# Patent

## Title
Succinic acid production in a eukaryotic cell

## Abstract

The present invention relates to a recombinant eukaryotic cell selected from a yeast of a filamentous fungus comprising a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyzes the conversion of fumaric acid to succinic acid. The invention further relates to a process for the production of succinic acid wherein the eukaryotic cell according to the present invention is used.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of U.S. application Ser. No. 12/743,106, filed May 14, 2010, which is a §371 National Stage Application of International Application No. PCT/EP2008/065583, filed Nov. 14, 2008, which claims priority to European Application No. 07121117.1, filed Nov. 20, 2007, European Application No. 07121120.5, filed Nov. 20, 2007, European Application No. 07121113.0, filed Nov. 20, 2007, European Application No. 08156961.8, filed May 27, 2008, European Application No. 08156960.0, filed May 27, 2008, and European Application No. 08156959.2, filed May 27, 2008, the content of all of which are hereby incorporated herein by reference in their entireties.


BACKGROUND
Field of the Invention
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.
Description of Related Art
Succinic acid is a potential precursor for numerous chemicals. For example, succinic acid can be converted into 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. 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). Despite the improvements that have been made in the fermentative production of succinic acid, there remains a need for improved microorganisms for the fermentative production of succinic acid.
SUMMARY
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.
Surprisingly it was found that 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. Preferably, 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.



DETAILED DESCRIPTION OF THE DRAWINGS
FIGS. 1-23 represent embodiments as described herein.



DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT
As used herein, a recombinant eukaryotic cell according to the present invention 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 (3rd edition), Cold Spring Harbor Laboratory Press.
The term “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.
The term “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 FADH2-dependent fumarate reductase, wherein FADH2 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.
Preferably, 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. Preferably, 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. in a solution comprising about 1 M salt, preferably 6×SSC (sodium chloride, sodium citrate) or any other solution having a comparable ionic strength, and washing at 65° C. in a solution comprising about 0.1 M salt, or less, preferably 0.2×SSC or any other solution having a comparable ionic strength. Preferably, 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 about 90% or more sequence identity.
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. Preferably, 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%.
To increase the likelihood that an introduced enzyme(s) is/are expressed in active form in a eukaryotic cell of the invention, the corresponding encoding nucleotide sequence may be adapted to optimise its codon usage to that of the chosen eukaryote host cell. Several methods for 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.
The term “gene”, as used herein, 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.
The term “nucleic acid” as used herein, 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.
The terms “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. The terms “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.
The term “enzyme” as used herein is defined as a protein which catalyses a (bio)chemical reaction in a cell.
Usually, the 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.
As used herein, the term “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. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
As used herein, the term “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. Preferably, 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.
Usually a 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).
In a preferred embodiment, a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase may be overexpressed to achieve a sufficient production of succinic acid by the cell.
There are various means available in the art for overexpression of nucleotide sequences encoding enzymes in a eukaryotic cell of the invention. In particular, 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. Preferably, 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. If the eukaryotic cell is of fungal origin, a suitable episomal nucleic acid construct may e.g. be based on the yeast 2μ or pKD1 plasmids (Gleer et al., 1991, Biotechnology 9: 968-975), or the AMA plasmids (Fierro et al., 1995, Curr Genet. 29:482-489). Alternatively, 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. Preferably, the NADH-dependent fumarate reductase is a heterologous enzyme, which may be derived from any suitable origin, for instance bacteria, fungi, protozoa or plants. Preferably, the cell according to the invention comprises hetereologous a NAD(H)-dependent fumarate reductase, preferably derived from a Trypanosoma sp, for instance a Trypanosomal brucei. 
In a preferred embodiment the nucleotide sequence encoding a NAD(H)-dependent fumarate reductase is expressed in the cytosol. Surprisingly, cytosolic activity of the enzyme resulted in an increased productivity of succinic acid by the eukaryotic cell.
In the event that the 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 Schlüter et al, Nucleic acid Research 2007, 35, D815-D822.
Preferably, 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.
Preferably, 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. Preferably 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. More preferably, 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 bailii. 
In addition to a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid, recombinant eukaryotic cell according to the present invention 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. It may for example be favourable to introduce, genetically modify and/or overexpress 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). 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.
Preferably 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. Preferably, 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.
In another preferred embodiment, 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. Surprisingly it was found that 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. Preferably, 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. Preferably, 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. In one embodiment the PEP carboxykinase of Actinobacillus succinogenes (PCKa) has been modified to replace EGY at position 120-122 with a DAF amino acid sequence. Preferably, a eukaryotic cell according to the present invention 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. Surprisingly it was found that the concomitant (over)expression of a PYC and a PEP carboxykinase as described herein resulted in at least 1.5 increase in succinic acid production.
In another preferred embodiment 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. A cytosolic MDH may be any suitable homologous or heterologous malate dehydrogenase. The MDH may be a S. cerevisiae MDH3 or S. cerevisiae MDH1. Preferably, the MDH lacks a peroxisomal or mitochondrial targeting signal in order to localize the enzyme in the cytosol. Alternatively, 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. It is known that the transcription of MDH2 is repressed and Mdh2p is degraded upon addition of glucose to glucose-starved cells. 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). Preferably, a eukaryotic cell according to the present invention 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. Preferably the malate dehydrogenase comprises SEQ ID NO: 19 or SEQ ID NO: 21. Preferably, the activity of malate dehydrogenase is increased by overexpressing the encoding nucleotide sequence by known methods in the art.
Preferably, 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. Preferably, 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. Preferably, the fumarase comprises SEQ ID NO: 23. Preferably 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.
In another embodiment, a eukaryotic cell according to the present invention 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. Preferably 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. Preferably, 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. Preferably the MAE comprises SEQ ID NO: 36. Surprisingly, it was found that 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. Preferably, the malic acid transporter is derived from Schizosaccharomyces pombe. 
In a preferred embodiment a eukaryotic cell according to the present invention 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. Surprisingly, it found that 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.
In another preferred embodiment 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.
Preferably, 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. Preferably, 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.
Preferably, 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. Surprisingly, it was found that 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.
In another preferred embodiment 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.
Preferably, 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.
Preferably, 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. Hence, a preferred host organism expresses enzymes such as cellulases (endocellulases and exocellulases) and hemicellulases (e.g. endo- and exo-xylanases, arabinases) necessary for the conversion of cellulose into glucose monomers and hemicellulose into xylose and arabinose monomers, pectinases able to convert pectines into glucuronic acid and galacturonic acid or amylases to convert starch into glucose monomers. Preferably, 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.
In another aspect, 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.
It was found advantageous to use a eukaryotic cell according to the invention in the process for the production of succinic acid, because most eukaryotic cells do not require sterile conditions for propagation and are insensitive to bacteriophage infections.
Preferably, 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. Preferably, 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. Preferably, in a process under oxygen-limited conditions, 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. Preferably, 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.
Preferably, succinic acid is recovered from the fermentation broth by a suitable method known in the art, for instance by crystallisation and ammonium precipitation.
Preferably, 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.
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.
Genetic Modifications
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 (3rd 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, WO90/14423, EP-A-0481008, EP-A-0635 574 and U.S. Pat. No. 6,265,186.
The following examples are for illustrative purposes only and are not to be construed as limiting the invention.
DESCRIPTION OF THE FIGURES
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. CPO denotes codon pair optimized.
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. 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 (X, PCKm, MDH3, FUMR) and SUC-169 (▴, PCKm, delta12NMDH2, FUMR, FRDm1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae. All data represent averages of 3 independent growth experiments of SUC-148, 149, 150, 151, 152, 154 and SUC-169 and averages of 6 independent growth experiments of SUC-101.
FIG. 18: Plasmid map of pGBS416MAE-1, containing malate permease from Schizosaccharomyces pombe (SpMAE1) for expression in Saccharomyces cerevisiae. The synthetic gene construct Eno1 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.
EXAMPLES
Example 1
Cloning of Fumarate Reductases from Trypanosoma Brucei in Aspergillus Niger 
1.1. Expression Constructs
Mitochondrial fumarate reductase m1 (FRDm1) [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
(www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1
(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).
It was shown that FRDm1 recombinant protein lacking the 68 N-terminal residues, relocalized to the cytosol of the procyclic trypanosomes (Coustou et al., J Biol Chem. 2005 Apr. 29; 280(17):16559-70). These results indicate that the predicted N-terminal signal motif of FRDm1 is required for targeting to the mitochondrion. The first 68 amino acids were removed from SEQ ID NO: 1 (corresponding to nucleotide sequence SEQ ID NO: 2) and a new methionine amino acid was reintroduced, which resulted in SEQ ID NO: 3. 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).
Likewise, glycosomal fumarate reductase (FRDg) [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 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.
1.2. Transformation of A. Niger 
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.
The expression constructs are co-transformed to strain A. niger WT-1 according to the method described by Tilburn, J. et al. (1983) Gene 26, 205-221 and Kelly, J. & Hynes, M. (1985) EMBO J., 4, 475-479 with the following modifications:

    
    
        Spores are germinated and cultivated for 16 hours at 30 degrees Celsius in a shake flask placed in a rotary shaker at 300 rpm in Aspergillus minimal medium (100 ml). Aspergillus minimal medium contains per liter: 6 g NaNO3, 0.52 g KCl, 1.52 g KH2PO4, 1.12 ml 4 M KOH, 0.52 g MgSO4.7H2O, 10 g glucose, 1 g casaminoacids, 22 mg ZnSO4.7H2O, 11 mg H3BO3, 5 mg FeSO4.7H2O, 1.7 mg CoCl2.6H2O, 1.6 mg CuSO4.5H2O, 5 mg MnCl2.2H2O, 1.5 mg Na2MoO4.2H2O, 50 mg EDTA, 2 mg riboflavin, 2 mg thiamine-HCl, 2 mg nicotinamide, 1 mg pyridoxine-HCL, 0.2 mg panthotenic acid, 4 g biotin, 10 ml Penicillin (5000 UI/ml) Streptomycin (5000 UG/ml) solution (Gibco).
        Novozym 234TM (Novo Industries) instead of helicase is used for the preparation of protoplasts;
        After protoplast formation (60-90 minutes), 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 CaCl2) is added. 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;
        To 200 microliter of the protoplast suspension, 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 CaCl2) is added;
        After incubation of the DNA-protoplast suspension for 10 minutes at room temperature, 1.5 ml PEG solution (60% PEG 4000 (Merck), 10 mM Tris-HCl pH 7.5, 50 mM CaCl2) is added slowly, with repeated mixing of the tubes. After incubation for 20 minutes at room temperature, suspensions are diluted with 5 ml 1.2 M sorbitol, mixed by inversion and centrifuged for 10 minutes at 4000 rpm at room temperature. 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. In case of acetamide selection the medium contains 10 mM acetamide as the sole nitrogen source and 1 M sucrose as osmoticum and C-source. Alternatively, 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.

1.3. Shake Flask Growth of A. Niger 

    
    


In total 10 transformants are selected for each construct and the presence of the construct is confirmed by PCR using primers specific for the constructs. Subsequently spores are inoculated in 100 ml Aspergillus minimal enriched medium comprising 100 g/l glucose. Strains are grown in an incubator at 250 rotations per minute for four days at 34 degrees Celsius. The supernatant of the culture medium is analysed for oxalic acid, malic acid, fumaric acid and succinic acid formation by HPLC and compared to a non transformed strain.
1.4 HPLC Analysis
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.
Example 2A
Cloning of Fumarate Reductases from Trypanosoma Brucei in Saccharomyces Cerevisiae 
2A.1. Expression Constructs
Mitochondrial fumarate reductase m1 (FRDm1) [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).
Likewise, 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. 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 2B
Cloning of Fumarate Reductases from Trypanosoma Brucei in Saccharomyces Cerevisiae 
2B.1. Expression Constructs
In a similar way as disclosed in 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).
Likewise, 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).
2B.2. Transformation and Microtiterplates (MTP's) Growth Experiments
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. The following numbers of individual transformants were inoculated in duplo in 250 microliters Verduyn medium comprising 2% glucose in 96 deep-well MTP's and pre-cultured at 30 degrees Celsius, 550 rpm, and a humidity of 80% in an Infors Microplate shaking incubator: 12 pGBS416FRD-1 (FRDm1), 12 pGBS416FRE-1 (FRDg) and 24 pRS416 empty vector control transformants. After 3 days, 25 microliters of the pre-culture present in the wells of the MTP plates was transferred to new 96 deep-well MTP's containing Verduyn medium containing glucose and CaCO3 (end-concentrations: glucose 10%, CaCO3 1% w/v in a total volume of 250 microliters). After 3 and 7 days of growth at 30° C., 550 rpm, and a humidity of 80% in an Infors Microplate shaking incubator, the MTP's were centrifuged for 2 minutes at 2000 rpm, and 200 microliters of supernatant was harvested using the Multimek 96 (Beckman). The supernatant was analyzed by HPLC as described in Example 1.4 for the presence succinic acid. The results are shown in Table 1.







TABLE 1







Effect of introduction of mitochondrial (FRDm1) and


glycosomal fumarate reductase (FRDg) from T. brucei in


S. cerevisiae on the succinic acid production levels


after 3 and 7 days of incubation









S. cerevisiae
Succinic acid (mg/l)
Succinic acid (mg/l)


comprising plasmid:
after 3 days
after 7 days





Empty vector pRS416
138 ± 18 (n = 48)
203 ± 48 (n = 48)


pGBS4 16FRD-1 (FRDm1)
340 ± 65 (n = 24)
399 ± 72 (n = 24)


pGBS4 16FRE-1 (FRDg)
489 ± 30 (n = 24)
516 ± 57 (n = 24)









The results in Table 1 show that introduction and overexpression of mitochondrial fumarate reductase (FRDm1) from T. brucei resulted in increased succinic acid production levels (2.47 fold, p=6.96E-14, Student's t-test, after 3 days incubation and 1.97 fold, p=8.63E-14, Student's t-test after 7 days incubation).
Likewise, introduction and overexpression of glycosomal fumarate reductase (FRDg) from T. brucei resulted in increased succinic acid production levels (3.55 fold, p=5.08E-32, Student's t-test, after 3 days incubation and a 2.55 fold increase, p=8.63E-25, Student's t-test after 7 days incubation).
Example 2C
Expression of PEP Carboxykinase from Actinobacillus succinogenes or Mannheimia succiniciproducens and Malate Dehydrogenase from Saccharomyces cerevisiae and Fumarase from Rhizopus oryzae and Fumarate Reductase from Trypanosoma brucei in Saccharomyces cerevisiae 
2C.1 Gene Sequences
Phosphoenolpyruvate Carboxykinase:
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
(www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1
(www.cbs.dtu.dk/services/TargetP/) Emanuelsson, O. et al. (2007) Nature Protocols 2, 953-971. Analysis as described by Schluter et al., (2007) NAR, 35, D815-D822 revealed a putative PTS2 signal sequence at position 115-123. The A. succinogenes sequence was modified to resemble the Mannheimia succiniciproducens protein sequence by replacing the amino acids EGY at position 120-122 with DAF resulting in amino acid sequence SEQ ID NO: 14 (nucleotide sequence SEQ ID NO: 15). SEQ ID NO: 14 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TAA in the resulting nucleotide sequence SEQ ID NO: 16 was modified to TAAG. 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).
Likewise phosphoenolpyruvate carboxykinase [E.C. 4.1.1.49], GenBank accession number 52426348, from Mannheimia succiniciproducens was analysed for the presence of signal sequences as described in Schlüter et al., (2007) NAR, 35, D815-D822. The sequence as shown in SEQ ID NO: 17 required no modifications. SEQ ID NO: 17 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TAA in the resulting sequence SEQ ID NO: 18 was modified to TAAG. 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).
Malate Dehydrogenase
Cytoplasmic malate dehydrogenase (Mdh2p) [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). To avoid glucose-induced degradation of Mdh2, the nucleotides encoding the first 12 amino acids were removed, and a new methionine amino acid was introduced (SEQ ID NO: 19) for overexpression of Mdh2 in S. cerevisiae. SEQ ID NO: 19 was subjected to the codon-pair method as disclosed in WO2008/000632 for S. cerevisiae. The stop codon TAA in the resulting in SEQ ID NO: 20, was modified to TAAG. 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 (Mdh3p) [E.C. 1.1.1.37], GenBank accession number 1431095, was analysed for peroxisomal targeting in filamentous fungi using the PTS1 predictor
(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 downstream of stop codon), and convenient restriction sites were added. The resulting sequence SEQ ID NO: 32 was synthesised at Sloning (Puchheim, Germany).

Fumarase:

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 (www.cbs.dtu.dk/services/SignalP/) Bendtsen, J. et al. (2004) Mol. Biol., 340:783-795 and TargetP 1.1 (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. cerevisiae, the first 23 amino acids were removed from FumR and a methionine amino acid was reintroduced resulting in SEQ ID NO: 23. 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).
Fumarate Reductase:
Gene sequences of mitochondrial fumarate reductase (FRDm1) and glycosomal fumarate reductase (FRDg) from T. brucei were designed and synthesized as described under 2A.1.
2C.2. Construction of Expression Constructs
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. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-1. Subsequently, pGBK414PPK-1 was restricted with AscI and NotI. To create pGBS414PPK-2, 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 pGBS414PPK-1 vector. The ligation mix was used for transformation of E. coli TOP10 (Invitrogen) resulting in the yeast expression construct pGBS414PPK-2 (FIG. 10). To create pGBS414PPK-3, 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. Subsequently, pGBK414PEK-1 was restricted with AscI and NotI. To create pGBS414PEK-2, 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). To create pGBS414PEK-3, 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. Subsequently, pGBK415FUM-1 was restricted with AscI and NotI. To create pGBS415FUM-2, 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). To create pGBS415FUM-3, an AscI/NotI restriction fragment consisting of peroxisomal malate dehydrogenase from S. 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).
2C.3. S. cerevisiae Strains
Different combinations of plasmids pGBS414PPK-1, pGBS414 PPK-2, pGBS414PPK-3, pGBS414PEK-1, pGBS414PEK-2, pGBS414PEK-3, pGBS415FUM-2, pGBS415-FUM-3 were transformed into S. cerevisiae strain CEN.PK113-6B (MATA ura3-52 leu2-112 trp1-289), resulting in the yeast strains depicted in Table 2. In addition to the mentioned plasmids, pRS416 (empty vector) was transformed to create prototrophic yeast strains. 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.







TABLE 2







Yeast strains constructed for Example 2C.










Name
Background
Plasmids
Genes





SUC-148
CEN.PK113-6B
pGBS414PPK-2
PCKa, FRDm1




pGBS415FUM-3
FUMR, MDH3




pRS416 (empty vector)


SUC-149
CEN.PK113-6B
pGBS414PPK-3
PCKa, FRDg




pGBS415FUM-3
FUMR, MDH3




pRS416 (empty vector)


SUC-150
CEN.PK113-6B
pGBS414PEK-2
PCKm, FRDm1




pGBS415FUM-3
FUMR, MDH3




pRS416 (empty vector)


SUC-151
CEN.PK113-6B
pGBS414PEK-3
PCKm, FRDg




pGBS415FUM-3
FUMR, MDH3




pRS416 (empty vector)


SUC-152
CEN.PK113-6B
PGBS414PPK-1
PCKa




pGBS415FUM-3
FUMR, MDH3




pRS416 (empty vector)


SUC-154
CEN.PK113-6B
PGBS414PEK-1
PCKm




pGBS415FUM-3
FUMR, MDH3




pRS416 (empty vector)


SUC-169
CEN.PK113-6B
pGBS414PEK-2
PCKm, FRDm1




pGBS415FUM-2
FUMR, Δ12NMDH2




pRS416 (empty vector)


SUC-101
CEN.PK113-6B
pRS414 (empty vector)




pRS415 (empty vector)




pRS415 (empty vector)









2C.4. Growth Experiments and Succinic Acid Production

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).
Strains transformed with empty vectors (control strain) produced up to 0.3 g/L succinic acid. Overexpression of PEP carboxykinase from M. succiniciproducens PCKm), peroxisomal malate dehydrogenase (MDH3) from S. cerevisiae and fumarase from R. oryzae (FUMR) resulted in production of 0.9 g/L succinic acid production. Overexpression of PEP carboxykinase from A. succinogenes (PCKa), MDH3 and FUMR resulted in a slight increase in succinic acid production to 1.0 g/L.
These results show that in S. cerevisiae as described increased succinic acid production about 3 times.
Additional overexpression of mitochondrial fumarate reductase (FRDm1) from T. brucei further increased succinic acid production levels; overexpression of PCKa, MDH3, FUMR, FRDm1 resulted in production of 2.6 g/L succinic acid, and overexpression of PCKm, MDH3, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid. Overexpression of delta12NMDH2 in combination with PCKm, FUMR and FRDm1 resulted in production of 2.7 g/L succinic acid, indicating that similar levels of succinic acid were produced using either truncated MDH2 or MDH3. Additional overexpression of glycosomal fumarate reductase (FRDg) 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 overexpression of PCKm, MDH3, FUMR and FRDg resulted in slightly lower production of 3.6 g/L succinic acid.
The results show addition of NAD(H) dependent fumarate reductase as disclosed herein in a S. cerevisiae comprising a genetic modification of PCKa/m, MDH3 and FUMR significantly increased succinic acid production levels.
Overexpression of FRDg had a more positive effect on succinic acid production levels in S. cerevisiae compared to overexpression of FRDm1 in S. cerevisiae. 
Example 2D
Effect of Overexpression of a Dicarboxylic Acid Transporter on Succinic Acid Production in Succinic Acid Producing S. cerevisiae Cells
2D.1. Gene Sequences
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. In the ENO1 promotor, 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).
2D.2. Construction of Expression Constructs
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.
2D.3. S. cerevisiae Strains
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. Strains SUC-101 is described in Table 2.







TABLE 3







Yeast strains constructed for Example 2D.










Name
Background
Plasmids
Genes





SUC-132
CEN.PK113-6B
pGBS414PEK-2
PCKm, FRDm1




pGBS415FUM-2
FUMR, Δ12NMDH2




pRS416 (empty vector)


SUC-194
CEN.PK113-6B
pGBS414PEK-2
PCKm, FRDm1




pGBS415FUM-2
FUMR, Δ12NMDH2




pRS416MAE-1
SpMAE1









2D.4. Growth Experiments and Succinic Acid Production in Wildtype CEN.PK Strains

Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% glucose (w/v) as carbon source. In the production medium 10% glucose (w/v) was used as carbon source.
Strains transformed with empty vectors (control strain) 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.
The results show that insertion of a malate transporter in a S. cerevisiae comprising the genetic modifications as described herein further increased succinic acid production at least 1.5 times.
Example 2E
Effect of a Dicarboxylic Acid Transporter in S. cerevisiae Comprising a Deletion of the Genes Alcohol Dehydrogenase 1 and 2 (Adh1, Adh2) and the Gene Glycerol-3-Phosphate Dehydrogenase 1 (Gpd1) on Succinic Acid Production Levels
2E.1. Gene Sequences
Described under 2D.1.
2E.2. Construction of Expression Constructs
Described under 2D.2.
2E.3. S. cerevisiae Strains
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.







TABLE 4







Yeast strains constructed for Example 2E.










Name
Background
Plasmids
Genes





SUC-200
CEN.PK113-6B
PGBS414PPK-3
PCKa, FRDg



adh1::lox adh2::lox
pGBS415FUM-3
FUMR, MDH3



gpd1::Kanlox
pGBS416MAE-1
SpMAE1


SUC-201
CEN.PK113-6B
PGBS414PPK-3
PCKa, FRDg



adh1::lox adh2::lox
pGBS415FUM-3
FUMR, MDH3



gpd1::Kanlox
pRS416 (empty vector)


SUC-103
CEN.PK113-6B
pRS414 (empty vector)



adh1::lox adh2::lox
pRS415 (empty vector)



gpd1::Kanlox
pRS415 (empty vector)









2E.4. Growth Experiments and Succinic Acid Production in CEN.PK Strains Deleted for the Genes Alcohol Dehydrogenase 1 and 2 (Adh1, Adh2) and the Gene Glycerol-3-Phosphate Dehydrogenase 1 (Gpd1)

Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% galactose (w/v) as carbon source. 5% galactose (w/v) was added to the production medium at t=0, 3 and 7 days.
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).
The results show that overexpression of a malate transporter in s S. cerevisiea comprising a deletion of alcohol dehydrogenase and glycerol-3-phosphate dehydrogenase genes resulted in a significant increase in succinic acid production levels. In addition it was shown that deletion of the gene adh1, adh2 and gpd1 (SUC 103) resulted in increased succinic acid production levels as compare to a wild type strain (SUC 101, Table 2).
Example 2F
Cloning of Phosphoenolpyruvate Carboxykinase from Actinobacillus succinogenes, Pyruvate Carboxylase from Saccharomyces cerevisiae, Malate Dehydrogenase from Saccharomyces cerevisiae, Fumarase from Rhizopus oryzae in Saccharomyces cerevisiae and Fumarate Reductase from Trypanosoma Brucei 
2F.1. Gene Sequences
Gene sequences of PEP carboxykinase from A. succinogenes, malate dehydrogenase from S. cerevisiae, fumarase from R. oryzae and fumarate reductase from T. brucei are described under 2F.1. Cytoplasmic pyruvate carboxylase from Saccharomyces cerevisiae (Pyc2p) [E.C. 6.4.1.1.], GenBank accession number 1041734, SEQ ID NO: 41, is encoded by the nucleotide sequence SEQ ID NO: 42. Genomic DNA from S. cerevisiae strain CEN.PK113-5D (MA TA 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.
2F.2. Construction of Expression Constructs
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). Construction of expression vectors pGBS414PPK-3 and pGBS415FUM-3 is described under 2C.2. 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).
2F.3. S. cerevisiae Strains
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.







TABLE 5







Yeast strains constructed for Example 2F.










Name
Background
Plasmids
Genes





SUC-226
CEN.PK113-6B
PGBS414PPK-3
PCKa, FRDg




pGBS415FUM-3
FUMR, MDH3




p426GPD (empty vector)


SUC-227
CEN.PK113-6B
PGBS414PPK-3
PCKa, FRDg




pGBS415FUM-3
FUMR, MDH3




pGBS426PYC-2
PYC2


SUC-228
CEN.PK113-6B
pGBS414FRE-1
FRDg




pGBS415FUM-3
FUMR, MDH3




pGBS426PYC-2
PYC2


SUC-230
CEN.PK113-6B
pGBS414FRE-1
FRDg




pGBS415FUM-3
FUMR, MDH3




p426GPD (empty vector)









2F.4. Growth Experiments and Succinic Acid Production

Growth parameters and sample analysis were performed as described under example 2C.4 with the following modifications: pre-culturing was performed using 2% glucose (w/v) as carbon source. In the production medium 10% glucose (w/v) was used as carbon source.
As depicted in FIG. 23 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), and additional overexpression of PYC2 increased succinic acid production up to 3.7 g/L (strain SUC-228). Surprisingly, overexpression of both PCKa and PYC2 (SUC-227) resulted 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. These results show a synergistic effect of combined overexpression of both PEP carboxykinase from A. succinogenes (PCKa) and pyruvate carboxylase from S. cerevisiae (PYC2) on succinic acid production levels in S. cerevisiae. 
Example 3
Inactivation of Succinate Dehydrogenase Encoding Genes in Aspergillus Niger 
3.1. Identification
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.
3.2. Inactivation of the sdhA Gene in Aspergillus niger. 
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. Growing colonies were diagnosed by PCR for integration at the sdhA locus and candidate strains tested by Southern analyses for deletion of the sdhA gene. Deletion of the sdhA gene was detectable by the  ̃2,2 kb size reduction of DNA fragments (4.6 kb wild-type fragment versus 2.4 kb for a successful deletion of SDHA) covering the entire locus and hybridized to appropriate probes. Approximately 9 strains showed a removal of the genomic sdhA gene from a pool of approximately 96 initial transformants.
Strain dSDHA was selected as a representative strain with the sdhA gene inactivated. The succinic acid production of dSDHA was determined in microtiterplates as described in Example 4.
Example 4
Cloning of FRDm from Trypanosoma brucei in Aspergillus niger dSDHA
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. These spores were resuspended using the Multimek 96 (Beckman) in 100 microliters minimal enriched Aspergillus medium containing 10% glucose. Subsequently 2 MTP with 170 micoliters minimal enriched Aspergillus medium containing 10% glucose and 1% CaCO3 were inoculated with 30 microliters of the spore suspension. Likewise, A. niger strains dSDHA and CBS513.88 were inoculated in the MTP's. These MTP's were incubated for 5 days at 34 degrees Celsius 80% humidity. After 5 days 160 microliters were harvested using the Multimek 96 (Beckman) and succinic acid was determined by HPLC as described in Example 1.4. The results are shown in Table 6.







TABLE 6







Effect of deletion of succinate dehydrogenase (SDHA) and insertion


of mitochondrial fumarate reductase (FRDm1) from T. brucei in


A. niger on succinic acid production levels.










A. niger strain
Succinic acid mg/l














CBS513.88
38



dSDHA
50



dSDHA, + gGBTOPAn1
583



(FRDm1)










Table 6 clearly shows an increased production of succinic acid by A. niger that comprises mitochondrial fumarate reductase from T. brucei

## Claims

1. A recombinant eukaryotic cell selected from the group consisting of a yeast and a filamentous fungus comprising and expressing a heterologous nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid wherein the NAD(H)-dependent fumarate reductase is active in the cytosol upon expression of the nucleotide sequence encoding the NAD(H)-dependent fumarate reductase, and wherein the heterologous nucleotide sequence encoding the NAD(H)-dependent fumarate reductase comprises an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 3 and/or SEQ ID NO: 6.

2. The cell according to claim 1, wherein the heterologous nucleotide sequence encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 95% sequence identity with the amino acid sequence of SEQ ID NO: 3 and/or SEQ ID NO: 6.

3. The cell according to claim 1, wherein the NAD(H)-dependent fumarate reductase is derived from a Trypanosoma sp.

4. The cell according to claim 1, wherein the cell further overexpresses a nucleotide sequence encoding a pyruvate carboxylase.

5. The cell according to claim 1, further comprising a nucleotide sequence encoding a heterologous phosphoenolpyruvate carboxykinase.

6. The cell according to claim 1, further comprising a nucleotide sequence encoding a malate dehydrogenase active in the cytosol upon expression of the nucleotide sequence encoding malate dehydrogenase.

7. The cell according to claim 1, further comprising a nucleotide sequence encoding an enzyme that catalyses the conversion of malic acid to fumaric acid in the cytosol, upon expression of the nucleotide sequence encoding enzyme that catalyses the conversion of malic acid to fumaric acid.

8. The cell according to claim 1, further comprising a nucleotide sequence encoding a dicarboxylic acid transporter.

9. The cell according to claim 1, wherein at least one gene of said cell encoding alcohol dehydrogenase is not functional.

10. The cell according to claim 1, wherein at least one gene of said cell encoding glycerol-3-phosphate dehydrogenase is not functional.

11. The cell according to claim 1, wherein at least one gene of said cell encoding succinate dehydrogenase is not functional.

12. The cell according to claim 1, which is an Aspergillus, optionally an Aspergillus niger.

13. The cell according to claim 1, which is a Saccharomyces, optionally a Saccharomyces cerevisiae.

14. A process for preparing succinic acid, comprising fermenting a eukaryotic cell according to claim 1, in a suitable fermentation medium, wherein succinic acid is prepared.

15. The process according to claim 14, wherein the succinic acid prepared is used for the production of a pharmaceutical, cosmetic, food, feed or chemical product.

16. The cell according to claim 1, wherein the heterologous nucleotide sequence encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 97% sequence identity with the amino acid sequence of SEQ ID NO:3 and/or SEQ ID NO:6.

17. The cell according to claim 1, further comprising a nucleotide sequence encoding a phosphoenolpyruvate carboxylase.