-10-14
CA2875319A1 (en) 2009-05-28
CN101978063A (zh) 2011-02-16
BRPI0819273B8 (pt) 2022-07-19
ES2522622T3 (es) 2014-11-17
JP2014236739A (ja) 2014-12-18
BRPI0819273A2 (pt) 2014-10-14
CN101903522A (zh) 2010-12-01
CN101903522B (zh) 2015-08-05
EP2220232B1 (en) 2014-09-03
JP5641938B2 (ja) 2014-12-17
ATE555203T1 (de) 2012-05-15
EP2220232A1 (en) 2010-08-25
EP2220233A1 (en) 2010-08-25
ES2383473T3 (es) 2012-06-21
EA032726B1 (ru) 2019-07-31
CA2704654A1 (en) 2009-05-28
BRPI0819275B8 (pt) 2022-07-19
US9340804B2 (en) 2016-05-17

Similar Documents

Publication Publication Date Title
US9689005B2 (en) Succinic acid production in a eukaryotic cell
US8735112B2 (en) Dicarboxylic acid production in a recombinant yeast
US20110104771A1 (en) Process for the production of a dicarboxylic acid
EP2495304A1 (en) Dicarboxylic acid production in a yeast cell
US20150291986A1 (en) Itaconic acid and itaconate methylester production
US20140045230A1 (en) Dicarboxylic acid production in a filamentous fungus
US20170191089A1 (en) Itaconic acid and itaconate methylester and dimethylester production
CA3008963A1 (en) Host cells for dicarboxylic acid production
WO2015181311A1 (en) Process for producing itaconic acid and itaconic acid esters under anaerobic conditions

Legal Events

Date Code Title Description
AS Assignment

Owner name: DSM IP ASSETS B.V., NETHERLANDS

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:VERWAAL, RENE;WU, LIANG;DAMVELD, ROBBERTUS ANTONIUS;AND OTHERS;REEL/FRAME:024387/0761

Effective date: 20100412

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION