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 catalyses 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.

Description

[0001] 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.

[0002] 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.

[0003] 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.

[0004] 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.

[0005] 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).

[0006] 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.

[0007] The aim of the present invention is an alternative microorganism for the production of succinic acid.

[0008] 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.

[0009] 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.

[0010] 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.

[0011] 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.sup.rd edition), Cold Spring Harbor Laboratory Press.

[0012] 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.

[0013] 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.

[0014] A NAD(H)-dependent fumarate reductase according to the present invention uses NAD(H) as a cofactor, whereas most eukaryotic cells comprise a FADH.sub.2-dependent fumarate reductase, wherein FADH.sub.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.sup.+ and influence the redox balance in the cell.

[0015] 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.

[0016] 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.

[0017] 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.

[0018] 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.degree. C. in a solution comprising about 1 M salt, preferably 6.times.SSC (sodium chloride, sodium citrate) or any other solution having a comparable ionic strength, and washing at 65.degree. C. in a solution comprising about 0.1 M salt, or less, preferably 0.2.times.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.

[0019] 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.degree. C. in a solution comprising about 1 M salt, preferably 6.times.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.times.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%.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] The term "enzyme" as used herein is defined as a protein which catalyses a (bio)chemical reaction in a cell.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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).

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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 2.mu. 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.

[0036] 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 Trypanosoma brucei.

[0037] 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.

[0038] 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 Schluter et al, Nucleic acid Research 2007, 35, D815-D822.

[0039] 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.

[0040] 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.

[0041] 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 bailiff.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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. Preferaby 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In another preferred embodiment a eukaryotic cell according to the present invention comprises reduced activity of enzymes that convert NAD(H) to NAD.sup.+ compared to the activity of these enzymes in a wild-type cell.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] A suitable temperature at which the process according to the present invention may be carried out is between 5 and 60.degree. C., preferably between 10 and 50.degree. C., more preferably between 15 and 35.degree. C., more preferably between 18.degree. C. and 30.degree. C. The skilled man in the art knows which optimal temperatures are suitable for fermenting a specific eukaryotic cell.

[0067] Preferably, succinic acid is recovered from the fermentation broth by a suitable method known in the art, for instance by crystallisation and ammonium precipitation.

[0068] 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.

[0069] The present invention also relates to a fermentation broth comprising a succinic acid obtainable by a process according to the present invention.

[0070] 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.

[0071] Genetic Modifications

[0072] 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.sup.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.

[0073] The following examples are for illustrative purposes only and are not to be construed as limiting the invention.

DESCRIPTION OF THE FIGURES

[0074] FIG. 1. Map of the pGBTOP-11 vector used for expression of fumarate reductase in A. niger

[0075] 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.

[0076] 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.

[0077] FIG. 4: Plasmid map of pDEL-SDHA

[0078] FIG. 5: Map of plasmid pGBTPAn1, for overexpression FRDm1 in A. niger.

[0079] FIG. 6: Replacement scheme of sdhA

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] FIG. 17: Succinic acid levels in strains SUC-101 (.largecircle., empty vectors control), SUC-148 (.box-solid., overexpression of PCKa, MDH3, FUMR, FRDm1), SUC-149 (.quadrature., PCKa, MDH3, FUMR, FRDg), SUC-150 (.diamond-solid., PCKm, MDH3, FUMR, FRDm1), SUC-151 (.diamond., PCKm, MDH3, FUMR, FRDg), SUC-152 ( , PCKa, MDH3, FUMR), SUC-154 (.times., PCKm, MDH3, FUMR) and SUC-169 (.tangle-solidup., 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.

[0091] 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.

[0092] FIG. 19: Succinic acid levels in strains SUC-101 (.largecircle., empty vectors control), SUC-169 (.tangle-solidup., PCKm, delta12NMDH2, FUMR, FRDm1) and SUC-194 (.box-solid., 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.

[0093] FIG. 20: Succinic acid levels in strains SUC-103 (.largecircle., adh1/2 and gpd1 deletion mutant; empty vectors control), SUC-201 (.quadrature., adh1/2 and gpd1 deletion mutant; PCKa, MDH3, FUMR, FRDg) and SUC-200 (.box-solid., adh1/2 and gpd1 deletion mutant; PCKa, MDH3, FUMR, FRDg, SpMAE1). All overexpressed genes were codon pair optimized for expression in S. cerevisiae.

[0094] 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.

[0095] 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.

[0096] FIG. 23: Succinic acid levels in strains SUC-226 (.quadrature., PCKa, MDH3, FUMR, FRDg), -227 (.tangle-solidup., PYC2, PCKa, MDH3, FUMR, FRDg), SUC-228 (.box-solid., PYC2, MDH3, FUMR, FRDg) and SUC-230 (.largecircle., 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

[0097] Mitochondrial fumarate reductase ml (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 (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).

[0098] 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).

[0099] 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 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.

1.2. Transformation of A. Niger

[0100] 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.

[0101] 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:

[0102] 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 litre: 6 g NaNO.sub.3, 0.52 g KCl, 1.52 g KH.sub.2PO.sub.4, 1.12 ml 4 M KOH, 0.52 g MgSO.sub.4.7H.sub.2O, 10 g glucose, 1 g casaminoacids, 22 mg ZnSO.sub.4.7H.sub.2O, 11 mg H.sub.3BO.sub.3, 5 mg FeSO.sub.4.7H.sub.2O, 1.7 mg CoCl.sub.2.6H.sub.2O, 1.6 mg CuSO.sub.4.5H.sub.2O, 5 mg MnCl.sub.2.2H.sub.2O, 1.5 mg Na.sub.2MoO.sub.4.2H.sub.2O, 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 IU/ml) Streptomycin (5000 UG/ml) solution (Gibco).

[0103] Novozym 234.TM. (Novo Industries) instead of helicase is used for the preparation of protoplasts;

[0104] 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 CaCl.sub.2) 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;

[0105] 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 CaCl.sub.2) is added;

[0106] 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 CaCl.sub.2) 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

[0107] 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

[0108] 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

[0109] 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).

[0110] 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).

[0111] 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.degree. 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

28.1. Expression Constructs

[0112] 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).

[0113] 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).

28.2. Transformation and Microtiterplates (MTP's) Growth Experiments

[0114] 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 microlitres 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 microlitres 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 CaCO.sub.3 (end-concentrations: glucose 10%, CaCO3 1% w/v in a total volume of 250 microlitres). After 3 and 7 days of growth at 30.degree. 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-US-00001 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 comprising Succinic acid (mg/l) Succinic acid (mg/l) plasmid: after 3 days after 7 days Empty vector pRS416 138 .+-. 18 (n = 48) 203 .+-. 48 (n = 48) pGBS416FRD-1 (FRDm1) 340 .+-. 65 (n = 24) 399 .+-. 72 (n = 24) pGBS416FRE-1 (FRDg) 489 .+-. 30 (n = 24) 516 .+-. 57 (n = 24)

[0115] 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).

[0116] 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:

[0117] 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. 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).

[0118] 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 Schluter 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

[0119] 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).

[0120] 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 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:

[0121] 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. 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:

[0122] 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

[0123] 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).

[0124] 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).

[0125] 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

[0126] 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-US-00002 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, .DELTA.12NMDH2 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

[0127] 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.degree. 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.degree. 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).

[0128] 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.

[0129] These results show that in S. cerevisiae as decribed increased succinic acid production about 3 times.

[0130] 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 overexexpression of PCKm, MDH3, FUMR and FRDg resulted in slightly lower production of 3.6 g/L succinic acid.

[0131] 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.

[0132] 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

[0133] 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

[0134] 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

[0135] 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.

[0136] 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-US-00003 TABLE 3 Yeast strains constructed for Example 2D. Name Background Plasmids Genes SUC-132 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR, .DELTA.12NMDH2 pRS416 (empty vector) SUC-194 CEN.PK113-6B pGBS414PEK-2 PCKm, FRDm1 pGBS415FUM-2 FUMR, .DELTA.12NMDH2 pRS416MAE-1 SpMAE1

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

[0137] 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.

[0138] 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.

[0139] 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

[0140] Described under 2D.1.

2E.2. Construction of Expression Constructs

[0141] Described under 2D.2.

2E.3. S. Cerevisiae Strains

[0142] 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-US-00004 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)

[0143] 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.

[0144] 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).

[0145] The results show that overexpression of a malate transporter in 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

[0146] 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 (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.

2F.2. Construction of Expression Constructs

[0147] 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

[0148] 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-US-00005 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

[0149] 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.

[0150] 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

[0151] 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.

[0152] 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 .about.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.

[0153] 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.

Example 4

Cloning of FRDm from Trypanosoma Brucei in Aspergillus Niger dSDHA

[0154] 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 microlitres minimal enriched Aspergillus medium containing 10% glucose. Subsequently 2 MTP with 170 micolitres minimal enriched Aspergillus medium containing 10% glucose and 1% CaCO3 were inoculated with 30 microlitres 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 Celsius80% humidity. After 5 days 160 microlitres 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-US-00006 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)

[0155] Table 6 clearly shows an increased production of succinic acid by A. niger that comprises mitochondrial fumarate reductase from T. brucei

Sequence CWU 1

1

4411232PRTTrypanosoma brucei 1Met Leu Ser Thr Lys Gln Leu Leu Leu Arg Ala Thr Ser Ala Leu Val1 5 10 15Ala Gly Ser Ser Gly Val Ala Arg Asp Ser Pro Ser Leu Val Gly Asp 20 25 30Pro Cys Asp Ser Val Ser Pro Thr Arg Val Val Trp Gly Arg Phe Phe 35 40 45Lys Ser Leu Ala Pro Pro Ala Pro Ser Val Val Ser Cys Gln Lys Arg 50 55 60Phe Thr Ser His Gly Ala Asp Gly Ile Ser Ser Ala Ser Ile Val Val65 70 75 80Thr Asp Pro Glu Ala Ala Ala Lys Lys Arg Asp Arg Met Ala Arg Glu 85 90 95Leu Leu Ser Ser Asn Ser Gly Leu Cys Gln Glu Asp Glu Pro Thr Ile 100 105 110Ile Asn Leu Lys Gly Leu Glu His Thr Ile Pro Tyr Arg Leu Ala Val 115 120 125Val Leu Cys Asn Ser Arg Ser Thr Gly Glu Phe Glu Ala Lys Ala Ala 130 135 140Glu Ile Leu Arg Lys Ala Phe His Met Val Asp Tyr Ser Leu Asn Cys145 150 155 160Phe Asn Pro Glu Ser Glu Leu Ser Arg Val Asn Ser Leu Pro Val Gly 165 170 175Glu Lys His Gln Met Ser Glu Asp Leu Arg His Val Met Glu Cys Thr 180 185 190Ile Ser Val His His Ser Ser Gly Met Gly Phe Asp Pro Ala Ala Gly 195 200 205Pro Ile Ile Ser Arg Leu Arg Gly Ala Met Arg Asp His Asn Asp Met 210 215 220Ser Asp Ile Ser Val Thr Glu Ala Glu Val Glu Leu Phe Ser Leu Ala225 230 235 240Gln Ser Phe Asp Val Asp Leu Glu Glu Gly Thr Ile Ala Arg Lys His 245 250 255Ser Glu Ala Arg Leu Asp Leu Gly Gly Val Asn Lys Gly Tyr Thr Val 260 265 270Asp Tyr Val Val Asp His Leu Arg Ala Ala Gly Met Pro Asn Val Leu 275 280 285Phe Glu Trp Gly Gly Asp Ile Arg Ala Ser Gly Arg Asn Ile Lys Gly 290 295 300Asn Leu Trp Ala Val Ala Ile Lys Arg Pro Pro Ser Val Glu Glu Val305 310 315 320Ile Arg Arg Ala Lys Gly Lys Met Leu Lys Met Gly Glu Glu Glu Gln 325 330 335Glu Glu Lys Asp Asp Asp Ser Pro Ser Leu Leu His Val Val Glu Leu 340 345 350Asp Asp Glu Ala Leu Cys Thr Ser Gly Asp Tyr Glu Asn Val Leu Tyr 355 360 365His Pro Lys His Gly Val Ala Gly Ser Ile Phe Asp Trp Gln Arg Arg 370 375 380Gly Leu Leu Ser Pro Glu Glu Gly Ala Leu Ala Gln Val Ser Val Lys385 390 395 400Cys Tyr Ser Ala Met Tyr Ala Asp Ala Leu Ala Thr Val Cys Leu Val 405 410 415Lys Arg Asp Ala Val Arg Ile Arg Tyr Leu Leu Glu Gly Trp Arg Tyr 420 425 430Val Arg Ser Arg Val Thr Asn Tyr Phe Ala Tyr Thr Arg Gln Gly Glu 435 440 445Arg Leu Ala His Met His Glu Ile Ala Gln Glu Thr Arg Glu Leu Arg 450 455 460Glu Ile Arg Ile Ala Gly Ser Leu Pro Ser Arg Ile Val Ile Val Gly465 470 475 480Gly Gly Leu Ala Gly Leu Ser Ala Ala Ile Glu Ala Ala Ser Cys Gly 485 490 495Ala Gln Val Ile Leu Met Glu Lys Glu Gly Arg Ile Gly Gly Asn Ser 500 505 510Ala Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr Arg Thr Gln Ala 515 520 525Lys Ser Asp Ile Leu Asp Gly Gly Lys Tyr Phe Glu Arg Asp Thr Phe 530 535 540Leu Ser Gly Val Gly Gly Thr Thr Asp Pro Ala Leu Val Lys Val Leu545 550 555 560Ser Val Lys Ser Gly Asp Ala Ile Gly Trp Leu Thr Ser Leu Gly Val 565 570 575Pro Leu Ser Val Leu Ser Gln Leu Gly Gly His Ser Phe Lys Arg Thr 580 585 590His Arg Ala Pro Asp Lys Thr Asp Gly Thr Pro Leu Pro Ile Gly His 595 600 605Thr Ile Met Arg Thr Leu Glu Asp His Ile Arg Asn Asn Leu Ser Glu 610 615 620Arg Val Thr Ile Met Thr His Val Ser Val Thr Glu Leu Leu His Glu625 630 635 640Thr Asp Thr Thr Pro Asp Gly Ala Ser Glu Val Arg Val Thr Gly Val 645 650 655Arg Tyr Arg Asp Leu Ser Asp Val Asp Gly Gln Pro Ser Lys Leu Leu 660 665 670Ala Asp Ala Val Val Leu Ala Thr Gly Gly Phe Ser Asn Asp Arg Glu 675 680 685Glu Asn Ser Leu Leu Cys Lys Tyr Ala Pro His Leu Ala Ser Phe Pro 690 695 700Thr Thr Asn Gly Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala Thr705 710 715 720Ser Val Gly Ala Lys Leu Val Asp Met Asp Lys Val Gln Leu His Pro 725 730 735Thr Gly Leu Ile Asp Pro Lys Asp Pro Ala Asn Thr Thr Lys Ile Leu 740 745 750Gly Pro Glu Ala Leu Arg Gly Ser Gly Gly Ile Leu Leu Asn Lys Gln 755 760 765Gly Lys Arg Phe Val Asn Glu Leu Asp Leu Arg Ser Val Val Ser Lys 770 775 780Ala Ile Asn Thr Gln Gly Asn Glu Tyr Pro Gly Ser Gly Gly Cys Tyr785 790 795 800Phe Ala Tyr Cys Val Leu Asn Glu Asp Ala Thr Asn Leu Phe Cys Gly 805 810 815Gly Ala Leu Gly Phe Tyr Gly Lys Lys Leu Gly Leu Phe Gln Arg Ala 820 825 830Glu Thr Val Glu Glu Leu Ala Lys Leu Ile Gly Cys Asp Glu Gly Glu 835 840 845Leu Arg Asp Thr Leu Glu Lys Tyr Glu Thr Cys Ser Lys Ala Lys Val 850 855 860Ala Cys Pro Val Thr Gly Lys Val Val Phe Pro Cys Val Val Gly Thr865 870 875 880Arg Gly Pro Tyr Asn Val Ala Phe Val Thr Pro Ser Ile His Tyr Thr 885 890 895Met Gly Gly Cys Leu Ile Ser Pro Ala Ala Glu Val Leu Gln Glu Tyr 900 905 910Lys Gly Leu Asn Ile Leu Glu Asn His Arg Pro Ile Arg Cys Leu Phe 915 920 925Gly Ala Gly Glu Val Thr Gly Gly Val His Gly Gly Asn Arg Leu Gly 930 935 940Gly Asn Ser Leu Leu Glu Cys Val Val Phe Gly Lys Ile Ala Gly Asp945 950 955 960Arg Ala Ala Thr Ile Leu Gln Lys Arg Glu Ile Ala Leu Ser Lys Thr 965 970 975Ser Trp Thr Ser Val Val Val Arg Glu Ser Arg Ser Gly Glu Gln Phe 980 985 990Gly Thr Gly Ser Arg Val Leu Arg Phe Asn Leu Pro Gly Ala Leu Gln 995 1000 1005Arg Thr Gly Leu Asn Leu Gly Glu Phe Val Ala Ile Arg Gly Glu 1010 1015 1020Trp Asp Gly Gln Gln Leu Val Gly Tyr Phe Ser Pro Ile Thr Leu 1025 1030 1035Pro Glu Asp Leu Gly Thr Ile Ser Leu Leu Val Arg Ala Asp Lys 1040 1045 1050Gly Thr Leu Lys Glu Trp Ile Cys Ala Leu Arg Pro Gly Asp Ser 1055 1060 1065Val Glu Ile Lys Ala Cys Gly Gly Leu Arg Ile Asp Gln Asp Pro 1070 1075 1080Val Lys Lys Cys Leu Leu Phe Arg Asn Arg Pro Ile Thr Arg Phe 1085 1090 1095Ala Leu Val Ala Ala Gly Thr Gly Val Ala Pro Met Leu Gln Val 1100 1105 1110Ile Arg Ala Ala Leu Lys Lys Pro Tyr Val Asp Thr Leu Glu Ser 1115 1120 1125Ile Arg Leu Ile Tyr Ala Ala Glu Glu Tyr Asp Thr Leu Thr Tyr 1130 1135 1140Arg Ser Ile Leu Gln Arg Phe Ala Glu Glu Phe Pro Asp Lys Phe 1145 1150 1155Val Cys Asn Phe Val Leu Asn Asn Pro Pro Glu Gly Trp Thr Gly 1160 1165 1170Gly Val Gly Phe Val Asn Lys Lys Ser Leu Gln Lys Val Leu Gln 1175 1180 1185Pro Pro Ser Ser Glu Pro Leu Ile Val Val Cys Gly Pro Pro Val 1190 1195 1200Met Gln Arg Asp Val Lys Asn Glu Leu Leu Ser Met Gly Tyr Asp 1205 1210 1215Lys Glu Leu Val His Thr Val Asp Gly Glu Ser Gly Thr Leu 1220 1225 123023698DNATrypanosoma brucei 2atgctctcaa cgaagcaact tctccttcga gccacatctg cattagtggc gggaagctct 60ggagttgcgc gagacagccc ttcgcttgtc ggcgaccctt gcgactcggt ttcaccaacg 120cgggtcgtat gggggcgctt cttcaaatcc ctagcgccac ccgctccctc ggttgtttca 180tgtcaaaagc gttttacgtc ccatggcgcc gatggtattt cctcggcttc gattgttgtc 240actgacccgg aggcggcagc aaagaagcgt gaccgcatgg cgcgcgagtt gctctcaagt 300aatagtggtc tttgtcaaga agatgaaccc actatcatta acttaaaggg gttggagcac 360acgattccgt acaggctcgc cgtggttctt tgtaactcgc gctctacagg tgaattcgaa 420gcaaaggcag ctgagatttt gcgaaaggca tttcacatgg tggactactc cctcaattgt 480ttcaatcctg aaagcgagtt gtcgcgtgtc aactctctgc cggtgggtga gaagcatcaa 540atgtcggagg atctccggca cgtgatggag tgcaccatca gtgtacatca ctccagcgga 600atgggcttcg acccggcggc aggtccaatt atcagccgac ttcggggggc aatgagggac 660cacaacgaca tgtccgacat ttccgtaacg gaagccgagg tagagctctt ctccttagcg 720caaagttttg acgtggacct cgaggaggga acaatagctc gcaagcactc tgaagcgagg 780cttgatcttg gtggtgtgaa caaaggctac acagttgatt atgtagtgga tcatcttcgt 840gcggccggta tgccaaacgt gctctttgag tggggcgggg atattcgagc gtcgggtagg 900aacatcaaag gaaacctatg ggcagttgct atcaaacgac cgccatctgt ggaggaggtg 960attcggcgcg ccaaagggaa aatgttaaaa atgggggagg aggagcagga agagaaggac 1020gatgattctc catccctgct tcatgtggtg gagcttgatg atgaagccct ttgcaccagt 1080ggtgactacg aaaacgtttt gtatcatcca aagcatggag tggcggggag catttttgac 1140tggcagcgaa gggggctact atctcctgag gaaggggcac tcgctcaagt gtctgtgaaa 1200tgttatagcg caatgtacgc tgatgctctg gcaacagtgt gccttgtgaa gcgtgatgct 1260gtgaggattc gctacttatt agagggctgg cgttacgttc gaagtcgtgt gacgaattac 1320tttgcctata cccgtcaggg cgagcggtta gcacatatgc acgagatagc gcaagaaaca 1380cgggagctac gtgaaatacg gattgccggg agtttgccct ccagaattgt tattgtgggt 1440ggaggtctag cgggcctttc agcggccatc gaagccgcaa gttgtggtgc acaagtcata 1500ctcatggaaa aggaaggaag aatcgggggg aacagcgcaa aggctacatc aggtattaat 1560gggtggggga cgcgtacgca ggcaaagtca gatattctcg acggtggaaa gtattttgag 1620cgtgacactt ttctctctgg cgttggcggt actaccgatc ctgccctcgt caaagtgctc 1680tcagttaaga gtggggacgc aattggttgg cttacttctc ttggtgtgcc actcagtgtc 1740ctctcgcaac ttggtggcca cagtttcaag cgaacccacc gtgccccgga caaaacggac 1800gggacacccc taccaattgg tcatacgatc atgagaaccc tcgaggatca catccgtaac 1860aacctctctg agcgagtaac gattatgaca catgtgtccg tgaccgagtt attgcacgaa 1920accgatacaa cacctgatgg cgcctccgaa gtgcgtgtta cgggtgtaag atacagggac 1980ctctccgatg tggatggcca gccatcaaaa ttgcttgcgg atgccgtcgt tcttgcaact 2040ggtggtttct ccaatgaccg tgaagaaaat tcactgctct gcaagtatgc gcctcacctg 2100gccagttttc caacgacaaa tggcccctgg gcgaccggtg acggggttaa actcgcaaca 2160tcggttggtg caaagcttgt ggatatggat aaggttcagc tacaccccac agggcttatc 2220gatccaaagg atcccgcgaa cacaacgaag attctcggcc cggaggcact ccgaggttca 2280ggtgggatat tactcaacaa gcaaggaaag cgcttcgtga atgaacttga cctccgctct 2340gttgtatcca aggcaattaa tacgcagggt aatgaatacc ctggatccgg tggatgttac 2400tttgcgtact gcgtgctcaa cgaagatgca acaaacctct tctgtggcgg tgcactgggg 2460ttctacggaa agaagcttgg tttgttccag cgtgctgaga ctgtggaaga gttggccaaa 2520ctgattggct gtgacgaagg tgaattacgg gatacgcttg aaaagtatga aacttgcagc 2580aaggccaaag ttgcgtgccc tgtgacgggg aaggtagtat tcccttgtgt ggtgggtaca 2640agggggccgt acaatgttgc ttttgtcacg ccttccattc attacacaat gggtggctgc 2700ctcatttcac cggctgctga agttcttcag gagtacaaag gtttaaatat tctggaaaac 2760catagaccga ttcgatgctt gtttggtgcc ggtgaagtga cgggtggtgt gcacggtggt 2820aaccgccttg gtggtaattc gctcttggaa tgtgtggtat tcgggaaaat tgcgggtgac 2880cgtgccgcaa caatacttca aaaacgtgag atagccctct ccaagacgag ttggacttcc 2940gttgttgtac gtgagtcccg ctccggcgaa cagttcggga ccggctctcg tgttcttcgt 3000tttaacctac ctggggcgct gcagcgcaca ggtctcaatc tgggcgaatt tgtggccatc 3060cgtggcgagt gggacggcca acaacttgtt ggttacttca gtccaattac actaccagag 3120gaccttggca ctatctccct tctggttcgt gccgacaagg gcacattgaa ggaatggatc 3180tgcgccttgc gaccgggcga ctccgtcgaa atcaaagcgt gtggaggtct tcgtattgat 3240caagacccgg taaagaagtg tctgctgttt cgtaaccggc ctattacgcg gtttgctctt 3300gtcgcggcag ggactggtgt cgcgcccatg ttgcaggtta ttcgtgcggc actcaagaag 3360ccttacgtgg acacgttgga aagcatccgt cttatatacg ccgcagaaga gtacgacaca 3420ttgacgtatc gctcaatttt gcagcggttt gcggaagagt tccccgacaa gttcgtctgc 3480aacttcgttc ttaacaaccc acccgaaggg tggacaggtg gagtggggtt tgtcaacaaa 3540aaatccctgc agaaggtgct gcaaccgcca tcgagtgagc cgctgattgt tgtgtgtgga 3600ccgcccgtga tgcagcgcga cgtgaagaat gagttactga gcatgggtta tgacaaagag 3660ctcgttcata cggttgacgg cgagtcggga acgctgta 369831164PRTArtificial sequenceFRDm Trypanosoma lacking 68 aa targeting signal 3Met Ala Asp Gly Ile Ser Ser Ala Ser Ile Val Val Thr Asp Pro Glu1 5 10 15Ala Ala Ala Lys Lys Arg Asp Arg Met Ala Arg Glu Leu Leu Ser Ser 20 25 30Asn Ser Gly Leu Cys Gln Glu Asp Glu Pro Thr Ile Ile Asn Leu Lys 35 40 45Gly Leu Glu His Thr Ile Pro Tyr Arg Leu Ala Val Val Leu Cys Asn 50 55 60Ser Arg Ser Thr Gly Glu Phe Glu Ala Lys Ala Ala Glu Ile Leu Arg65 70 75 80Lys Ala Phe His Met Val Asp Tyr Ser Leu Asn Cys Phe Asn Pro Glu 85 90 95Ser Glu Leu Ser Arg Val Asn Ser Leu Pro Val Gly Glu Lys His Gln 100 105 110Met Ser Glu Asp Leu Arg His Val Met Glu Cys Thr Ile Ser Val His 115 120 125His Ser Ser Gly Met Gly Phe Asp Pro Ala Ala Gly Pro Ile Ile Ser 130 135 140Arg Leu Arg Gly Ala Met Arg Asp His Asn Asp Met Ser Asp Ile Ser145 150 155 160Val Thr Glu Ala Glu Val Glu Leu Phe Ser Leu Ala Gln Ser Phe Asp 165 170 175Val Asp Leu Glu Glu Gly Thr Ile Ala Arg Lys His Ser Glu Ala Arg 180 185 190Leu Asp Leu Gly Gly Val Asn Lys Gly Tyr Thr Val Asp Tyr Val Val 195 200 205Asp His Leu Arg Ala Ala Gly Met Pro Asn Val Leu Phe Glu Trp Gly 210 215 220Gly Asp Ile Arg Ala Ser Gly Arg Asn Ile Lys Gly Asn Leu Trp Ala225 230 235 240Val Ala Ile Lys Arg Pro Pro Ser Val Glu Glu Val Ile Arg Arg Ala 245 250 255Lys Gly Lys Met Leu Lys Met Gly Glu Glu Glu Gln Glu Glu Lys Asp 260 265 270Asp Asp Ser Pro Ser Leu Leu His Val Val Glu Leu Asp Asp Glu Ala 275 280 285Leu Cys Thr Ser Gly Asp Tyr Glu Asn Val Leu Tyr His Pro Lys His 290 295 300Gly Val Ala Gly Ser Ile Phe Asp Trp Gln Arg Arg Gly Leu Leu Ser305 310 315 320Pro Glu Glu Gly Ala Leu Ala Gln Val Ser Val Lys Cys Tyr Ser Ala 325 330 335Met Tyr Ala Asp Ala Leu Ala Thr Val Cys Leu Val Lys Arg Asp Ala 340 345 350Val Arg Ile Arg Tyr Leu Leu Glu Gly Trp Arg Tyr Val Arg Ser Arg 355 360 365Val Thr Asn Tyr Phe Ala Tyr Thr Arg Gln Gly Glu Arg Leu Ala His 370 375 380Met His Glu Ile Ala Gln Glu Thr Arg Glu Leu Arg Glu Ile Arg Ile385 390 395 400Ala Gly Ser Leu Pro Ser Arg Ile Val Ile Val Gly Gly Gly Leu Ala 405 410 415Gly Leu Ser Ala Ala Ile Glu Ala Ala Ser Cys Gly Ala Gln Val Ile 420 425 430Leu Met Glu Lys Glu Gly Arg Ile Gly Gly Asn Ser Ala Lys Ala Thr 435 440 445Ser Gly Ile Asn Gly Trp Gly Thr Arg Thr Gln Ala Lys Ser Asp Ile 450 455 460Leu Asp Gly Gly Lys Tyr Phe Glu Arg Asp Thr Phe Leu Ser Gly Val465 470 475 480Gly Gly Thr Thr Asp Pro Ala Leu Val Lys Val Leu Ser Val Lys Ser 485 490 495Gly Asp Ala Ile Gly Trp Leu Thr Ser Leu Gly Val Pro Leu Ser Val 500 505 510Leu Ser Gln Leu Gly Gly His Ser Phe Lys Arg Thr His Arg Ala Pro 515 520 525Asp Lys Thr Asp Gly Thr Pro Leu Pro Ile Gly His Thr Ile Met Arg 530 535 540Thr Leu Glu Asp His Ile Arg Asn Asn Leu Ser Glu Arg Val Thr Ile545 550 555 560Met Thr His Val Ser Val Thr Glu Leu Leu His Glu Thr Asp Thr Thr 565 570 575Pro Asp Gly Ala Ser Glu Val Arg Val Thr Gly Val Arg Tyr Arg Asp 580 585 590Leu Ser Asp Val Asp Gly Gln Pro Ser Lys Leu Leu Ala Asp Ala Val 595 600 605Val Leu

Ala Thr Gly Gly Phe Ser Asn Asp Arg Glu Glu Asn Ser Leu 610 615 620Leu Cys Lys Tyr Ala Pro His Leu Ala Ser Phe Pro Thr Thr Asn Gly625 630 635 640Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala Thr Ser Val Gly Ala 645 650 655Lys Leu Val Asp Met Asp Lys Val Gln Leu His Pro Thr Gly Leu Ile 660 665 670Asp Pro Lys Asp Pro Ala Asn Thr Thr Lys Ile Leu Gly Pro Glu Ala 675 680 685Leu Arg Gly Ser Gly Gly Ile Leu Leu Asn Lys Gln Gly Lys Arg Phe 690 695 700Val Asn Glu Leu Asp Leu Arg Ser Val Val Ser Lys Ala Ile Asn Thr705 710 715 720Gln Gly Asn Glu Tyr Pro Gly Ser Gly Gly Cys Tyr Phe Ala Tyr Cys 725 730 735Val Leu Asn Glu Asp Ala Thr Asn Leu Phe Cys Gly Gly Ala Leu Gly 740 745 750Phe Tyr Gly Lys Lys Leu Gly Leu Phe Gln Arg Ala Glu Thr Val Glu 755 760 765Glu Leu Ala Lys Leu Ile Gly Cys Asp Glu Gly Glu Leu Arg Asp Thr 770 775 780Leu Glu Lys Tyr Glu Thr Cys Ser Lys Ala Lys Val Ala Cys Pro Val785 790 795 800Thr Gly Lys Val Val Phe Pro Cys Val Val Gly Thr Arg Gly Pro Tyr 805 810 815Asn Val Ala Phe Val Thr Pro Ser Ile His Tyr Thr Met Gly Gly Cys 820 825 830Leu Ile Ser Pro Ala Ala Glu Val Leu Gln Glu Tyr Lys Gly Leu Asn 835 840 845Ile Leu Glu Asn His Arg Pro Ile Arg Cys Leu Phe Gly Ala Gly Glu 850 855 860Val Thr Gly Gly Val His Gly Gly Asn Arg Leu Gly Gly Asn Ser Leu865 870 875 880Leu Glu Cys Val Val Phe Gly Lys Ile Ala Gly Asp Arg Ala Ala Thr 885 890 895Ile Leu Gln Lys Arg Glu Ile Ala Leu Ser Lys Thr Ser Trp Thr Ser 900 905 910Val Val Val Arg Glu Ser Arg Ser Gly Glu Gln Phe Gly Thr Gly Ser 915 920 925Arg Val Leu Arg Phe Asn Leu Pro Gly Ala Leu Gln Arg Thr Gly Leu 930 935 940Asn Leu Gly Glu Phe Val Ala Ile Arg Gly Glu Trp Asp Gly Gln Gln945 950 955 960Leu Val Gly Tyr Phe Ser Pro Ile Thr Leu Pro Glu Asp Leu Gly Thr 965 970 975Ile Ser Leu Leu Val Arg Ala Asp Lys Gly Thr Leu Lys Glu Trp Ile 980 985 990Cys Ala Leu Arg Pro Gly Asp Ser Val Glu Ile Lys Ala Cys Gly Gly 995 1000 1005Leu Arg Ile Asp Gln Asp Pro Val Lys Lys Cys Leu Leu Phe Arg 1010 1015 1020Asn Arg Pro Ile Thr Arg Phe Ala Leu Val Ala Ala Gly Thr Gly 1025 1030 1035Val Ala Pro Met Leu Gln Val Ile Arg Ala Ala Leu Lys Lys Pro 1040 1045 1050Tyr Val Asp Thr Leu Glu Ser Ile Arg Leu Ile Tyr Ala Ala Glu 1055 1060 1065Glu Tyr Asp Thr Leu Thr Tyr Arg Ser Ile Leu Gln Arg Phe Ala 1070 1075 1080Glu Glu Phe Pro Asp Lys Phe Val Cys Asn Phe Val Leu Asn Asn 1085 1090 1095Pro Pro Glu Gly Trp Thr Gly Gly Val Gly Phe Val Asn Lys Lys 1100 1105 1110Ser Leu Gln Lys Val Leu Gln Pro Pro Ser Ser Glu Pro Leu Ile 1115 1120 1125Val Val Cys Gly Pro Pro Val Met Gln Arg Asp Val Lys Asn Glu 1130 1135 1140Leu Leu Ser Met Gly Tyr Asp Lys Glu Leu Val His Thr Val Asp 1145 1150 1155Gly Glu Ser Gly Thr Leu 116041142PRTTrypanosoma brucei 4Met Val Asp Gly Arg Ser Ser Ala Ser Ile Val Ala Val Asp Pro Glu1 5 10 15Arg Ala Ala Arg Glu Arg Asp Ala Ala Ala Arg Ala Leu Leu Gln Asp 20 25 30Ser Pro Leu His Thr Thr Met Gln Tyr Ala Thr Ser Gly Leu Glu Leu 35 40 45Thr Val Pro Tyr Ala Leu Lys Val Val Ala Ser Ala Asp Thr Phe Asp 50 55 60Arg Ala Lys Glu Val Ala Asp Glu Val Leu Arg Cys Ala Trp Gln Leu65 70 75 80Ala Asp Thr Val Leu Asn Ser Phe Asn Pro Asn Ser Glu Val Ser Leu 85 90 95Val Gly Arg Leu Pro Val Gly Gln Lys His Gln Met Ser Ala Pro Leu 100 105 110Lys Arg Val Met Ala Cys Cys Gln Arg Val Tyr Asn Ser Ser Ala Gly 115 120 125Cys Phe Asp Pro Ser Thr Ala Pro Val Ala Lys Ala Leu Arg Glu Ile 130 135 140Ala Leu Gly Lys Glu Arg Asn Asn Ala Cys Leu Glu Ala Leu Thr Gln145 150 155 160Ala Cys Thr Leu Pro Asn Ser Phe Val Ile Asp Phe Glu Ala Gly Thr 165 170 175Ile Ser Arg Lys His Glu His Ala Ser Leu Asp Leu Gly Gly Val Ser 180 185 190Lys Gly Tyr Ile Val Asp Tyr Val Ile Asp Asn Ile Asn Ala Ala Gly 195 200 205Phe Gln Asn Val Phe Phe Asp Trp Gly Gly Asp Cys Arg Ala Ser Gly 210 215 220Met Asn Ala Arg Asn Thr Pro Trp Val Val Gly Ile Thr Arg Pro Pro225 230 235 240Ser Leu Asp Met Leu Pro Asn Pro Pro Lys Glu Ala Ser Tyr Ile Ser 245 250 255Val Ile Ser Leu Asp Asn Glu Ala Leu Ala Thr Ser Gly Asp Tyr Glu 260 265 270Asn Leu Ile Tyr Thr Ala Asp Asp Lys Pro Leu Thr Cys Thr Tyr Asp 275 280 285Trp Lys Gly Lys Glu Leu Met Lys Pro Ser Gln Ser Asn Ile Ala Gln 290 295 300Val Ser Val Lys Cys Tyr Ser Ala Met Tyr Ala Asp Ala Leu Ala Thr305 310 315 320Ala Cys Phe Ile Lys Arg Asp Pro Ala Lys Val Arg Gln Leu Leu Asp 325 330 335Gly Trp Arg Tyr Val Arg Asp Thr Val Arg Asp Tyr Arg Val Tyr Val 340 345 350Arg Glu Asn Glu Arg Val Ala Lys Met Phe Glu Ile Ala Thr Glu Asp 355 360 365Ala Glu Met Arg Lys Arg Arg Ile Ser Asn Thr Leu Pro Ala Arg Val 370 375 380Ile Val Val Gly Gly Gly Leu Ala Gly Leu Ser Ala Ala Ile Glu Ala385 390 395 400Ala Gly Cys Gly Ala Gln Val Val Leu Met Glu Lys Glu Ala Lys Leu 405 410 415Gly Gly Asn Ser Ala Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr 420 425 430Arg Ala Gln Ala Lys Ala Ser Ile Val Asp Gly Gly Lys Tyr Phe Glu 435 440 445Arg Asp Thr Tyr Lys Ser Gly Ile Gly Gly Asn Thr Asp Pro Ala Leu 450 455 460Val Lys Thr Leu Ser Met Lys Ser Ala Asp Ala Ile Gly Trp Leu Thr465 470 475 480Ser Leu Gly Val Pro Leu Thr Val Leu Ser Gln Leu Gly Gly His Ser 485 490 495Arg Lys Arg Thr His Arg Ala Pro Asp Lys Lys Asp Gly Thr Pro Leu 500 505 510Pro Ile Gly Phe Thr Ile Met Lys Thr Leu Glu Asp His Val Arg Gly 515 520 525Asn Leu Ser Gly Arg Ile Thr Ile Met Glu Asn Cys Ser Val Thr Ser 530 535 540Leu Leu Ser Glu Thr Lys Glu Arg Pro Asp Gly Thr Lys Gln Ile Arg545 550 555 560Val Thr Gly Val Glu Phe Thr Gln Ala Gly Ser Gly Lys Thr Thr Ile 565 570 575Leu Ala Asp Ala Val Ile Leu Ala Thr Gly Gly Phe Ser Asn Asp Lys 580 585 590Thr Ala Asp Ser Leu Leu Arg Glu His Ala Pro His Leu Val Asn Phe 595 600 605Pro Thr Thr Asn Gly Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala 610 615 620Gln Arg Leu Gly Ala Gln Leu Val Asp Met Asp Lys Val Gln Leu His625 630 635 640Pro Thr Gly Leu Ile Asn Pro Lys Asp Pro Ala Asn Pro Thr Lys Phe 645 650 655Leu Gly Pro Glu Ala Leu Arg Gly Ser Gly Gly Val Leu Leu Asn Lys 660 665 670Gln Gly Lys Arg Phe Val Asn Glu Leu Asp Leu Arg Ser Val Val Ser 675 680 685Lys Ala Ile Met Glu Gln Gly Ala Glu Tyr Pro Gly Ser Gly Gly Ser 690 695 700Met Phe Ala Tyr Cys Val Leu Asn Ala Ala Ala Gln Lys Leu Phe Gly705 710 715 720Val Ser Ser His Glu Phe Tyr Trp Lys Lys Met Gly Leu Phe Val Lys 725 730 735Ala Asp Thr Met Arg Asp Leu Ala Ala Leu Ile Gly Cys Pro Val Glu 740 745 750Ser Val Gln Gln Thr Leu Glu Glu Tyr Glu Arg Leu Ser Ile Ser Gln 755 760 765Arg Ser Cys Pro Ile Thr Arg Lys Ser Val Tyr Pro Cys Val Leu Gly 770 775 780Thr Lys Gly Pro Tyr Tyr Val Ala Phe Val Thr Pro Ser Ile His Tyr785 790 795 800Thr Met Gly Gly Cys Leu Ile Ser Pro Ser Ala Glu Ile Gln Met Lys 805 810 815Asn Thr Ser Ser Arg Ala Pro Leu Ser His Ser Asn Pro Ile Leu Gly 820 825 830Leu Phe Gly Ala Gly Glu Val Thr Gly Gly Val His Gly Gly Asn Arg 835 840 845Leu Gly Gly Asn Ser Leu Leu Glu Cys Val Val Phe Gly Arg Ile Ala 850 855 860Gly Asp Arg Ala Ser Thr Ile Leu Gln Arg Lys Ser Ser Ala Leu Ser865 870 875 880Phe Lys Val Trp Thr Thr Val Val Leu Arg Glu Val Arg Glu Gly Gly 885 890 895Val Tyr Gly Ala Gly Ser Arg Val Leu Arg Phe Asn Leu Pro Gly Ala 900 905 910Leu Gln Arg Ser Gly Leu Ser Leu Gly Gln Phe Ile Ala Ile Arg Gly 915 920 925Asp Trp Asp Gly Gln Gln Leu Ile Gly Tyr Tyr Ser Pro Ile Thr Leu 930 935 940Pro Asp Asp Leu Gly Met Ile Asp Ile Leu Ala Arg Ser Asp Lys Gly945 950 955 960Thr Leu Arg Glu Trp Ile Ser Ala Leu Glu Pro Gly Asp Ala Val Glu 965 970 975Met Lys Ala Cys Gly Gly Leu Val Ile Glu Arg Arg Leu Ser Asp Lys 980 985 990His Phe Val Phe Met Gly His Ile Ile Asn Lys Leu Cys Leu Ile Ala 995 1000 1005Gly Gly Thr Gly Val Ala Pro Met Leu Gln Ile Ile Lys Ala Ala 1010 1015 1020Phe Met Lys Pro Phe Ile Asp Thr Leu Glu Ser Val His Leu Ile 1025 1030 1035Tyr Ala Ala Glu Asp Val Thr Glu Leu Thr Tyr Arg Glu Val Leu 1040 1045 1050Glu Glu Arg Arg Arg Glu Ser Arg Gly Lys Phe Lys Lys Thr Phe 1055 1060 1065Val Leu Asn Arg Pro Pro Pro Leu Trp Thr Asp Gly Val Gly Phe 1070 1075 1080Ile Asp Arg Gly Ile Leu Thr Asn His Val Gln Pro Pro Ser Asp 1085 1090 1095Asn Leu Leu Val Ala Ile Cys Gly Pro Pro Val Met Gln Arg Ile 1100 1105 1110Val Lys Ala Thr Leu Lys Thr Leu Gly Tyr Asn Met Asn Leu Val 1115 1120 1125Arg Thr Val Asp Glu Thr Glu Pro Ser Gly Ser Ser Lys Ile 1130 1135 114053429DNATrypanosoma brucei 5atggtagacg ggcgatcttc tgcatcaatt gttgccgttg atcccgaaag ggctgcgcgt 60gagcgcgacg cagcagcgcg tgcccttctt caagacagtc cgctacacac gaccatgcaa 120tatgcaacgt ctggtcttga gcttaccgtt ccctatgcac ttaaggtggt tgccagtgct 180gacaccttcg atcgcgctaa ggaggttgcc gatgaggtgc tacgctgcgc atggcaactc 240gccgacaccg tgttgaacag tttcaacccg aacagtgagg tttcactcgt gggtcgcctg 300cctgtggggc agaagcacca aatgtctgct ccactcaagc gtgtgatggc atgctgccag 360cgtgtgtata actcatcggc tggatgtttt gatccctcca cagcacccgt cgcaaaggcg 420ctgcgtgaga ttgcactggg gaaggagcgg aacaatgctt gtctggaggc acttactcaa 480gcgtgtacgc ttcccaacag ttttgtgatc gatttcgaag ctggaactat cagccgtaag 540cacgagcatg cgtctctgga cctaggtggg gttagcaaag gttatatcgt tgattatgtc 600attgataata tcaatgctgc tggatttcaa aacgtttttt ttgactgggg tggagactgc 660cgtgcgagtg gtatgaatgc gcgcaatacc ccgtgggttg ttggtataac tcgccctccg 720tcccttgata tgctccctaa cccgccaaag gaggcgtcgt atatcagcgt tatctctctc 780gacaacgagg cccttgccac gagtggcgat tatgaaaact taatatacac cgctgatgat 840aaacccctta cctgcactta tgactggaag gggaaggaac tgatgaaacc ttctcagtcc 900aatatcgcgc aggtatcggt taaatgttat agcgccatgt acgctgacgc gcttgcgact 960gcgtgtttca taaagcggga tcccgcgaag gttcgacagc tgctggacgg ttggcgttac 1020gtgcgtgata cagtgagaga ttacagggtc tacgttcgtg aaaatgagcg agtagcgaag 1080atgtttgaga tcgccacaga ggatgcggaa atgaggaaga ggcggatcag caacacactt 1140cccgctcgtg tcattgtggt gggcggtggt cttgcgggtt tgtccgcggc catcgaagct 1200gcaggatgcg gtgctcaggt tgtgcttatg gagaaggagg cgaagctcgg aggcaacagc 1260gccaaggcga catctggtat caacggatgg ggcacacgtg ctcaggcgaa ggcaagcatt 1320gtggatggtg ggaaatactt cgagcgtgac acatacaagt ctggtatcgg gggtaacacc 1380gatcctgccc ttgtgaagac actttctatg aaaagtgctg acgctattgg gtggctgacc 1440tcgttgggtg taccgctgac ggtattgtca cagcttgggg gtcacagccg caagcgcaca 1500catcgggcac cggataagaa agatggtaca cctctaccta tcggatttac aatcatgaaa 1560accctcgagg atcacgtgcg tggtaacctt tctggccgca tcaccataat ggaaaactgc 1620agtgtaacgt cgttgctcag tgagacgaag gaacggccag atggcactaa acagatacga 1680gttactggtg tggagttcac gcaggctggc agtgggaaga cgaccatact tgcagatgct 1740gtcatccttg ccactggtgg attttctaac gacaaaactg cagactccct gcttcgtgag 1800cacgccccgc acttggtcaa cttccctacg acgaatggcc cgtgggcgac aggtgatggc 1860gtgaaacttg cacagcgact tggcgctcaa ctggtggata tggacaaggt ccagttgcat 1920ccgacaggcc tcatcaaccc gaaggatcca gcgaacccta caaagttcct tggacctgag 1980gcgctacgtg gatccggtgg cgttttgttg aacaagcaag gcaagcgctt cgttaatgaa 2040cttgacctcc gttctgtggt atcgaaagcc atcatggaac agggtgcgga atatcctgga 2100tcgggtggta gcatgttcgc ctactgtgtg ttgaatgctg cggcgcagaa gctctttggt 2160gtcagctcac acgagttcta ctggaagaag atgggtctct tcgtgaaggc tgacaccatg 2220agggacctcg ctgcactcat tgggtgccca gtggaatctg tgcagcagac gctggaggag 2280tacgagcggc tctccatatc acagcgttcc tgccccatca cgcgcaaaag cgtctatccg 2340tgcgtgctcg gcactaaggg cccctactac gtcgccttcg tgacaccttc gattcactac 2400acaatgggtg gatgtctcat ctcgccttct gctgaaatac aaatgaagaa cacatcatca 2460cgcgctccac tgagtcacag caacccaatc ctcgggttat ttggtgccgg tgaggtaacg 2520ggtggtgtgc acggtgggaa ccggttgggc ggcaattcgc tgcttgagtg cgtcgtgttt 2580gggagaattg cgggtgatcg ggcctcgacc atccttcaga ggaagtcctc agcactttcc 2640ttcaaggtgt ggacgaccgt ggtgctgcgt gaagtacgcg aaggtggtgt gtacggtgct 2700gggtcccgcg tgcttcgctt taatttaccc ggggcgctgc aacggtctgg tctgagcctc 2760ggccaattta tcgcaattcg tggtgattgg gacggtcagc agttgatcgg ttattacagt 2820cccatcacgc tgccagatga tcttggcatg atcgatatac tcgcccgcag tgataagggg 2880acgctgaggg agtggatttc cgctctggag ccgggtgacg ctgtggagat gaaggcatgc 2940ggtggtctgg tgattgagcg ccgcttaagc gataagcact ttgtgttcat gggacacatt 3000atcaacaagc tttgtctaat tgctggtgga acgggtgtgg caccgatgct gcaaataatc 3060aaagcagcct ttatgaaacc cttcattgac acattggaga gcgttcatct catctatgcc 3120gcggaggacg tgacggagtt gacgtatcgc gaggtgctgg aggagcgccg tcgtgagtca 3180cgtggaaagt tcaagaaaac gtttgtcctc aaccggcccc cgcccctatg gactgatggt 3240gttggcttca tcgaccgggg catcctcaca aatcatgtgc agccgccatc tgacaacctg 3300ctggtggcca tatgcggacc accggtaatg cagcgcattg taaaggcgac cctgaagact 3360ttgggctaca acatgaacct tgtgaggact gtggatgaaa cggagccgag cggctcatcc 3420aaaatttga 342961139PRTArtificial sequenceFRDg lacking 3 aa C-terminal targeting signal 6Met Val Asp Gly Arg Ser Ser Ala Ser Ile Val Ala Val Asp Pro Glu1 5 10 15Arg Ala Ala Arg Glu Arg Asp Ala Ala Ala Arg Ala Leu Leu Gln Asp 20 25 30Ser Pro Leu His Thr Thr Met Gln Tyr Ala Thr Ser Gly Leu Glu Leu 35 40 45Thr Val Pro Tyr Ala Leu Lys Val Val Ala Ser Ala Asp Thr Phe Asp 50 55 60Arg Ala Lys Glu Val Ala Asp Glu Val Leu Arg Cys Ala Trp Gln Leu65 70 75 80Ala Asp Thr Val Leu Asn Ser Phe Asn Pro Asn Ser Glu Val Ser Leu 85 90 95Val Gly Arg Leu Pro Val Gly Gln Lys His Gln Met Ser Ala Pro Leu 100 105 110Lys Arg Val Met Ala Cys Cys Gln Arg Val Tyr Asn Ser Ser Ala Gly 115 120 125Cys Phe Asp Pro Ser Thr Ala Pro Val Ala Lys Ala Leu Arg Glu Ile 130 135 140Ala Leu Gly Lys Glu Arg Asn Asn Ala Cys Leu Glu Ala Leu Thr Gln145 150 155 160Ala Cys Thr Leu Pro Asn Ser Phe Val Ile Asp Phe Glu Ala Gly Thr 165 170 175Ile Ser Arg Lys His Glu His Ala Ser Leu Asp Leu

Gly Gly Val Ser 180 185 190Lys Gly Tyr Ile Val Asp Tyr Val Ile Asp Asn Ile Asn Ala Ala Gly 195 200 205Phe Gln Asn Val Phe Phe Asp Trp Gly Gly Asp Cys Arg Ala Ser Gly 210 215 220Met Asn Ala Arg Asn Thr Pro Trp Val Val Gly Ile Thr Arg Pro Pro225 230 235 240Ser Leu Asp Met Leu Pro Asn Pro Pro Lys Glu Ala Ser Tyr Ile Ser 245 250 255Val Ile Ser Leu Asp Asn Glu Ala Leu Ala Thr Ser Gly Asp Tyr Glu 260 265 270Asn Leu Ile Tyr Thr Ala Asp Asp Lys Pro Leu Thr Cys Thr Tyr Asp 275 280 285Trp Lys Gly Lys Glu Leu Met Lys Pro Ser Gln Ser Asn Ile Ala Gln 290 295 300Val Ser Val Lys Cys Tyr Ser Ala Met Tyr Ala Asp Ala Leu Ala Thr305 310 315 320Ala Cys Phe Ile Lys Arg Asp Pro Ala Lys Val Arg Gln Leu Leu Asp 325 330 335Gly Trp Arg Tyr Val Arg Asp Thr Val Arg Asp Tyr Arg Val Tyr Val 340 345 350Arg Glu Asn Glu Arg Val Ala Lys Met Phe Glu Ile Ala Thr Glu Asp 355 360 365Ala Glu Met Arg Lys Arg Arg Ile Ser Asn Thr Leu Pro Ala Arg Val 370 375 380Ile Val Val Gly Gly Gly Leu Ala Gly Leu Ser Ala Ala Ile Glu Ala385 390 395 400Ala Gly Cys Gly Ala Gln Val Val Leu Met Glu Lys Glu Ala Lys Leu 405 410 415Gly Gly Asn Ser Ala Lys Ala Thr Ser Gly Ile Asn Gly Trp Gly Thr 420 425 430Arg Ala Gln Ala Lys Ala Ser Ile Val Asp Gly Gly Lys Tyr Phe Glu 435 440 445Arg Asp Thr Tyr Lys Ser Gly Ile Gly Gly Asn Thr Asp Pro Ala Leu 450 455 460Val Lys Thr Leu Ser Met Lys Ser Ala Asp Ala Ile Gly Trp Leu Thr465 470 475 480Ser Leu Gly Val Pro Leu Thr Val Leu Ser Gln Leu Gly Gly His Ser 485 490 495Arg Lys Arg Thr His Arg Ala Pro Asp Lys Lys Asp Gly Thr Pro Leu 500 505 510Pro Ile Gly Phe Thr Ile Met Lys Thr Leu Glu Asp His Val Arg Gly 515 520 525Asn Leu Ser Gly Arg Ile Thr Ile Met Glu Asn Cys Ser Val Thr Ser 530 535 540Leu Leu Ser Glu Thr Lys Glu Arg Pro Asp Gly Thr Lys Gln Ile Arg545 550 555 560Val Thr Gly Val Glu Phe Thr Gln Ala Gly Ser Gly Lys Thr Thr Ile 565 570 575Leu Ala Asp Ala Val Ile Leu Ala Thr Gly Gly Phe Ser Asn Asp Lys 580 585 590Thr Ala Asp Ser Leu Leu Arg Glu His Ala Pro His Leu Val Asn Phe 595 600 605Pro Thr Thr Asn Gly Pro Trp Ala Thr Gly Asp Gly Val Lys Leu Ala 610 615 620Gln Arg Leu Gly Ala Gln Leu Val Asp Met Asp Lys Val Gln Leu His625 630 635 640Pro Thr Gly Leu Ile Asn Pro Lys Asp Pro Ala Asn Pro Thr Lys Phe 645 650 655Leu Gly Pro Glu Ala Leu Arg Gly Ser Gly Gly Val Leu Leu Asn Lys 660 665 670Gln Gly Lys Arg Phe Val Asn Glu Leu Asp Leu Arg Ser Val Val Ser 675 680 685Lys Ala Ile Met Glu Gln Gly Ala Glu Tyr Pro Gly Ser Gly Gly Ser 690 695 700Met Phe Ala Tyr Cys Val Leu Asn Ala Ala Ala Gln Lys Leu Phe Gly705 710 715 720Val Ser Ser His Glu Phe Tyr Trp Lys Lys Met Gly Leu Phe Val Lys 725 730 735Ala Asp Thr Met Arg Asp Leu Ala Ala Leu Ile Gly Cys Pro Val Glu 740 745 750Ser Val Gln Gln Thr Leu Glu Glu Tyr Glu Arg Leu Ser Ile Ser Gln 755 760 765Arg Ser Cys Pro Ile Thr Arg Lys Ser Val Tyr Pro Cys Val Leu Gly 770 775 780Thr Lys Gly Pro Tyr Tyr Val Ala Phe Val Thr Pro Ser Ile His Tyr785 790 795 800Thr Met Gly Gly Cys Leu Ile Ser Pro Ser Ala Glu Ile Gln Met Lys 805 810 815Asn Thr Ser Ser Arg Ala Pro Leu Ser His Ser Asn Pro Ile Leu Gly 820 825 830Leu Phe Gly Ala Gly Glu Val Thr Gly Gly Val His Gly Gly Asn Arg 835 840 845Leu Gly Gly Asn Ser Leu Leu Glu Cys Val Val Phe Gly Arg Ile Ala 850 855 860Gly Asp Arg Ala Ser Thr Ile Leu Gln Arg Lys Ser Ser Ala Leu Ser865 870 875 880Phe Lys Val Trp Thr Thr Val Val Leu Arg Glu Val Arg Glu Gly Gly 885 890 895Val Tyr Gly Ala Gly Ser Arg Val Leu Arg Phe Asn Leu Pro Gly Ala 900 905 910Leu Gln Arg Ser Gly Leu Ser Leu Gly Gln Phe Ile Ala Ile Arg Gly 915 920 925Asp Trp Asp Gly Gln Gln Leu Ile Gly Tyr Tyr Ser Pro Ile Thr Leu 930 935 940Pro Asp Asp Leu Gly Met Ile Asp Ile Leu Ala Arg Ser Asp Lys Gly945 950 955 960Thr Leu Arg Glu Trp Ile Ser Ala Leu Glu Pro Gly Asp Ala Val Glu 965 970 975Met Lys Ala Cys Gly Gly Leu Val Ile Glu Arg Arg Leu Ser Asp Lys 980 985 990His Phe Val Phe Met Gly His Ile Ile Asn Lys Leu Cys Leu Ile Ala 995 1000 1005Gly Gly Thr Gly Val Ala Pro Met Leu Gln Ile Ile Lys Ala Ala 1010 1015 1020Phe Met Lys Pro Phe Ile Asp Thr Leu Glu Ser Val His Leu Ile 1025 1030 1035Tyr Ala Ala Glu Asp Val Thr Glu Leu Thr Tyr Arg Glu Val Leu 1040 1045 1050Glu Glu Arg Arg Arg Glu Ser Arg Gly Lys Phe Lys Lys Thr Phe 1055 1060 1065Val Leu Asn Arg Pro Pro Pro Leu Trp Thr Asp Gly Val Gly Phe 1070 1075 1080Ile Asp Arg Gly Ile Leu Thr Asn His Val Gln Pro Pro Ser Asp 1085 1090 1095Asn Leu Leu Val Ala Ile Cys Gly Pro Pro Val Met Gln Arg Ile 1100 1105 1110Val Lys Ala Thr Leu Lys Thr Leu Gly Tyr Asn Met Asn Leu Val 1115 1120 1125Arg Thr Val Asp Glu Thr Glu Pro Ser Gly Ser 1130 113573498DNAArtificial sequenceFRDm1 codon optimised for A. niger 7atgggtgccg atggtatctc ctctgcctcc attgtcgtca ccgaccccga ggctgctgcc 60aagaagcgtg accgcatggc ccgtgagctc ctctcctcca actccggtct ttgccaggag 120gatgagccca ccatcatcaa cctgaagggt ctggaacaca ccatccccta ccgtcttgct 180gttgtccttt gcaactctcg cagcactggt gaattcgagg ccaaggctgc tgagatcctc 240cgcaaggctt tccacatggt tgactactct ctgaactgct tcaaccccga gtccgagctc 300tcccgtgtca acagcttgcc tgtcggtgag aagcaccaga tgagcgaaga tctgcgccac 360gtcatggagt gcaccatctc cgtccaccac tcctctggca tgggtttcga ccctgctgct 420ggtcccatca tctcccgtct gcgtggtgcc atgcgcgacc acaacgacat gtccgacatc 480tccgtcaccg aggctgaggt tgagctgttc tcgctagcgc agtcgttcga tgttgacctc 540gaggagggca ccattgctcg caagcactcc gaggctcgcc tcgaccttgg tggtgtcaac 600aagggctaca ctgttgacta cgtggtggac cacctccgcg ctgctggcat gcccaacgtc 660ctgttcgaat ggggtggtga catccgtgcc tccggccgca acatcaaggg caacctctgg 720gctgttgcca tcaagcgccc tccctccgtt gaggaggtca tccgccgtgc caagggcaag 780atgctcaaga tgggtgaaga agaacaggag gagaaggatg atgactctcc cagccttctg 840cacgttgttg agctcgatga tgaggccctc tgcacctccg gtgactacga gaacgtcctc 900taccacccca agcacggtgt tgctggcagc atcttcgact ggcagcgccg tggtctgctg 960tctcctgagg agggtgctct tgctcaggtt tccgtcaagt gctactctgc catgtacgcc 1020gatgcccttg ccaccgtctg cctggtcaag cgtgatgccg tccgtatccg ctacctcctg 1080gaaggctggc gctacgtgcg ctctcgtgtc accaactact tcgcctacac ccgccagggt 1140gagcgtcttg ctcacatgca cgaaattgcc caggagactc gtgagctccg tgagatccgc 1200attgctggct ccctcccctc ccgtatcgtc atcgtcggtg gtggtctggc cggtctgtct 1260gctgccattg aggctgcctc ctgcggtgct caggtcatcc tgatggagaa ggagggtcgt 1320attggtggca actctgccaa ggccacctcc ggtatcaacg gctggggtac tcgcactcag 1380gccaagtccg acatcctgga tggcggcaag tacttcgagc gtgacacctt cctgagcggt 1440gttggtggta ccactgaccc tgctctggtc aaggtcctct ccgtcaagtc cggtgatgcc 1500attggctggt tgaccagcct tggtgttcct ctttctgttc tctcccagct gggtggtcac 1560tctttcaagc gtacccaccg tgctcctgac aagactgatg gcactcctct ccccatcggt 1620cacaccatca tgcgcaccct cgaggaccac atccgcaaca acctgagcga acgtgtcacc 1680atcatgaccc acgtttccgt cactgagctc ctccacgaga ctgacaccac tcccgatggt 1740gcctccgagg tccgtgtcac cggtgtccgc taccgtgacc tctccgatgt tgacggccag 1800cccagcaagc tccttgccga tgccgttgtc cttgccactg gtggtttctc caacgaccgc 1860gaggagaaca gcttgctttg caagtacgcc ccccacctgg cctccttccc caccaccaac 1920ggcccttggg ccactggtga tggtgtcaag ctggccacct ccgtcggtgc caagctcgtc 1980gacatggaca aggtccagct gcaccccact ggcttgattg accccaagga ccccgccaac 2040accaccaaga tcctgggccc cgaggctctc cgtggcagcg gtggtatcct gctcaacaag 2100cagggcaagc gcttcgtcaa cgagcttgac ctccgcagcg ttgtctccaa ggccatcaac 2160actcagggca acgaataccc cggcagcggt ggctgctact tcgcctactg cgtgttgaac 2220gaagatgcca ccaacctgtt ctgcggtggt gctcttggat tctacggcaa gaagcttggt 2280ctgttccagc gtgctgagac tgttgaggag cttgccaagt tgattggctg cgatgagggc 2340gagctccgtg acaccctcga gaagtacgag acttgctcga aggccaaggt tgcctgcccc 2400gtgaccggca aggtcgtgtt cccctgcgtt gttggtaccc gtggtcccta caacgtcgct 2460ttcgtcaccc cctccatcca ctacaccatg ggtggctgct tgatttctcc tgctgctgag 2520gtcctccagg aatacaaggg tctgaacatc ctggagaacc accgtcccat tcgctgcttg 2580ttcggtgctg gtgaagtcac cggtggtgtc cacggtggca accgcctggg tggcaactcc 2640ctcctcgagt gcgttgtgtt cggcaagatc gctggtgacc gtgctgccac cattctccag 2700aagcgcgaaa ttgccctctc caagaccagc tggacctccg tcgtcgtccg cgagtcccgc 2760tctggcgagc agttcggtac cggctctcgt gtcctccgct tcaacctgcc cggtgctctc 2820cagcgcactg gtctgaacct gggtgagttc gtcgccatcc gtggtgaatg ggatggccag 2880cagctggtcg gctacttctc ccccatcacc ctccccgaag atcttggtac catctccctc 2940ctggtccgtg ccgacaaggg caccctcaag gaatggatat gtgccctccg ccccggtgac 3000agcgttgaga tcaaggcctg cggtggtctg cgtatcgacc aggaccctgt caagaagtgc 3060ttgctattcc gcaaccgccc catcacccgc ttcgctcttg ttgctgctgg tactggtgtt 3120gctcccatgc tccaggtcat ccgtgctgct ctcaagaagc cctacgtgga tacattggag 3180tccatccgtc tgatctacgc tgctgaagaa tacgacaccc tgacctaccg ctccatcctc 3240cagcgcttcg ctgaggagtt ccccgacaag ttcgtctgca acttcgtcct caacaaccct 3300cctgaaggct ggactggtgg tgttggtttc gtcaacaaga agtccctcca gaaggtcctc 3360cagcctccta gctctgagcc tctgattgtc gtctgcggtc ctcctgtcat gcagcgtgat 3420gtcaagaacg agctcctcag catgggctac gacaaggagc ttgtccacac cgttgacggc 3480gagtctggca ccctataa 349883420DNAArtificial sequenceFRDg gene optimised for A. niger 8atggtcgatg gccgctcctc cgcctccatt gttgctgttg accccgagcg tgctgctcgt 60gagcgtgatg ctgctgctcg tgccctcctc caggactctc ccttgcacac caccatgcag 120tacgccacct ccggtctgga attgactgtt ccctacgccc tcaaggttgt tgcctccgcc 180gacaccttcg accgtgccaa ggaggttgcc gatgaggtcc tccgctgcgc ctggcagctg 240gccgacaccg tcctcaactc tttcaacccc aacagcgaag tctctctggt cggccgcctc 300cccgtcggtc agaagcacca gatgagcgct cctctcaagc gtgtcatggc ctgctgccag 360cgtgtctaca acagctctgc tggctgcttc gaccccagca ctgctcctgt tgccaaggcc 420ctccgtgaga tcgctcttgg caaggagcgc aacaacgcct gcttggaggc tcttactcag 480gcctgcaccc tccccaactc gttcgtcatt gacttcgagg ctggcaccat ctcccgcaag 540cacgaacacg cctccctcga tcttggtggt gtcagcaagg gctacatcgt cgactacgtc 600attgacaaca tcaacgctgc tggtttccag aacgttttct tcgactgggg tggtgactgc 660cgtgcctccg gcatgaacgc ccgcaacacc ccctgggttg ttggtatcac ccgccccccg 720tcattggaca tgcttcccaa ccctcccaag gaggccagct acatctccgt catctccctc 780gacaacgagg ctcttgccac cagcggtgac tacgagaacc tgatctacac tgccgatgac 840aagcctctga cctgcaccta cgactggaag ggcaaggagc tcatgaagcc cagccagtcc 900aacattgccc aggtcagcgt caagtgctac tctgccatgt acgccgatgc ccttgccact 960gcttgcttca tcaagcgtga ccccgccaag gtccgccagc tgttggatgg ctggcgctac 1020gtgcgcgaca ccgtccgtga ctaccgtgtc tacgtgcgcg agaacgagcg tgttgccaag 1080atgttcgaaa ttgccactga ggatgccgag atgcgcaagc gccgtatctc caacaccctc 1140cctgctcgtg tcattgttgt tggtggtggt ctggctggtc tttctgctgc cattgaggct 1200gctggctgcg gtgctcaggt tgtcctgatg gagaaggagg ccaagctcgg tggcaactcc 1260gccaaggcca cctccggtat caacggctgg ggtactcgtg ctcaggccaa ggcctccatc 1320gtcgatggcg gcaagtactt cgagcgtgac acctacaagt ccggtatcgg tggcaacacc 1380gaccctgctc tggtcaagac cctgagcatg aagtccgccg atgccattgg ctggttgacc 1440agccttggtg ttcctcttac tgtcctttct cagctgggtg gccactctcg caagcgcacc 1500caccgtgctc ctgacaagaa ggacggcacc cccctcccca tcggtttcac catcatgaaa 1560actctcgagg accacgtccg tggcaacctg tctggccgta tcaccatcat ggagaactgc 1620tcggtgacct cgctactctc cgagactaag gagcgccccg atggcaccaa gcagatccgt 1680gtcaccggtg ttgagttcac ccaggctggc tctggcaaga ccaccatcct ggccgatgcc 1740gtcatcctgg ccactggtgg tttctccaac gacaagactg ccgactcgct actccgcgaa 1800cacgctcccc acctggtcaa cttccccacc accaacggcc cctgggcgac tggtgatggt 1860gtcaagctgg cccagcgtct gggtgctcag ctcgtcgaca tggacaaggt ccagctccac 1920cccactggtc tgatcaaccc caaggaccct gccaacccca ccaagttcct tggacctgag 1980gctctccgtg gctccggtgg tgtccttctg aacaagcagg gcaagcgctt cgtcaacgag 2040ctcgatctcc gcagcgttgt ctccaaggcc atcatggagc agggtgctga ataccccggc 2100agcggtggca gcatgttcgc ctactgcgtt ctcaacgctg ctgctcagaa gctgttcggt 2160gtctcctccc acgaattcta ctggaagaag atgggtctgt tcgtcaaggc cgacaccatg 2220cgtgatcttg ctgctctgat cggttgcccc gttgagagcg tgcagcagac cctggaagaa 2280tacgagcgcc tctccatctc ccagcgctct tgccccatca cccgcaagtc ggtgtaccct 2340tgcgtgcttg gcaccaaggg tccctactac gtggctttcg tcaccccctc catccactac 2400accatgggtg gctgcttgat ctctccttct gctgagatcc agatgaagaa cacctcctcc 2460cgtgctcctc tctcccactc caaccccatc ctcggtctgt tcggtgctgg tgaagtcact 2520ggtggtgtcc acggtggcaa ccgtcttggt ggcaactccc tcctcgagtg cgttgtgttc 2580ggccgtatcg ctggtgaccg tgccagcacc atcctccagc gcaagagctc tgctctctcc 2640ttcaaggtct ggaccactgt tgtcctccgc gaagtccgcg agggtggtgt ctacggtgct 2700ggctctcgtg tcctccgctt caacctcccc ggtgctctcc agcgctccgg tctgtctctt 2760ggccagttca ttgccatccg tggtgactgg gatggccagc agctcattgg ctactactct 2820cccatcaccc tccccgatga tcttggaatg atcgacatcc tggctcgctc cgacaagggt 2880accctccgcg aatggatctc cgctctggag cccggtgatg ccgttgagat gaaggcctgc 2940ggtggtctgg tcattgagcg tcgtctgtcc gacaagcact tcgtgttcat gggtcacatc 3000atcaacaagc tctgcttgat tgccggtggt actggtgttg ctcccatgct tcagatcatc 3060aaggctgctt tcatgaagcc cttcattgac accctcgagt ccgtccacct gatctacgct 3120gctgaggatg tcactgagct gacctaccgt gaggtccttg aggagcgccg ccgcgagtcc 3180cgtggcaagt tcaagaaaac cttcgtcctg aaccgccctc ctcctctctg gactgatggt 3240gttggtttca ttgaccgtgg tatcctgacc aaccacgtcc agcctccctc cgacaaccta 3300ttagtggcca tctgcggtcc tcctgtcatg cagcgcattg tcaaggccac tctcaagacc 3360ctaggataca acatgaacct ggtccgcact gttgatgaga ctgagccctc cggatcataa 342093498DNAArtificial sequenceFRDm1 gene optimsied for S. cerevisiae 9atgggtgctg atggtatttc ttctgcttcc attgttgtta ctgacccaga agctgctgcc 60aagaagcgtg acagaatggc cagagaattg ttgtcctcca actctggtct atgtcaagaa 120gatgaaccaa ccatcatcaa cttaaagggt ttggaacaca ccattccata cagattggcc 180gttgttttgt gtaactccag atccactggt gaattcgaag ccaaggctgc tgaaatcttg 240agaaaggctt tccacatggt tgactactct ttgaattgtt tcaacccaga atctgaattg 300tcccgtgtca actctttacc agtcggtgaa aagcaccaaa tgtccgaaga tctaagacat 360gtcatggaat gtaccatttc tgtccaccac tcctctggta tgggtttcga cccagctgct 420ggtccaatca tctccagatt gagaggtgcc atgagagatc acaacgacat gtccgatatc 480tccgtcactg aagctgaagt tgaattattc tctttggctc aatctttcga tgtcgacttg 540gaagaaggta ctattgccag aaagcactct gaagccagat tggatttggg tggtgtcaac 600aagggttaca ctgttgacta cgttgttgac catttgagag ctgctggtat gccaaacgtc 660ttgttcgaat ggggtggtga tatcagagct tctggtagaa acatcaaggg taacttgtgg 720gctgttgcca tcaagcgtcc accatctgtt gaagaagtta tccgtcgtgc caagggtaag 780atgttaaaga tgggtgaaga agaacaagaa gaaaaggacg atgactctcc atctttgttg 840cacgttgttg aattggatga cgaagctttg tgtacctctg gtgactacga aaacgtctta 900taccatccaa agcacggtgt tgctggttcc attttcgact ggcaacgtcg tggtttattg 960tctccagaag aaggtgcttt agctcaagtt tccgtcaaat gttactctgc catgtacgct 1020gatgctttgg ccactgtttg tttggtcaag agagatgctg tcagaatcag atacttgttg 1080gaaggttgga gatacgtcag atctcgtgtc accaactact tcgcttacac cagacaaggt 1140gaaagattgg ctcacatgca cgaaattgct caagaaacca gagaattaag agaaatcaga 1200attgctggtt ctttgccatc cagaattgtt atcgtcggtg gtggtttggc tggtctatcc 1260gctgccattg aagctgcttc ttgtggtgct caagtcattt tgatggaaaa ggaaggtaga 1320attggtggta actctgccaa ggctacctct ggtatcaacg gttggggtac cagaacccaa 1380gccaagtctg atatcttgga tggtggtaag tactttgaaa gagacacttt cttgtccggt 1440gtcggtggta ccactgaccc agctttggtc aaggtcttgt ccgtcaaatc tggtgacgct 1500atcggttggt taacttcttt gggtgtccca ttgtccgttt tgtctcaatt gggtggtcac 1560tctttcaaga gaactcacag agctccagac aagactgatg gtactccatt accaattggt 1620cacaccatca tgagaacttt ggaagatcat atcagaaaca acttgtctga aagagttacc 1680atcatgaccc acgtttctgt tactgaattg ttgcacgaaa ctgacaccac tccagatggt 1740gcttctgaag ttcgtgtcac cggtgtccgt tacagagact tgtctgatgt cgatggtcaa 1800ccttccaaac tattggctga cgctgttgtt ttggccactg gtggtttctc caacgacaga 1860gaagaaaact ctttgttgtg taaatacgct cctcatttgg cttctttccc aactaccaac 1920ggtccatggg ctactggtga cggtgtcaaa ttggccacct ccgttggtgc caagttggtt 1980gacatggaca aggttcaatt gcacccaact ggtttgattg acccaaagga cccagctaac 2040accactaaga tcttgggtcc agaagctttg agaggttctg gtggtatttt gttgaacaag

2100caaggtaaga gattcgtcaa cgaattggac ttgagatccg ttgtttccaa ggccattaac 2160actcaaggta acgaataccc aggttctggt ggttgttact ttgcttactg tgtcttaaac 2220gaagatgcta ccaacttatt ctgtggtggt gctttgggtt tctacggtaa gaaattaggt 2280ttgttccaaa gagctgaaac tgttgaagaa ttggccaaat tgattggttg tgacgaaggt 2340gaattgagag acactttgga aaaatacgaa acctgttcca aggccaaggt tgcttgtcca 2400gtcactggta aggttgtttt cccatgtgtt gtcggtacca gaggtccata caatgttgct 2460ttcgtcactc catccatcca ctacaccatg ggtggttgtt tgatctctcc agctgctgaa 2520gtcttgcaag aatacaaggg tttgaatatc ttggaaaacc acagaccaat cagatgtttg 2580ttcggtgctg gtgaagtcac tggtggtgtc cacggtggta acagattagg tggtaactct 2640ctattggaat gtgttgtctt tggtaagatt gctggtgaca gagctgccac tatcttgcaa 2700aagagagaaa ttgctttgtc caagacctcc tggacctctg ttgttgtcag agaatccaga 2760tctggtgaac aattcggtac cggttccaga gttttgagat tcaacttgcc aggtgcttta 2820caaagaaccg gtttgaactt gggtgaattc gttgccatca gaggtgaatg ggatggtcaa 2880caattagtcg gttacttctc tccaatcact ttgccagaag atttgggtac catctctttg 2940ttggtcagag ctgacaaggg tactttgaag gaatggatct gtgctttgcg tccaggtgac 3000tccgttgaaa tcaaggcttg tggtggtcta agaattgacc aagatccagt caagaaatgt 3060ttgttgttca gaaacagacc aattaccaga tttgctttgg ttgctgctgg taccggtgtt 3120gctccaatgt tgcaagttat cagagctgct ttgaagaagc catacgtcga cactttggaa 3180tccatcagat tgatctacgc tgctgaagaa tatgacactt taacctacag atctatcttg 3240caaagatttg ctgaagaatt cccagacaaa ttcgtttgta acttcgtctt aaacaaccct 3300ccagaaggtt ggaccggtgg tgttggtttc gtcaacaaga aatctttgca aaaggttttg 3360caaccacctt cttctgaacc attgattgtt gtttgtggtc cacctgttat gcaaagagat 3420gtcaaaaatg aattgttgtc catgggttac gacaaggaat tggttcacac tgtcgatggt 3480gaatctggta ccttgtaa 3498103420DNAArtificial sequenceFRDg gene optimised for S. cerevisiae 10atggttgatg gtagatcttc tgcttccatt gttgccgttg acccagaaag agctgccaga 60gaaagagatg ctgctgccag agctttgttg caagactctc cattgcacac caccatgcaa 120tacgctacct ctggtttgga attgactgtt ccatacgctt tgaaggttgt tgcttctgct 180gacactttcg acagagccaa ggaagttgct gatgaagtct tgagatgtgc ctggcaattg 240gctgacaccg ttttgaactc tttcaaccca aactctgaag tctctttagt cggtagatta 300ccagtcggtc aaaagcatca aatgtctgct ccattgaaac gtgtcatggc ttgttgtcaa 360agagtctaca actcctctgc tggttgtttc gacccatcca ctgctccagt tgccaaggct 420ttgagagaaa ttgctttggg taaggaaaga aacaatgctt gtttggaagc tttgactcaa 480gcttgtacct tgccaaactc tttcgtcatt gatttcgaag ctggtactat ctccagaaag 540cacgaacacg cttctttgga tttgggtggt gtttccaagg gttacatcgt cgattacgtc 600attgacaaca tcaatgctgc tggtttccaa aacgttttct ttgactgggg tggtgactgt 660cgtgcctccg gtatgaacgc cagaaacact ccatgggttg tcggtatcac tagacctcct 720tccttggaca tgttgccaaa ccctccaaag gaagcttctt acatctccgt catctctttg 780gacaatgaag ctttggctac ctctggtgat tacgaaaact tgatctacac tgctgacgat 840aaaccattga cctgtaccta cgattggaaa ggtaaggaat tgatgaagcc atctcaatcc 900aatatcgctc aagtttccgt caagtgttac tctgccatgt acgctgacgc tttggctacc 960gcttgtttca tcaagcgtga cccagccaag gtcagacaat tgttggatgg ttggagatac 1020gttagagaca ccgtcagaga ttaccgtgtc tacgtcagag aaaacgaaag agttgccaag 1080atgttcgaaa ttgccactga agatgctgaa atgagaaaga gaagaatttc caacacttta 1140ccagctcgtg tcattgttgt tggtggtggt ttggctggtt tgtccgctgc cattgaagct 1200gctggttgtg gtgctcaagt tgttttgatg gaaaaggaag ccaagttggg tggtaactct 1260gccaaggcta cctctggtat caacggttgg ggtactagag ctcaagctaa ggcttccatt 1320gtcgatggtg gtaagtactt cgaaagagat acctacaagt ctggtatcgg tggtaacacc 1380gatccagctt tggttaagac tttgtccatg aaatctgctg acgctatcgg ttggttgact 1440tctctaggtg ttccattgac tgttttgtcc caattaggtg gtcactccag aaagagaact 1500cacagagctc cagacaagaa ggatggtact ccattgccaa ttggtttcac catcatgaaa 1560actttagaag atcatgttag aggtaacttg tccggtagaa tcaccatcat ggaaaactgt 1620tccgttacct ctttgttgtc tgaaaccaag gaaagaccag acggtaccaa gcaaatcaga 1680gttaccggtg tcgaattcac tcaagctggt tctggtaaga ccaccatttt ggctgatgct 1740gttatcttgg ccaccggtgg tttctccaac gacaagactg ctgattcttt gttgagagaa 1800catgccccac acttggttaa cttcccaacc accaacggtc catgggctac tggtgatggt 1860gtcaagttgg ctcaaagatt aggtgctcaa ttggtcgata tggacaaggt tcaattgcac 1920ccaactggtt tgatcaaccc aaaggaccca gccaacccaa ccaaattctt gggtccagaa 1980gctctaagag gttctggtgg tgttttgttg aacaaacaag gtaagagatt tgtcaacgaa 2040ttggatttga gatctgttgt ttccaaggcc atcatggaac aaggtgctga atacccaggt 2100tctggtggtt ccatgtttgc ttactgtgtc ttgaacgctg ctgctcaaaa attgtttggt 2160gtttcctctc acgaattcta ctggaagaag atgggtttgt tcgtcaaggc tgacaccatg 2220agagacttgg ctgctttgat tggttgtcca gttgaatccg ttcaacaaac tttagaagaa 2280tacgaaagat tatccatctc tcaaagatct tgtccaatta ccagaaaatc tgtttaccca 2340tgtgttttgg gtaccaaagg tccatactat gtcgcctttg tcactccatc tatccactac 2400accatgggtg gttgtttgat ttctccatct gctgaaatcc aaatgaagaa cacttcttcc 2460agagctccat tgtcccactc caacccaatc ttgggtttat tcggtgctgg tgaagtcacc 2520ggtggtgtcc acggtggtaa cagattaggt ggtaactctt tgttggaatg tgttgttttc 2580ggtagaattg ccggtgacag agcttctacc attttgcaaa gaaagtcctc tgctttgtct 2640ttcaaggtct ggaccactgt tgttttgaga gaagtcagag aaggtggtgt ctacggtgct 2700ggttcccgtg tcttgagatt caacttacca ggtgctctac aaagatctgg tctatccttg 2760ggtcaattca ttgccatcag aggtgactgg gacggtcaac aattgattgg ttactactct 2820ccaatcactt tgccagacga tttgggtatg attgacattt tggccagatc tgacaagggt 2880actttacgtg aatggatctc tgctttggaa ccaggtgacg ctgtcgaaat gaaggcttgt 2940ggtggtttgg tcatcgaaag aagattatct gacaagcact tcgttttcat gggtcacatt 3000atcaacaagc tatgtttgat tgctggtggt accggtgttg ctccaatgtt gcaaatcatc 3060aaggccgctt tcatgaagcc attcatcgac actttggaat ccgtccactt gatctacgct 3120gctgaagatg tcactgaatt gacttacaga gaagttttgg aagaacgtcg tcgtgaatcc 3180agaggtaaat tcaagaaaac tttcgttttg aacagacctc ctccattatg gactgacggt 3240gtcggtttca tcgaccgtgg tatcttgacc aaccacgttc aaccaccatc tgacaactta 3300ttggttgcca tctgtggtcc accagttatg caaagaattg tcaaggccac tttaaagact 3360ttaggttaca acatgaactt ggtcagaacc gttgacgaaa ctgaaccatc tggaagttaa 342011898DNAArtificial sequenceGPDA promotor 11tcagcgtcca attcgagctc tgtacagtga ccggtgactc tttctggcat gcggagacac 60ggacggtcgc agagaggagg gctgagtaat aagcgcactc atgtcagctc tggcgctctg 120aggtgcagtg gatgattatt aatccgggac cggccgcccc tccgccccga agtggaaagg 180ctggtgtgcc cctcgttgac caagaatcta ttgcatcatc ggagaatatg gagcttcatc 240gaatcaccgg cagtaagcga aggagaatgt gaagccaggg gtgtatagcc gtcggcgaaa 300tagcatgcca ttaacctagg tacagaagtc caattgcttc cgatctggta aaagattcac 360gagatagtac cttctccgaa gtaggtagag cgagtacccg gcgcgtaagc tccctaattg 420gcccatccgg catctgtagg gcgtccaaat atcgtgcctc tcctgctttg cccggtgtat 480gaaaccggaa aggccgctca ggagctggcc agcggcgcag accgggaaca caagctggca 540gtcgacccat ccggtgctct gcactcgacc tgctgaggtc cctcagtccc tggtaggcag 600ctttgccccg tctgtccgcc cggtgtgtcg gcggggttga caaggtcgtt gcgtcagtcc 660aacatttgtt gccatatttt cctgctctcc ccaccagctg ctcttttctt ttctctttct 720tttcccatct tcagtatatt catcttccca tccaagaacc tttatttccc ctaagtaagt 780actttgctac atccatactc catccttccc atcccttatt cctttgaacc tttcagttcg 840agctttccca cttcatcgca gcttgactaa cagctacccc gcttgagcca ccgtcaaa 898121000DNAArtificial sequenceTDH3 promotor 12ctattttcga ggaccttgtc accttgagcc caagagagcc aagatttaaa ttttcctatg 60acttgatgca aattcccaaa gctaataaca tgcaagacac gtacggtcaa gaagacatat 120ttgacctctt aacaggttca gacgcgactg cctcatcagt aagacccgtt gaaaagaact 180tacctgaaaa aaacgaatat atactagcgt tgaatgttag cgtcaacaac aagaagttta 240atgacgcgga ggccaaggca aaaagattcc ttgattacgt aagggagtta gaatcatttt 300gaataaaaaa cacgcttttt cagttcgagt ttatcattat caatactgcc atttcaaaga 360atacgtaaat aattaatagt agtgattttc ctaactttat ttagtcaaaa aattagcctt 420ttaattctgc tgtaacccgt acatgcccaa aatagggggc gggttacaca gaatatataa 480catcgtaggt gtctgggtga acagtttatt cctggcatcc actaaatata atggagcccg 540ctttttaagc tggcatccag aaaaaaaaag aatcccagca ccaaaatatt gttttcttca 600ccaaccatca gttcataggt ccattctctt agcgcaacta cagagaacag gggcacaaac 660aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc tgcctggagt aaatgatgac 720acaaggcaat tgacccacgc atgtatctat ctcattttct tacaccttct attaccttct 780gctctctctg atttggaaaa agctgaaaaa aaaggttgaa accagttccc tgaaattatt 840cccctacttg actaataagt atataaagac ggtaggtatt gattgtaatt ctgtaaatct 900atttcttaaa cttcttaaat tctactttta tagttagtct tttttttagt tttaaaacac 960caagaactta gtttcgaata aacacacata aacaaacaaa 100013500DNAArtificial sequenceTDH3 terminator 13gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt tatgacttag 60tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag ctttgtgttt 120tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata tctgtagtag 180atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg cgctcttaat 240aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt ccgccaggat 300aacgattctg aagttactct tagcgttcct atcggtacag ccatcaaatc atgcctataa 360atcatgccta tatttgcgtg cagtcagtat catctacatg aaaaaaactc ccgcaatttc 420ttatagaata cgttgaaaat taaatgtacg cgccaagata agataacata tatctagatg 480cagtaatata cacagattcc 50014538PRTArtificial sequenceA. succinogenes PEP carboxykinase wherein EGY at position 120-122 is replaced by DAF 14Met Thr Asp Leu Asn Lys Leu Val Lys Glu Leu Asn Asp Leu Gly Leu1 5 10 15Thr Asp Val Lys Glu Ile Val Tyr Asn Pro Ser Tyr Glu Gln Leu Phe 20 25 30Glu Glu Glu Thr Lys Pro Gly Leu Glu Gly Phe Asp Lys Gly Thr Leu 35 40 45Thr Thr Leu Gly Ala Val Ala Val Asp Thr Gly Ile Phe Thr Gly Arg 50 55 60Ser Pro Lys Asp Lys Tyr Ile Val Cys Asp Glu Thr Thr Lys Asp Thr65 70 75 80Val Trp Trp Asn Ser Glu Ala Ala Lys Asn Asp Asn Lys Pro Met Thr 85 90 95Gln Glu Thr Trp Lys Ser Leu Arg Glu Leu Val Ala Lys Gln Leu Ser 100 105 110Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Ser Glu Lys 115 120 125His Arg Ile Gly Val Arg Met Val Thr Glu Val Ala Trp Gln Ala His 130 135 140Phe Val Lys Asn Met Phe Ile Arg Pro Thr Asp Glu Glu Leu Lys Asn145 150 155 160Phe Lys Ala Asp Phe Thr Val Leu Asn Gly Ala Lys Cys Thr Asn Pro 165 170 175Asn Trp Lys Glu Gln Gly Leu Asn Ser Glu Asn Phe Val Ala Phe Asn 180 185 190Ile Thr Glu Gly Ile Gln Leu Ile Gly Gly Thr Trp Tyr Gly Gly Glu 195 200 205Met Lys Lys Gly Met Phe Ser Met Met Asn Tyr Phe Leu Pro Leu Lys 210 215 220Gly Val Ala Ser Met His Cys Ser Ala Asn Val Gly Lys Asp Gly Asp225 230 235 240Val Ala Ile Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser 245 250 255Thr Asp Pro Lys Arg Gln Leu Ile Gly Asp Asp Glu His Gly Trp Asp 260 265 270Glu Ser Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys Thr Ile 275 280 285Asn Leu Ser Gln Glu Asn Glu Pro Asp Ile Tyr Gly Ala Ile Arg Arg 290 295 300Asp Ala Leu Leu Glu Asn Val Val Val Arg Ala Asp Gly Ser Val Asp305 310 315 320Phe Asp Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr Pro Ile 325 330 335Tyr His Ile Asp Asn Ile Val Arg Pro Val Ser Lys Ala Gly His Ala 340 345 350Thr Lys Val Ile Phe Leu Thr Ala Asp Ala Phe Gly Val Leu Pro Pro 355 360 365Val Ser Lys Leu Thr Pro Glu Gln Thr Glu Tyr Tyr Phe Leu Ser Gly 370 375 380Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Val Thr Glu Pro Thr385 390 395 400Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu His Pro 405 410 415Ile Gln Tyr Ala Asp Val Leu Val Glu Arg Met Lys Ala Ser Gly Ala 420 425 430Glu Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys Arg Ile 435 440 445Ser Ile Lys Asp Thr Arg Gly Ile Ile Asp Ala Ile Leu Asp Gly Ser 450 455 460Ile Glu Lys Ala Glu Met Gly Glu Leu Pro Ile Phe Asn Leu Ala Ile465 470 475 480Pro Lys Ala Leu Pro Gly Val Asp Pro Ala Ile Leu Asp Pro Arg Asp 485 490 495Thr Tyr Ala Asp Lys Ala Gln Trp Gln Val Lys Ala Glu Asp Leu Ala 500 505 510Asn Arg Phe Val Lys Asn Phe Val Lys Tyr Thr Ala Asn Pro Glu Ala 515 520 525Ala Lys Leu Val Gly Ala Gly Pro Lys Ala 530 535151617DNAArtificial sequencent. A. succinogenes PEP carboxykinase encoding DAF instead of EGY 15atgactgact taaacaaact cgttaaagaa cttaatgact tagggcttac cgatgttaag 60gaaattgtgt ataacccgag ttatgaacaa cttttcgagg aagaaaccaa accgggtttg 120gagggtttcg ataaagggac gttaaccacg cttggcgcgg ttgccgtcga tacggggatt 180tttaccggtc gttcaccgaa agataaatat atcgtttgcg atgaaactac gaaagacacc 240gtttggtgga acagcgaagc ggcgaaaaac gataacaaac cgatgacgca agaaacttgg 300aaaagtttga gagaattagt ggcgaaacaa ctttccggta aacgtttatt cgtggtagac 360gcattctgcg gcgccagtga aaaacaccgt atcggtgtgc gtatggttac tgaagtggca 420tggcaggcgc attttgtgaa aaacatgttt atccgaccga ccgatgaaga gttgaaaaat 480ttcaaagcgg attttaccgt gttaaacggt gctaaatgta ctaatccgaa ctggaaagaa 540caaggtttga acagtgaaaa ctttgtcgct ttcaatatta ccgaaggtat tcagcttatc 600ggcggtactt ggtacggcgg tgaaatgaaa aaaggtatgt tctcaatgat gaactacttc 660ctgccgttaa aaggtgtggc ttccatgcac tgttccgcca acgtaggtaa agacggtgac 720gtggctattt tcttcggttt atccggtacg ggtaaaacaa cgctttcgac cgatcctaaa 780cgccaattaa tcggtgatga cgaacacggt tgggatgaat ccggcgtatt taactttgaa 840ggcggttgtt acgcgaaaac cattaactta tctcaagaaa acgaaccgga tatttacggc 900gcaatccgtc gtgacgcatt attagaaaac gtcgtggttc gtgcagacgg ttccgttgac 960tttgacgacg gttcaaaaac agaaaatacc cgtgtttcat atccgattta ccacatcgac 1020aacatcgttc gtccggtatc gaaagccggt catgcaacca aagtgatttt cttaaccgcg 1080gacgcattcg gcgtattgcc gccggtttca aaactgactc cggaacaaac cgaatactac 1140ttcttatccg gctttactgc aaaattagcg ggtacggaac gcggcgtaac cgaaccgact 1200ccgacattct cggcctgttt cggtgcggca ttcttaagcc tgcatccgat tcaatatgcg 1260gacgtgttgg tcgaacgcat gaaagcctcc ggtgcggaag cttatttggt gaacaccggt 1320tggaacggca cgggtaaacg tatttcaatc aaagataccc gcggtattat cgatgcgatt 1380ttggacggtt caatcgaaaa agcggaaatg ggcgaattgc caatctttaa tttagcgatt 1440cctaaagcat taccgggtgt tgatcctgct attttggatc cgcgcgatac ttacgcagac 1500aaagcgcaat ggcaagttaa agcggaagat ttggcaaacc gtttcgtgaa aaactttgtg 1560aaatatacgg cgaatccgga agcggctaaa ttagttggcg ccggtccaaa agcataa 1617161617DNAArtificial sequenceCodon pair optimised A. succinogenes PEPCK for S. cerevisiae 16atgactgatt tgaacaaatt ggtcaaggaa ttgaatgatt tgggtttgac tgacgtcaag 60gaaattgtct acaacccatc ttacgaacaa ttattcgaag aagaaaccaa gccaggtttg 120gaaggtttcg acaagggtac tttgaccact ttaggtgctg ttgctgttga caccggtatt 180ttcaccggtc gttctccaaa ggacaaatac attgtttgtg atgaaaccac caaggacacc 240gtctggtgga actctgaagc tgccaagaac gataacaagc caatgactca agaaacctgg 300aaatctttga gagaattggt tgccaagcaa ttgtctggta agagattatt cgttgttgac 360gctttctgtg gtgcttctga aaagcacaga attggtgtca gaatggtcac tgaagttgct 420tggcaagctc atttcgtcaa gaacatgttc atcagaccaa ctgacgaaga attgaagaac 480ttcaaggctg acttcaccgt tttgaatggt gccaagtgta ccaacccaaa ctggaaggaa 540caaggtttga actctgaaaa ctttgttgct ttcaacatca ctgaaggtat ccaattgatt 600ggtggtacct ggtacggtgg tgaaatgaag aagggtatgt tctccatgat gaactatttc 660ttgccattga aaggtgttgc ttccatgcac tgttctgcca atgtcggtaa ggatggtgac 720gttgccatct tcttcggtct atccggtact ggtaagacca ctctatccac tgacccaaag 780agacaattga ttggtgatga cgaacacggt tgggacgaat ctggtgtctt taactttgaa 840ggtggttgtt acgccaagac catcaactta tctcaagaaa acgaaccaga tatctacggt 900gccatccgtc gtgatgcttt gttggaaaac gttgttgtca gagctgacgg ttctgttgac 960ttcgacgacg gttccaagac tgaaaacacc agagtttctt acccaatcta ccacattgac 1020aacattgtca gacctgtttc caaggctggt cacgctacca aggttatctt cttgactgct 1080gatgctttcg gtgtcttgcc acctgtttcc aaattgactc cagaacaaac cgaatactac 1140ttcttgtccg gtttcactgc caaattggct ggtactgaaa gaggtgtcac tgaaccaact 1200ccaactttct ctgcttgttt cggtgctgct ttcttatctt tgcacccaat ccaatacgct 1260gatgtcttgg ttgaaagaat gaaggcttct ggtgctgaag cttacttggt caacaccggt 1320tggaacggta ccggtaagag aatctccatc aaggatacca gaggtatcat tgatgctatc 1380ttggacggtt ccattgaaaa ggctgaaatg ggtgaattgc caatcttcaa cttggccatt 1440ccaaaggctt tgccaggtgt tgacccagcc atcttagatc caagagacac ctacgctgac 1500aaggctcaat ggcaagtcaa ggctgaagat ttggctaaca gattcgtcaa gaactttgtc 1560aaatacactg ctaacccaga agctgccaaa ttggttggtg ctggtccaaa ggcttaa 161717538PRTMannheimia succinicipoducens 17Met Thr Asp Leu Asn Gln Leu Thr Gln Glu Leu Gly Ala Leu Gly Ile1 5 10 15His Asp Val Gln Glu Val Val Tyr Asn Pro Ser Tyr Glu Leu Leu Phe 20 25 30Ala Glu Glu Thr Lys Pro Gly Leu Glu Gly Tyr Glu Lys Gly Thr Val 35 40 45Thr Asn Gln Gly Ala Val Ala Val Asn Thr Gly Ile Phe Thr Gly Arg 50 55 60Ser Pro Lys Asp Lys Tyr Ile Val Leu Asp Asp Lys Thr Lys Asp Thr65 70 75 80Val Trp Trp Thr Ser Glu Lys Val Lys Asn Asp Asn Lys Pro Met Ser 85 90 95Gln Asp Thr Trp Asn Ser Leu Lys Gly Leu Val Ala Asp Gln Leu Ser 100 105 110Gly Lys Arg Leu Phe Val Val Asp Ala Phe Cys Gly Ala Asn Lys Asp 115

120 125Thr Arg Leu Ala Val Arg Val Val Thr Glu Val Ala Trp Gln Ala His 130 135 140Phe Val Thr Asn Met Phe Ile Arg Pro Ser Ala Glu Glu Leu Lys Gly145 150 155 160Phe Lys Pro Asp Phe Val Val Met Asn Gly Ala Lys Cys Thr Asn Pro 165 170 175Asn Trp Lys Glu Gln Gly Leu Asn Ser Glu Asn Phe Val Ala Phe Asn 180 185 190Ile Thr Glu Gly Val Gln Leu Ile Gly Gly Thr Trp Tyr Gly Gly Glu 195 200 205Met Lys Lys Gly Met Phe Ser Met Met Asn Tyr Phe Leu Pro Leu Arg 210 215 220Gly Ile Ala Ser Met His Cys Ser Ala Asn Val Gly Lys Asp Gly Asp225 230 235 240Thr Ala Ile Phe Phe Gly Leu Ser Gly Thr Gly Lys Thr Thr Leu Ser 245 250 255Thr Asp Pro Lys Arg Gln Leu Ile Gly Asp Asp Glu His Gly Trp Asp 260 265 270Asp Glu Gly Val Phe Asn Phe Glu Gly Gly Cys Tyr Ala Lys Thr Ile 275 280 285Asn Leu Ser Ala Glu Asn Glu Pro Asp Ile Tyr Gly Ala Ile Lys Arg 290 295 300Asp Ala Leu Leu Glu Asn Val Val Val Leu Asp Asn Gly Asp Val Asp305 310 315 320Tyr Ala Asp Gly Ser Lys Thr Glu Asn Thr Arg Val Ser Tyr Pro Ile 325 330 335Tyr His Ile Gln Asn Ile Val Lys Pro Val Ser Lys Ala Gly Pro Ala 340 345 350Thr Lys Val Ile Phe Leu Ser Ala Asp Ala Phe Gly Val Leu Pro Pro 355 360 365Val Ser Lys Leu Thr Pro Glu Gln Thr Lys Tyr Tyr Phe Leu Ser Gly 370 375 380Phe Thr Ala Lys Leu Ala Gly Thr Glu Arg Gly Ile Thr Glu Pro Thr385 390 395 400Pro Thr Phe Ser Ala Cys Phe Gly Ala Ala Phe Leu Ser Leu His Pro 405 410 415Thr Gln Tyr Ala Glu Val Leu Val Lys Arg Met Gln Glu Ser Gly Ala 420 425 430Glu Ala Tyr Leu Val Asn Thr Gly Trp Asn Gly Thr Gly Lys Arg Ile 435 440 445Ser Ile Lys Asp Thr Arg Gly Ile Ile Asp Ala Ile Leu Asp Gly Ser 450 455 460Ile Asp Lys Ala Glu Met Gly Ser Leu Pro Ile Phe Asp Phe Ser Ile465 470 475 480Pro Lys Ala Leu Pro Gly Val Asn Pro Ala Ile Leu Asp Pro Arg Asp 485 490 495Thr Tyr Ala Asp Lys Ala Gln Trp Glu Glu Lys Ala Gln Asp Leu Ala 500 505 510Gly Arg Phe Val Lys Asn Phe Glu Lys Tyr Thr Gly Thr Ala Glu Gly 515 520 525Gln Ala Leu Val Ala Ala Gly Pro Lys Ala 530 535181617DNAArtificial sequencePEPcarboxykinase M. succiniciproducens cpo for S. cerevisiae 18atgaccgatt tgaaccaatt gactcaagaa ttgggtgctt tgggtattca cgatgtccaa 60gaagttgtct acaacccatc ttacgaattg ttgtttgctg aagaaaccaa gccaggtttg 120gaaggttacg aaaagggtac tgttaccaac caaggtgctg ttgctgtcaa caccggtatc 180ttcaccggtc gttctccaaa ggacaaatac attgtcttgg atgacaagac caaggacact 240gtctggtgga cttctgaaaa ggtcaagaac gacaacaaac caatgtccca agacacttgg 300aactctttaa agggtttagt cgctgaccaa ttgtctggta agagattatt cgttgtcgat 360gctttctgtg gtgccaacaa ggacaccaga ttagctgtca gagttgtcac tgaagttgct 420tggcaagctc acttcgttac caacatgttc atcagaccat ctgctgaaga attgaaaggt 480ttcaagccag atttcgttgt catgaacggt gccaaatgta ccaacccaaa ctggaaggaa 540caaggtttga actctgaaaa ctttgttgct ttcaacatca ctgaaggtgt tcaattgatt 600ggtggtacct ggtacggtgg tgaaatgaag aagggtatgt tctccatgat gaactacttc 660ttgccattga gaggtattgc ttccatgcac tgttctgcca atgtcggtaa ggacggtgac 720actgccatct tcttcggtct atccggtacc ggtaagacca ctttgtccac tgacccaaag 780agacaattga ttggtgatga cgaacacggt tgggatgacg aaggtgtttt caactttgaa 840ggtggttgtt acgccaagac catcaactta tctgctgaaa atgaaccaga tatctacggt 900gccatcaagc gtgacgctct attggaaaac gttgttgttt tggacaatgg tgacgtcgat 960tatgctgacg gttccaagac tgaaaacacc agagtttctt acccaatcta ccatattcaa 1020aacattgtca agccagtttc caaggctggt ccagctacca aagttatctt cttgtctgct 1080gatgctttcg gtgttttgcc tcctgtttcc aagttgactc cagaacaaac caagtactac 1140ttcttgtctg gtttcaccgc caagttggct ggtactgaaa gaggtatcac tgaaccaact 1200ccaactttct ctgcttgttt cggtgctgcc tttttgtctt tgcacccaac tcaatacgct 1260gaagttttgg tcaagagaat gcaagaatct ggtgctgaag cttacttggt caacactggt 1320tggaacggta ccggtaagag aatctccatc aaagatacca gaggtatcat cgatgccatc 1380ttggatggtt ccattgacaa ggctgaaatg ggttctttgc caattttcga tttctccatt 1440ccaaaggctt tgccaggtgt caacccagcc atcttagacc caagagacac ctacgctgac 1500aaagctcaat gggaagaaaa ggctcaagac ttggctggta gattcgtcaa gaacttcgaa 1560aaatacactg gtactgctga aggtcaagct ttggttgctg ctggtccaaa ggcctaa 161719365PRTArtificial sequenceMDH2 S. cerevisiae lacking first 12 a.a. 19Met Leu Lys Ile Ala Ile Leu Gly Ala Ala Gly Gly Ile Gly Gln Ser1 5 10 15Leu Ser Leu Leu Leu Lys Ala Gln Leu Gln Tyr Gln Leu Lys Glu Ser 20 25 30Asn Arg Ser Val Thr His Ile His Leu Ala Leu Tyr Asp Val Asn Gln 35 40 45Glu Ala Ile Asn Gly Val Thr Ala Asp Leu Ser His Ile Asp Thr Pro 50 55 60Ile Ser Val Ser Ser His Ser Pro Ala Gly Gly Ile Glu Asn Cys Leu65 70 75 80His Asn Ala Ser Ile Val Val Ile Pro Ala Gly Val Pro Arg Lys Pro 85 90 95Gly Met Thr Arg Asp Asp Leu Phe Asn Val Asn Ala Gly Ile Ile Ser 100 105 110Gln Leu Gly Asp Ser Ile Ala Glu Cys Cys Asp Leu Ser Lys Val Phe 115 120 125Val Leu Val Ile Ser Asn Pro Val Asn Ser Leu Val Pro Val Met Val 130 135 140Ser Asn Ile Leu Lys Asn His Pro Gln Ser Arg Asn Ser Gly Ile Glu145 150 155 160Arg Arg Ile Met Gly Val Thr Lys Leu Asp Ile Val Arg Ala Ser Thr 165 170 175Phe Leu Arg Glu Ile Asn Ile Glu Ser Gly Leu Thr Pro Arg Val Asn 180 185 190Ser Met Pro Asp Val Pro Val Ile Gly Gly His Ser Gly Glu Thr Ile 195 200 205Ile Pro Leu Phe Ser Gln Ser Asn Phe Leu Ser Arg Leu Asn Glu Asp 210 215 220Gln Leu Lys Tyr Leu Ile His Arg Val Gln Tyr Gly Gly Asp Glu Val225 230 235 240Val Lys Ala Lys Asn Gly Lys Gly Ser Ala Thr Leu Ser Met Ala His 245 250 255Ala Gly Tyr Lys Cys Val Val Gln Phe Val Ser Leu Leu Leu Gly Asn 260 265 270Ile Glu Gln Ile His Gly Thr Tyr Tyr Val Pro Leu Lys Asp Ala Asn 275 280 285Asn Phe Pro Ile Ala Pro Gly Ala Asp Gln Leu Leu Pro Leu Val Asp 290 295 300Gly Ala Asp Tyr Phe Ala Ile Pro Leu Thr Ile Thr Thr Lys Gly Val305 310 315 320Ser Tyr Val Asp Tyr Asp Ile Val Asn Arg Met Asn Asp Met Glu Arg 325 330 335Asn Gln Met Leu Pro Ile Cys Val Ser Gln Leu Lys Lys Asn Ile Asp 340 345 350Lys Gly Leu Glu Phe Val Ala Ser Arg Ser Ala Ser Ser 355 360 365201099DNAArtificial sequencecpo MDH2 S. cerevisiae lacking fisrt 12 a.a. 20atgttgaaga ttgccatctt gggtgctgct ggtggtatcg gtcaatcttt gtctttgttg 60ttgaaggctc aattgcaata ccaattgaag gaatccaaca gatctgttac ccacattcat 120ttggctttgt acgatgtcaa ccaagaagct atcaacggtg tcactgctga cttgtctcac 180atcgataccc caatctctgt ttcctctcac tctccagctg gtggtattga aaactgtttg 240cacaacgctt ccattgttgt cattccagcc ggtgttccaa gaaagccagg tatgacccgt 300gacgatttgt tcaacgtcaa tgccggtatc atctctcaat taggtgattc cattgctgaa 360tgttgtgact tgtccaaggt tttcgtcttg gttatctcca acccagtcaa ctctttggtt 420cctgttatgg tttccaacat cttgaagaac cacccacaat ccagaaactc tggtattgaa 480agaagaatca tgggtgtcac caaattggac attgtcagag cttccacttt cttgagagaa 540atcaacattg aatctggttt gactccaaga gtcaactcca tgccagatgt tccagttatc 600ggtggtcact ctggtgaaac tatcatccca ttattctctc aatctaactt cttgtccaga 660ttgaatgaag atcaattgaa atacttgatt caccgtgtcc aatacggtgg tgacgaagtt 720gtcaaggcca agaacggtaa gggttctgct actctatcca tggctcatgc cggttacaag 780tgtgttgtcc aattcgtttc tctattatta ggtaacattg aacaaatcca cggtacctac 840tacgttccat tgaaagatgc taacaacttc ccaattgctc caggtgctga ccaattattg 900ccattagtcg acggtgctga ctactttgcc atcccattga ccatcactac caagggtgtt 960tcttacgttg actacgatat cgtcaacaga atgaacgaca tggaaagaaa ccaaatgttg 1020cctatctgtg tttctcaatt gaagaagaac attgacaagg gtttggaatt cgttgcttcc 1080agatctgctt ccagttaag 109921340PRTArtificial sequenceMDH3 S. cerevisiae lacking C-terminal SKL 21Met Val Lys Val Ala Ile Leu Gly Ala Ser Gly Gly Val Gly Gln Pro1 5 10 15Leu Ser Leu Leu Leu Lys Leu Ser Pro Tyr Val Ser Glu Leu Ala Leu 20 25 30Tyr Asp Ile Arg Ala Ala Glu Gly Ile Gly Lys Asp Leu Ser His Ile 35 40 45Asn Thr Asn Ser Ser Cys Val Gly Tyr Asp Lys Asp Ser Ile Glu Asn 50 55 60Thr Leu Ser Asn Ala Gln Val Val Leu Ile Pro Ala Gly Val Pro Arg65 70 75 80Lys Pro Gly Leu Thr Arg Asp Asp Leu Phe Lys Met Asn Ala Gly Ile 85 90 95Val Lys Ser Leu Val Thr Ala Val Gly Lys Phe Ala Pro Asn Ala Arg 100 105 110Ile Leu Val Ile Ser Asn Pro Val Asn Ser Leu Val Pro Ile Ala Val 115 120 125Glu Thr Leu Lys Lys Met Gly Lys Phe Lys Pro Gly Asn Val Met Gly 130 135 140Val Thr Asn Leu Asp Leu Val Arg Ala Glu Thr Phe Leu Val Asp Tyr145 150 155 160Leu Met Leu Lys Asn Pro Lys Ile Gly Gln Glu Gln Asp Lys Thr Thr 165 170 175Met His Arg Lys Val Thr Val Ile Gly Gly His Ser Gly Glu Thr Ile 180 185 190Ile Pro Ile Ile Thr Asp Lys Ser Leu Val Phe Gln Leu Asp Lys Gln 195 200 205Tyr Glu His Phe Ile His Arg Val Gln Phe Gly Gly Asp Glu Ile Val 210 215 220Lys Ala Lys Gln Gly Ala Gly Ser Ala Thr Leu Ser Met Ala Phe Ala225 230 235 240Gly Ala Lys Phe Ala Glu Glu Val Leu Arg Ser Phe His Asn Glu Lys 245 250 255Pro Glu Thr Glu Ser Leu Ser Ala Phe Val Tyr Leu Pro Gly Leu Lys 260 265 270Asn Gly Lys Lys Ala Gln Gln Leu Val Gly Asp Asn Ser Ile Glu Tyr 275 280 285Phe Ser Leu Pro Ile Val Leu Arg Asn Gly Ser Val Val Ser Ile Asp 290 295 300Thr Ser Val Leu Glu Lys Leu Ser Pro Arg Glu Glu Gln Leu Val Asn305 310 315 320Thr Ala Val Lys Glu Leu Arg Lys Asn Ile Glu Lys Gly Lys Ser Phe 325 330 335Ile Leu Asp Ser 340221024DNAArtificial sequenceMDH3 S. cerevisiae lacking SKL encoding nt, cpo 22atggttaagg ttgccatctt aggtgcttct ggtggtgtcg gtcaaccatt atctctatta 60ttgaaattgt ctccatacgt ttctgaattg gctttgtacg atatcagagc tgctgaaggt 120attggtaagg atttgtccca catcaacacc aactcctctt gtgttggtta cgacaaggat 180tccatcgaaa acactttgtc caatgctcaa gttgtcttga ttccagctgg tgttccaaga 240aagccaggtt tgaccagaga tgatttgttc aagatgaacg ctggtatcgt taagtctttg 300gttactgctg tcggtaaatt tgccccaaac gctcgtatct tagtcatctc caaccctgtt 360aactctttgg ttccaattgc cgttgaaact ttgaagaaga tgggtaagtt caagccaggt 420aacgttatgg gtgtcaccaa cttggatttg gtcagagctg aaactttctt ggttgactac 480ttgatgttga agaacccaaa gatcggtcaa gaacaagaca agaccaccat gcacagaaag 540gtcaccgtca tcggtggtca ctctggtgaa accatcattc caatcatcac tgacaaatcc 600ttggttttcc aattggacaa gcaatacgaa catttcatcc acagagtcca attcggtggt 660gacgaaattg tcaaggccaa gcaaggtgcc ggttctgcta ccttgtccat ggctttcgct 720ggtgccaaat ttgctgaaga agtcttacgt tctttccaca acgaaaagcc agaaactgaa 780tctttgtctg ctttcgtcta cttgccaggt ttgaagaacg gtaagaaggc tcaacaatta 840gtcggtgaca actccattga atacttctct ttgccaattg ttttgagaaa cggttccgtt 900gtttccattg acacttctgt tttggaaaaa ttgtctccaa gagaagaaca attggtcaac 960actgctgtca aggaattgag aaagaacatt gaaaagggta agtctttcat cttggacagt 1020taag 102423472PRTArtificial sequenceFumarase R. oryzae lacking first 23 aa+ new M 23Met Ser Ser Ala Ser Ala Ala Leu Gln Lys Phe Arg Ala Glu Arg Asp1 5 10 15Thr Phe Gly Asp Leu Gln Val Pro Ala Asp Arg Tyr Trp Gly Ala Gln 20 25 30Thr Gln Arg Ser Leu Gln Asn Phe Asp Ile Gly Gly Pro Thr Glu Arg 35 40 45Met Pro Glu Pro Leu Ile Arg Ala Phe Gly Val Leu Lys Lys Ala Ala 50 55 60Ala Thr Val Asn Met Thr Tyr Gly Leu Asp Pro Lys Val Gly Glu Ala65 70 75 80Ile Gln Lys Ala Ala Asp Glu Val Ile Asp Gly Ser Leu Ile Asp His 85 90 95Phe Pro Leu Val Val Trp Gln Thr Gly Ser Gly Thr Gln Thr Lys Met 100 105 110Asn Val Asn Glu Val Ile Ser Asn Arg Ala Ile Glu Leu Leu Gly Gly 115 120 125Glu Leu Gly Ser Lys Ala Pro Val His Pro Asn Asp His Val Asn Met 130 135 140Ser Gln Ser Ser Asn Asp Thr Phe Pro Thr Ala Met His Val Ala Ala145 150 155 160Val Val Glu Ile His Gly Arg Leu Ile Pro Ala Leu Thr Thr Leu Arg 165 170 175Asp Ala Leu Gln Ala Lys Ser Ala Glu Phe Glu His Ile Ile Lys Ile 180 185 190Gly Arg Thr His Leu Gln Asp Ala Thr Pro Leu Thr Leu Gly Gln Glu 195 200 205Phe Ser Gly Tyr Thr Gln Gln Leu Thr Tyr Gly Ile Ala Arg Val Gln 210 215 220Gly Thr Leu Glu Arg Leu Tyr Asn Leu Ala Gln Gly Gly Thr Ala Val225 230 235 240Gly Thr Gly Leu Asn Thr Arg Lys Gly Phe Asp Ala Lys Val Ala Glu 245 250 255Ala Ile Ala Ser Ile Thr Gly Leu Pro Phe Lys Thr Ala Pro Asn Lys 260 265 270Phe Glu Ala Leu Ala Ala His Asp Ala Leu Val Glu Ala His Gly Ala 275 280 285Leu Asn Thr Val Ala Cys Ser Leu Met Lys Ile Ala Asn Asp Ile Arg 290 295 300Tyr Leu Gly Ser Gly Pro Arg Cys Gly Leu Gly Glu Leu Ser Leu Pro305 310 315 320Glu Asn Glu Pro Gly Ser Ser Ile Met Pro Gly Lys Val Asn Pro Thr 325 330 335Gln Cys Glu Ala Met Thr Met Val Cys Ala Gln Val Met Gly Asn Asn 340 345 350Thr Ala Ile Ser Val Ala Gly Ser Asn Gly Gln Phe Glu Leu Asn Val 355 360 365Phe Lys Pro Val Met Ile Lys Asn Leu Ile Gln Ser Ile Arg Leu Ile 370 375 380Ser Asp Ala Ser Ile Ser Phe Thr Lys Asn Cys Val Val Gly Ile Glu385 390 395 400Ala Asn Glu Lys Lys Ile Ser Ser Ile Met Asn Glu Ser Leu Met Leu 405 410 415Val Thr Ala Leu Asn Pro His Ile Gly Tyr Asp Lys Ala Ala Lys Cys 420 425 430Ala Lys Lys Ala His Lys Glu Gly Thr Thr Leu Lys Glu Ala Ala Leu 435 440 445Ser Leu Gly Tyr Leu Thr Ser Glu Glu Phe Asp Gln Trp Val Arg Pro 450 455 460Glu Asp Met Ile Ser Ala Lys Asp465 470241419DNAArtificial sequenceFumarase R. oryzae lacking nt encoding first aa + M 24atgtcctctg cttctgctgc tttgcaaaaa ttcagagctg aaagagatac cttcggtgac 60ttgcaagttc cagctgaccg ttactggggt gctcaaactc aaagatcttt gcaaaacttt 120gacattggtg gtccaactga aagaatgcca gaaccattaa tcagagcttt cggtgttttg 180aagaaggctg ctgccaccgt caacatgacc tacggtttgg acccaaaggt tggtgaagcc 240atccaaaagg ctgctgacga agttatcgat ggttctttga ttgaccattt cccattggtt 300gtctggcaaa ccggttctgg tactcaaacc aagatgaacg tcaatgaagt catctccaac 360agagccattg aattgttggg tggtgaatta ggttccaagg ctccagtcca cccaaacgat 420catgtcaaca tgtctcaatc ttccaacgac actttcccaa ctgccatgca cgttgctgcc 480gttgttgaaa ttcacggtag attgattcca gctttgacca ctttgagaga tgctttgcaa 540gccaaatctg ctgaattcga acacatcatc aagattggta gaacccactt gcaagatgct 600accccattga ctttaggtca agaattctcc ggttacactc aacaattgac ctacggtatt 660gctcgtgttc aaggtacttt ggaaagatta tacaacttgg ctcaaggtgg tactgctgtc 720ggtactggtt tgaacaccag aaagggtttc gatgccaagg ttgctgaagc cattgcttcc 780atcactggtt taccattcaa gaccgctcca aacaaattcg aagctttggc tgctcacgac 840gctttggttg aagctcacgg tgctttgaac accgttgctt gttctttgat gaagattgcc 900aacgatatcc gttacttggg ttctggtcca agatgtggtt taggtgaatt gtctctacca 960gaaaacgaac caggttcttc catcatgcca ggtaaggtca acccaactca atgtgaagct 1020atgaccatgg tttgtgctca agtcatgggt aacaacactg

ccatctctgt tgctggttcc 1080aacggtcaat tcgaattgaa tgtctttaaa ccagtcatga tcaagaactt gatccaatcc 1140atcagattaa tctctgacgc ttccatctct ttcaccaaga actgtgttgt cggtattgaa 1200gctaacgaaa agaagatctc ctccatcatg aacgaatctt tgatgttggt cactgctttg 1260aaccctcaca ttggttacga caaggctgcc aagtgtgcca agaaggctca caaggaaggt 1320accactttga aagaagctgc tctatctttg ggttacttga cctctgaaga attcgaccaa 1380tgggttagac ctgaggacat gatttctgcc aaggattaa 1419251000DNAArtificial sequenceTDH1 promotor 25cttccctttt acagtgcttc ggaaaagcac agcgttgtcc aagggaacaa tttttcttca 60agttaatgca taagaaatat ctttttttat gtttagctaa gtaaaagcag cttggagtaa 120aaaaaaaaat gagtaaattt ctcgatggat tagtttctca caggtaacat aacaaaaacc 180aagaaaagcc cgcttctgaa aactacagtt gacttgtatg ctaaagggcc agactaatgg 240gaggagaaaa agaaacgaat gtatatgctc atttacactc tatatcacca tatggaggat 300aagttgggct gagcttctga tccaatttat tctatccatt agttgctgat atgtcccacc 360agccaacact tgatagtatc tactcgccat tcacttccag cagcgccagt agggttgttg 420agcttagtaa aaatgtgcgc accacaagcc tacatgactc cacgtcacat gaaaccacac 480cgtggggcct tgttgcgcta ggaataggat atgcgacgaa gacgcttctg cttagtaacc 540acaccacatt ttcagggggt cgatctgctt gcttccttta ctgtcacgag cggcccataa 600tcgcgctttt tttttaaaag gcgcgagaca gcaaacagga agctcgggtt tcaaccttcg 660gagtggtcgc agatctggag actggatctt tacaatacag taaggcaagc caccatctgc 720ttcttaggtg catgcgacgg tatccacgtg cagaacaaca tagtctgaag aaggggggga 780ggagcatgtt cattctctgt agcagtaaga gcttggtgat aatgaccaaa actggagtct 840cgaaatcata taaatagaca atatattttc acacaatgag atttgtagta cagttctatt 900ctctctcttg cataaataag aaattcatca agaacttggt ttgatatttc accaacacac 960acaaaaaaca gtacttcact aaatttacac acaaaacaaa 100026500DNAArtificial sequenceTDH1 terminator 26ataaagcaat cttgatgagg ataatgattt ttttttgaat atacataaat actaccgttt 60ttctgctaga ttttgtgaag acgtaaataa gtacatatta ctttttaagc caagacaaga 120ttaagcatta actttaccct tttctcttct aagtttcaat actagttatc actgtttaaa 180agttatggcg agaacgtcgg cggttaaaat atattaccct gaacgtggtg aattgaagtt 240ctaggatggt ttaaagattt ttcctttttg ggaaataagt aaacaatata ttgctgcctt 300tgcaaaacgc acatacccac aatatgtgac tattggcaaa gaacgcatta tcctttgaag 360aggtggatac tgatactaag agagtctcta ttccggctcc acttttagtc cagagattac 420ttgtcttctt acgtatcaga acaagaaagc atttccaaag taattgcatt tgcccttgag 480cagtatatat atactaagaa 50027600DNAArtificial sequencesecond TDH3 promotor 27ttagtcaaaa aattagcctt ttaattctgc tgtaacccgt acatgcccaa aatagggggc 60gggttacaca gaatatataa catcgtaggt gtctgggtga acagtttatt cctggcatcc 120actaaatata atggagcccg ctttttaagc tggcatccag aaaaaaaaag aatcccagca 180ccaaaatatt gttttcttca ccaaccatca gttcataggt ccattctctt agcgcaacta 240cagagaacag gggcacaaac aggcaaaaaa cgggcacaac ctcaatggag tgatgcaacc 300tgcctggagt aaatgatgac acaaggcaat tgacccacgc atgtatctat ctcattttct 360tacaccttct attaccttct gctctctctg atttggaaaa agctgaaaaa aaaggttgaa 420accagttccc tgaaattatt cccctacttg actaataagt atataaagac ggtaggtatt 480gattgtaatt ctgtaaatct atttcttaaa cttcttaaat tctactttta tagttagtct 540tttttttagt tttaaaacac caagaactta gtttcgaata aacacacata aacaaacaaa 60028300DNAArtificial sequencesecond TDH3 terminator 28gtgaatttac tttaaatctt gcatttaaat aaattttctt tttatagctt tatgacttag 60tttcaattta tatactattt taatgacatt ttcgattcat tgattgaaag ctttgtgttt 120tttcttgatg cgctattgca ttgttcttgt ctttttcgcc acatgtaata tctgtagtag 180atacctgata cattgtggat gctgagtgaa attttagtta ataatggagg cgctcttaat 240aattttgggg atattggctt ttttttttaa agtttacaaa tgaatttttt ccgccaggat 300293148DNAArtificial sequenceTDH1p-PCKm-TDH1t synthetic construct 29ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg gaacaatttt 60tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa aagcagcttg 120gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg taacataaca 180aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa agggccagac 240taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata tcaccatatg 300gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt gctgatatgt 360cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc gccagtaggg 420ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg tcacatgaaa 480ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg cttctgctta 540gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt cacgagcggc 600ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct cgggtttcaa 660ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag gcaagccacc 720atctgcttct taggtgcatg cgacggtatc cacgtgcaga acaacatagt ctgaagaagg 780gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg accaaaactg 840gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt gtagtacagt 900tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga tatttcacca 960acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatga ctgatttgaa 1020caaattggtc aaggaattga atgatttggg tttgactgac gtcaaggaaa ttgtctacaa 1080cccatcttac gaacaattat tcgaagaaga aaccaagcca ggtttggaag gtttcgacaa 1140gggtactttg accactttag gtgctgttgc tgttgacacc ggtattttca ccggtcgttc 1200tccaaaggac aaatacattg tttgtgatga aaccaccaag gacaccgtct ggtggaactc 1260tgaagctgcc aagaacgata acaagccaat gactcaagaa acctggaaat ctttgagaga 1320attggttgcc aagcaattgt ctggtaagag attattcgtt gttgacgctt tctgtggtgc 1380ttctgaaaag cacagaattg gtgtcagaat ggtcactgaa gttgcttggc aagctcattt 1440cgtcaagaac atgttcatca gaccaactga cgaagaattg aagaacttca aggctgactt 1500caccgttttg aatggtgcca agtgtaccaa cccaaactgg aaggaacaag gtttgaactc 1560tgaaaacttt gttgctttca acatcactga aggtatccaa ttgattggtg gtacctggta 1620cggtggtgaa atgaagaagg gtatgttctc catgatgaac tatttcttgc cattgaaagg 1680tgttgcttcc atgcactgtt ctgccaatgt cggtaaggat ggtgacgttg ccatcttctt 1740cggtctatcc ggtactggta agaccactct atccactgac ccaaagagac aattgattgg 1800tgatgacgaa cacggttggg acgaatctgg tgtctttaac tttgaaggtg gttgttacgc 1860caagaccatc aacttatctc aagaaaacga accagatatc tacggtgcca tccgtcgtga 1920tgctttgttg gaaaacgttg ttgtcagagc tgacggttct gttgacttcg acgacggttc 1980caagactgaa aacaccagag tttcttaccc aatctaccac attgacaaca ttgtcagacc 2040tgtttccaag gctggtcacg ctaccaaggt tatcttcttg actgctgatg ctttcggtgt 2100cttgccacct gtttccaaat tgactccaga acaaaccgaa tactacttct tgtccggttt 2160cactgccaaa ttggctggta ctgaaagagg tgtcactgaa ccaactccaa ctttctctgc 2220ttgtttcggt gctgctttct tatctttgca cccaatccaa tacgctgatg tcttggttga 2280aagaatgaag gcttctggtg ctgaagctta cttggtcaac accggttgga acggtaccgg 2340taagagaatc tccatcaagg ataccagagg tatcattgat gctatcttgg acggttccat 2400tgaaaaggct gaaatgggtg aattgccaat cttcaacttg gccattccaa aggctttgcc 2460aggtgttgac ccagccatct tagatccaag agacacctac gctgacaagg ctcaatggca 2520agtcaaggct gaagatttgg ctaacagatt cgtcaagaac tttgtcaaat acactgctaa 2580cccagaagct gccaaattgg ttggtgctgg tccaaaggct taaggcccgg gcataaagca 2640atcttgatga ggataatgat ttttttttga atatacataa atactaccgt ttttctgcta 2700gattttgtga agacgtaaat aagtacatat tactttttaa gccaagacaa gattaagcat 2760taactttacc cttttctctt ctaagtttca atactagtta tcactgttta aaagttatgg 2820cgagaacgtc ggcggttaaa atatattacc ctgaacgtgg tgaattgaag ttctaggatg 2880gtttaaagat ttttcctttt tgggaaataa gtaaacaata tattgctgcc tttgcaaaac 2940gcacataccc acaatatgtg actattggca aagaacgcat tatcctttga agaggtggat 3000actgatacta agagagtctc tattccggct ccacttttag tccagagatt acttgtcttc 3060ttacgtatca gaacaagaaa gcatttccaa agtaattgca tttgcccttg agcagtatat 3120atatactaag aaggcgcgcc gcggccgc 3148303148DNAArtificial sequenceTDH1p-PCK1-TDH1t synthetic construct 30ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg gaacaatttt 60tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa aagcagcttg 120gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg taacataaca 180aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa agggccagac 240taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata tcaccatatg 300gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt gctgatatgt 360cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc gccagtaggg 420ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg tcacatgaaa 480ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg cttctgctta 540gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt cacgagcggc 600ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct cgggtttcaa 660ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag gcaagccacc 720atctgcttct taggtgcatg cgacggtatc cacgtgcaga acaacatagt ctgaagaagg 780gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg accaaaactg 840gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt gtagtacagt 900tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga tatttcacca 960acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatga ccgatttgaa 1020ccaattgact caagaattgg gtgctttggg tattcacgat gtccaagaag ttgtctacaa 1080cccatcttac gaattgttgt ttgctgaaga aaccaagcca ggtttggaag gttacgaaaa 1140gggtactgtt accaaccaag gtgctgttgc tgtcaacacc ggtatcttca ccggtcgttc 1200tccaaaggac aaatacattg tcttggatga caagaccaag gacactgtct ggtggacttc 1260tgaaaaggtc aagaacgaca acaaaccaat gtcccaagac acttggaact ctttaaaggg 1320tttagtcgct gaccaattgt ctggtaagag attattcgtt gtcgatgctt tctgtggtgc 1380caacaaggac accagattag ctgtcagagt tgtcactgaa gttgcttggc aagctcactt 1440cgttaccaac atgttcatca gaccatctgc tgaagaattg aaaggtttca agccagattt 1500cgttgtcatg aacggtgcca aatgtaccaa cccaaactgg aaggaacaag gtttgaactc 1560tgaaaacttt gttgctttca acatcactga aggtgttcaa ttgattggtg gtacctggta 1620cggtggtgaa atgaagaagg gtatgttctc catgatgaac tacttcttgc cattgagagg 1680tattgcttcc atgcactgtt ctgccaatgt cggtaaggac ggtgacactg ccatcttctt 1740cggtctatcc ggtaccggta agaccacttt gtccactgac ccaaagagac aattgattgg 1800tgatgacgaa cacggttggg atgacgaagg tgttttcaac tttgaaggtg gttgttacgc 1860caagaccatc aacttatctg ctgaaaatga accagatatc tacggtgcca tcaagcgtga 1920cgctctattg gaaaacgttg ttgttttgga caatggtgac gtcgattatg ctgacggttc 1980caagactgaa aacaccagag tttcttaccc aatctaccat attcaaaaca ttgtcaagcc 2040agtttccaag gctggtccag ctaccaaagt tatcttcttg tctgctgatg ctttcggtgt 2100tttgcctcct gtttccaagt tgactccaga acaaaccaag tactacttct tgtctggttt 2160caccgccaag ttggctggta ctgaaagagg tatcactgaa ccaactccaa ctttctctgc 2220ttgtttcggt gctgcctttt tgtctttgca cccaactcaa tacgctgaag ttttggtcaa 2280gagaatgcaa gaatctggtg ctgaagctta cttggtcaac actggttgga acggtaccgg 2340taagagaatc tccatcaaag ataccagagg tatcatcgat gccatcttgg atggttccat 2400tgacaaggct gaaatgggtt ctttgccaat tttcgatttc tccattccaa aggctttgcc 2460aggtgtcaac ccagccatct tagacccaag agacacctac gctgacaaag ctcaatggga 2520agaaaaggct caagacttgg ctggtagatt cgtcaagaac ttcgaaaaat acactggtac 2580tgctgaaggt caagctttgg ttgctgctgg tccaaaggcc taaggcccgg gcataaagca 2640atcttgatga ggataatgat ttttttttga atatacataa atactaccgt ttttctgcta 2700gattttgtga agacgtaaat aagtacatat tactttttaa gccaagacaa gattaagcat 2760taactttacc cttttctctt ctaagtttca atactagtta tcactgttta aaagttatgg 2820cgagaacgtc ggcggttaaa atatattacc ctgaacgtgg tgaattgaag ttctaggatg 2880gtttaaagat ttttcctttt tgggaaataa gtaaacaata tattgctgcc tttgcaaaac 2940gcacataccc acaatatgtg actattggca aagaacgcat tatcctttga agaggtggat 3000actgatacta agagagtctc tattccggct ccacttttag tccagagatt acttgtcttc 3060ttacgtatca gaacaagaaa gcatttccaa agtaattgca tttgcccttg agcagtatat 3120atatactaag aaggcgcgcc gcggccgc 3148312637DNAArtificial sequenceTDH3p-delta 12N MDH2-TDH3t synthetic construct 31ggatccggcg cgccctattt tcgaggacct tgtcaccttg agcccaagag agccaagatt 60taaattttcc tatgacttga tgcaaattcc caaagctaat aacatgcaag acacgtacgg 120tcaagaagac atatttgacc tcttaacagg ttcagacgcg actgcctcat cagtaagacc 180cgttgaaaag aacttacctg aaaaaaacga atatatacta gcgttgaatg ttagcgtcaa 240caacaagaag tttaatgacg cggaggccaa ggcaaaaaga ttccttgatt acgtaaggga 300gttagaatca ttttgaataa aaaacacgct ttttcagttc gagtttatca ttatcaatac 360tgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact ttatttagtc 420aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta 480cacagaatat ataacatcgt aggtgtctgg gtgaacagtt tattcctggc atccactaaa 540tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa 600tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacagaga 660acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcctg 720gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt ttcttacacc 780ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt 840tccctgaaat tattccccta cttgactaat aagtatataa agacggtagg tattgattgt 900aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt 960tagttttaaa acaccaagaa cttagtttcg aataaacaca cataaacaaa caaaatgttg 1020aagattgcca tcttgggtgc tgctggtggt atcggtcaat ctttgtcttt gttgttgaag 1080gctcaattgc aataccaatt gaaggaatcc aacagatctg ttacccacat tcatttggct 1140ttgtacgatg tcaaccaaga agctatcaac ggtgtcactg ctgacttgtc tcacatcgat 1200accccaatct ctgtttcctc tcactctcca gctggtggta ttgaaaactg tttgcacaac 1260gcttccattg ttgtcattcc agccggtgtt ccaagaaagc caggtatgac ccgtgacgat 1320ttgttcaacg tcaatgccgg tatcatctct caattaggtg attccattgc tgaatgttgt 1380gacttgtcca aggttttcgt cttggttatc tccaacccag tcaactcttt ggttcctgtt 1440atggtttcca acatcttgaa gaaccaccca caatccagaa actctggtat tgaaagaaga 1500atcatgggtg tcaccaaatt ggacattgtc agagcttcca ctttcttgag agaaatcaac 1560attgaatctg gtttgactcc aagagtcaac tccatgccag atgttccagt tatcggtggt 1620cactctggtg aaactatcat cccattattc tctcaatcta acttcttgtc cagattgaat 1680gaagatcaat tgaaatactt gattcaccgt gtccaatacg gtggtgacga agttgtcaag 1740gccaagaacg gtaagggttc tgctactcta tccatggctc atgccggtta caagtgtgtt 1800gtccaattcg tttctctatt attaggtaac attgaacaaa tccacggtac ctactacgtt 1860ccattgaaag atgctaacaa cttcccaatt gctccaggtg ctgaccaatt attgccatta 1920gtcgacggtg ctgactactt tgccatccca ttgaccatca ctaccaaggg tgtttcttac 1980gttgactacg atatcgtcaa cagaatgaac gacatggaaa gaaaccaaat gttgcctatc 2040tgtgtttctc aattgaagaa gaacattgac aagggtttgg aattcgttgc ttccagatct 2100gcttccagtt aaggcccggg cgtgaattta ctttaaatct tgcatttaaa taaattttct 2160ttttatagct ttatgactta gtttcaattt atatactatt ttaatgacat tttcgattca 2220ttgattgaaa gctttgtgtt ttttcttgat gcgctattgc attgttcttg tctttttcgc 2280cacatgtaat atctgtagta gatacctgat acattgtgga tgctgagtga aattttagtt 2340aataatggag gcgctcttaa taattttggg gatattggct ttttttttta aagtttacaa 2400atgaattttt tccgccagga taacgattct gaagttactc ttagcgttcc tatcggtaca 2460gccatcaaat catgcctata aatcatgcct atatttgcgt gcagtcagta tcatctacat 2520gaaaaaaact cccgcaattt cttatagaat acgttgaaaa ttaaatgtac gcgccaagat 2580aagataacat atatctagat gcagtaatat acacagattc cggccggccg cggccgc 2637321966DNAArtificial sequenceTDH3p-MDH3-TDH3t synthetic construct 32ggatccggcg cgccacgcgt ggccggcctt agtcaaaaaa ttagcctttt aattctgctg 60taacccgtac atgcccaaaa tagggggcgg gttacacaga atatataaca tcgtaggtgt 120ctgggtgaac agtttattcc tggcatccac taaatataat ggagcccgct ttttaagctg 180gcatccagaa aaaaaaagaa tcccagcacc aaaatattgt tttcttcacc aaccatcagt 240tcataggtcc attctcttag cgcaactaca gagaacaggg gcacaaacag gcaaaaaacg 300ggcacaacct caatggagtg atgcaacctg cctggagtaa atgatgacac aaggcaattg 360acccacgcat gtatctatct cattttctta caccttctat taccttctgc tctctctgat 420ttggaaaaag ctgaaaaaaa aggttgaaac cagttccctg aaattattcc cctacttgac 480taataagtat ataaagacgg taggtattga ttgtaattct gtaaatctat ttcttaaact 540tcttaaattc tacttttata gttagtcttt tttttagttt taaaacacca agaacttagt 600ttcgaataaa cacacataaa caaacaaaat ggttaaggtt gccatcttag gtgcttctgg 660tggtgtcggt caaccattat ctctattatt gaaattgtct ccatacgttt ctgaattggc 720tttgtacgat atcagagctg ctgaaggtat tggtaaggat ttgtcccaca tcaacaccaa 780ctcctcttgt gttggttacg acaaggattc catcgaaaac actttgtcca atgctcaagt 840tgtcttgatt ccagctggtg ttccaagaaa gccaggtttg accagagatg atttgttcaa 900gatgaacgct ggtatcgtta agtctttggt tactgctgtc ggtaaatttg ccccaaacgc 960tcgtatctta gtcatctcca accctgttaa ctctttggtt ccaattgccg ttgaaacttt 1020gaagaagatg ggtaagttca agccaggtaa cgttatgggt gtcaccaact tggatttggt 1080cagagctgaa actttcttgg ttgactactt gatgttgaag aacccaaaga tcggtcaaga 1140acaagacaag accaccatgc acagaaaggt caccgtcatc ggtggtcact ctggtgaaac 1200catcattcca atcatcactg acaaatcctt ggttttccaa ttggacaagc aatacgaaca 1260tttcatccac agagtccaat tcggtggtga cgaaattgtc aaggccaagc aaggtgccgg 1320ttctgctacc ttgtccatgg ctttcgctgg tgccaaattt gctgaagaag tcttacgttc 1380tttccacaac gaaaagccag aaactgaatc tttgtctgct ttcgtctact tgccaggttt 1440gaagaacggt aagaaggctc aacaattagt cggtgacaac tccattgaat acttctcttt 1500gccaattgtt ttgagaaacg gttccgttgt ttccattgac acttctgttt tggaaaaatt 1560gtctccaaga gaagaacaat tggtcaacac tgctgtcaag gaattgagaa agaacattga 1620aaagggtaag tctttcatct tggacagtta aggtgaattt actttaaatc ttgcatttaa 1680ataaattttc tttttatagc tttatgactt agtttcaatt tatatactat tttaatgaca 1740ttttcgattc attgattgaa agctttgtgt tttttcttga tgcgctattg cattgttctt 1800gtctttttcg ccacatgtaa tatctgtagt agatacctga tacattgtgg atgctgagtg 1860aaattttagt taataatgga ggcgctctta ataattttgg ggatattggc tttttttttt 1920aaagtttaca aatgaatttt ttccgccagg atgggcccgc ggccgc 1966332950DNAArtificial sequenceTDH1-FUMR-TDH1t synthetic construct 33ggatcccttc ccttttacag tgcttcggaa aagcacagcg ttgtccaagg gaacaatttt 60tcttcaagtt aatgcataag aaatatcttt ttttatgttt agctaagtaa aagcagcttg 120gagtaaaaaa aaaaatgagt aaatttctcg atggattagt ttctcacagg taacataaca 180aaaaccaaga aaagcccgct tctgaaaact acagttgact tgtatgctaa agggccagac 240taatgggagg agaaaaagaa acgaatgtat atgctcattt acactctata tcaccatatg 300gaggataagt tgggctgagc ttctgatcca atttattcta tccattagtt gctgatatgt 360cccaccagcc aacacttgat agtatctact cgccattcac ttccagcagc gccagtaggg 420ttgttgagct tagtaaaaat gtgcgcacca caagcctaca tgactccacg tcacatgaaa 480ccacaccgtg gggccttgtt gcgctaggaa taggatatgc gacgaagacg cttctgctta 540gtaaccacac cacattttca gggggtcgat ctgcttgctt cctttactgt cacgagcggc 600ccataatcgc gctttttttt taaaaggcgc gagacagcaa acaggaagct cgggtttcaa 660ccttcggagt ggtcgcagat ctggagactg gatctttaca atacagtaag gcaagccacc 720atctgcttct taggtgcatg cgacggtatc cacgtgcaga

acaacatagt ctgaagaagg 780gggggaggag catgttcatt ctctgtagca gtaagagctt ggtgataatg accaaaactg 840gagtctcgaa atcatataaa tagacaatat attttcacac aatgagattt gtagtacagt 900tctattctct ctcttgcata aataagaaat tcatcaagaa cttggtttga tatttcacca 960acacacacaa aaaacagtac ttcactaaat ttacacacaa aacaaaatgt cctctgcttc 1020tgctgctttg caaaaattca gagctgaaag agataccttc ggtgacttgc aagttccagc 1080tgaccgttac tggggtgctc aaactcaaag atctttgcaa aactttgaca ttggtggtcc 1140aactgaaaga atgccagaac cattaatcag agctttcggt gttttgaaga aggctgctgc 1200caccgtcaac atgacctacg gtttggaccc aaaggttggt gaagccatcc aaaaggctgc 1260tgacgaagtt atcgatggtt ctttgattga ccatttccca ttggttgtct ggcaaaccgg 1320ttctggtact caaaccaaga tgaacgtcaa tgaagtcatc tccaacagag ccattgaatt 1380gttgggtggt gaattaggtt ccaaggctcc agtccaccca aacgatcatg tcaacatgtc 1440tcaatcttcc aacgacactt tcccaactgc catgcacgtt gctgccgttg ttgaaattca 1500cggtagattg attccagctt tgaccacttt gagagatgct ttgcaagcca aatctgctga 1560attcgaacac atcatcaaga ttggtagaac ccacttgcaa gatgctaccc cattgacttt 1620aggtcaagaa ttctccggtt acactcaaca attgacctac ggtattgctc gtgttcaagg 1680tactttggaa agattataca acttggctca aggtggtact gctgtcggta ctggtttgaa 1740caccagaaag ggtttcgatg ccaaggttgc tgaagccatt gcttccatca ctggtttacc 1800attcaagacc gctccaaaca aattcgaagc tttggctgct cacgacgctt tggttgaagc 1860tcacggtgct ttgaacaccg ttgcttgttc tttgatgaag attgccaacg atatccgtta 1920cttgggttct ggtccaagat gtggtttagg tgaattgtct ctaccagaaa acgaaccagg 1980ttcttccatc atgccaggta aggtcaaccc aactcaatgt gaagctatga ccatggtttg 2040tgctcaagtc atgggtaaca acactgccat ctctgttgct ggttccaacg gtcaattcga 2100attgaatgtc tttaaaccag tcatgatcaa gaacttgatc caatccatca gattaatctc 2160tgacgcttcc atctctttca ccaagaactg tgttgtcggt attgaagcta acgaaaagaa 2220gatctcctcc atcatgaacg aatctttgat gttggtcact gctttgaacc ctcacattgg 2280ttacgacaag gctgccaagt gtgccaagaa ggctcacaag gaaggtacca ctttgaaaga 2340agctgctcta tctttgggtt acttgacctc tgaagaattc gaccaatggg ttagacctga 2400ggacatgatt tctgccaagg attaaggccc gggcataaag caatcttgat gaggataatg 2460attttttttt gaatatacat aaatactacc gtttttctgc tagattttgt gaagacgtaa 2520ataagtacat attacttttt aagccaagac aagattaagc attaacttta cccttttctc 2580ttctaagttt caatactagt tatcactgtt taaaagttat ggcgagaacg tcggcggtta 2640aaatatatta ccctgaacgt ggtgaattga agttctagga tggtttaaag atttttcctt 2700tttgggaaat aagtaaacaa tatattgctg cctttgcaaa acgcacatac ccacaatatg 2760tgactattgg caaagaacgc attatccttt gaagaggtgg atactgatac taagagagtc 2820tctattccgg ctccactttt agtccagaga ttacttgtct tcttacgtat cagaacaaga 2880aagcatttcc aaagtaattg catttgccct tgagcagtat atatatacta agaaggcgcg 2940ccgcggccgc 2950345037DNAArtificial sequenceTDH3p-FRDm1-TDH3t synthetic construct 34ggatccggcg cgccctattt tcgaggacct tgtcaccttg agcccaagag agccaagatt 60taaattttcc tatgacttga tgcaaattcc caaagctaat aacatgcaag acacgtacgg 120tcaagaagac atatttgacc tcttaacagg ttcagacgcg actgcctcat cagtaagacc 180cgttgaaaag aacttacctg aaaaaaacga atatatacta gcgttgaatg ttagcgtcaa 240caacaagaag tttaatgacg cggaggccaa ggcaaaaaga ttccttgatt acgtaaggga 300gttagaatca ttttgaataa aaaacacgct ttttcagttc gagtttatca ttatcaatac 360tgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact ttatttagtc 420aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta 480cacagaatat ataacatcgt aggtgtctgg gtgaacagtt tattcctggc atccactaaa 540tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa 600tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacagaga 660acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcctg 720gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt ttcttacacc 780ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt 840tccctgaaat tattccccta cttgactaat aagtatataa agacggtagg tattgattgt 900aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt 960tagttttaaa acaccaagaa cttagtttcg aataaacaca cataaacaaa caaaatgggt 1020gctgatggta tttcttctgc ttccattgtt gttactgacc cagaagctgc tgccaagaag 1080cgtgacagaa tggccagaga attgttgtcc tccaactctg gtctatgtca agaagatgaa 1140ccaaccatca tcaacttaaa gggtttggaa cacaccattc catacagatt ggccgttgtt 1200ttgtgtaact ccagatccac tggtgaattc gaagccaagg ctgctgaaat cttgagaaag 1260gctttccaca tggttgacta ctctttgaat tgtttcaacc cagaatctga attgtcccgt 1320gtcaactctt taccagtcgg tgaaaagcac caaatgtccg aagatctaag acatgtcatg 1380gaatgtacca tttctgtcca ccactcctct ggtatgggtt tcgacccagc tgctggtcca 1440atcatctcca gattgagagg tgccatgaga gatcacaacg acatgtccga tatctccgtc 1500actgaagctg aagttgaatt attctctttg gctcaatctt tcgatgtcga cttggaagaa 1560ggtactattg ccagaaagca ctctgaagcc agattggatt tgggtggtgt caacaagggt 1620tacactgttg actacgttgt tgaccatttg agagctgctg gtatgccaaa cgtcttgttc 1680gaatggggtg gtgatatcag agcttctggt agaaacatca agggtaactt gtgggctgtt 1740gccatcaagc gtccaccatc tgttgaagaa gttatccgtc gtgccaaggg taagatgtta 1800aagatgggtg aagaagaaca agaagaaaag gacgatgact ctccatcttt gttgcacgtt 1860gttgaattgg atgacgaagc tttgtgtacc tctggtgact acgaaaacgt cttataccat 1920ccaaagcacg gtgttgctgg ttccattttc gactggcaac gtcgtggttt attgtctcca 1980gaagaaggtg ctttagctca agtttccgtc aaatgttact ctgccatgta cgctgatgct 2040ttggccactg tttgtttggt caagagagat gctgtcagaa tcagatactt gttggaaggt 2100tggagatacg tcagatctcg tgtcaccaac tacttcgctt acaccagaca aggtgaaaga 2160ttggctcaca tgcacgaaat tgctcaagaa accagagaat taagagaaat cagaattgct 2220ggttctttgc catccagaat tgttatcgtc ggtggtggtt tggctggtct atccgctgcc 2280attgaagctg cttcttgtgg tgctcaagtc attttgatgg aaaaggaagg tagaattggt 2340ggtaactctg ccaaggctac ctctggtatc aacggttggg gtaccagaac ccaagccaag 2400tctgatatct tggatggtgg taagtacttt gaaagagaca ctttcttgtc cggtgtcggt 2460ggtaccactg acccagcttt ggtcaaggtc ttgtccgtca aatctggtga cgctatcggt 2520tggttaactt ctttgggtgt cccattgtcc gttttgtctc aattgggtgg tcactctttc 2580aagagaactc acagagctcc agacaagact gatggtactc cattaccaat tggtcacacc 2640atcatgagaa ctttggaaga tcatatcaga aacaacttgt ctgaaagagt taccatcatg 2700acccacgttt ctgttactga attgttgcac gaaactgaca ccactccaga tggtgcttct 2760gaagttcgtg tcaccggtgt ccgttacaga gacttgtctg atgtcgatgg tcaaccttcc 2820aaactattgg ctgacgctgt tgttttggcc actggtggtt tctccaacga cagagaagaa 2880aactctttgt tgtgtaaata cgctcctcat ttggcttctt tcccaactac caacggtcca 2940tgggctactg gtgacggtgt caaattggcc acctccgttg gtgccaagtt ggttgacatg 3000gacaaggttc aattgcaccc aactggtttg attgacccaa aggacccagc taacaccact 3060aagatcttgg gtccagaagc tttgagaggt tctggtggta ttttgttgaa caagcaaggt 3120aagagattcg tcaacgaatt ggacttgaga tccgttgttt ccaaggccat taacactcaa 3180ggtaacgaat acccaggttc tggtggttgt tactttgctt actgtgtctt aaacgaagat 3240gctaccaact tattctgtgg tggtgctttg ggtttctacg gtaagaaatt aggtttgttc 3300caaagagctg aaactgttga agaattggcc aaattgattg gttgtgacga aggtgaattg 3360agagacactt tggaaaaata cgaaacctgt tccaaggcca aggttgcttg tccagtcact 3420ggtaaggttg ttttcccatg tgttgtcggt accagaggtc catacaatgt tgctttcgtc 3480actccatcca tccactacac catgggtggt tgtttgatct ctccagctgc tgaagtcttg 3540caagaataca agggtttgaa tatcttggaa aaccacagac caatcagatg tttgttcggt 3600gctggtgaag tcactggtgg tgtccacggt ggtaacagat taggtggtaa ctctctattg 3660gaatgtgttg tctttggtaa gattgctggt gacagagctg ccactatctt gcaaaagaga 3720gaaattgctt tgtccaagac ctcctggacc tctgttgttg tcagagaatc cagatctggt 3780gaacaattcg gtaccggttc cagagttttg agattcaact tgccaggtgc tttacaaaga 3840accggtttga acttgggtga attcgttgcc atcagaggtg aatgggatgg tcaacaatta 3900gtcggttact tctctccaat cactttgcca gaagatttgg gtaccatctc tttgttggtc 3960agagctgaca agggtacttt gaaggaatgg atctgtgctt tgcgtccagg tgactccgtt 4020gaaatcaagg cttgtggtgg tctaagaatt gaccaagatc cagtcaagaa atgtttgttg 4080ttcagaaaca gaccaattac cagatttgct ttggttgctg ctggtaccgg tgttgctcca 4140atgttgcaag ttatcagagc tgctttgaag aagccatacg tcgacacttt ggaatccatc 4200agattgatct acgctgctga agaatatgac actttaacct acagatctat cttgcaaaga 4260tttgctgaag aattcccaga caaattcgtt tgtaacttcg tcttaaacaa ccctccagaa 4320ggttggaccg gtggtgttgg tttcgtcaac aagaaatctt tgcaaaaggt tttgcaacca 4380ccttcttctg aaccattgat tgttgtttgt ggtccacctg ttatgcaaag agatgtcaaa 4440aatgaattgt tgtccatggg ttacgacaag gaattggttc acactgtcga tggtgaatct 4500ggtaccttgt aaggcccggg cgtgaattta ctttaaatct tgcatttaaa taaattttct 4560ttttatagct ttatgactta gtttcaattt atatactatt ttaatgacat tttcgattca 4620ttgattgaaa gctttgtgtt ttttcttgat gcgctattgc attgttcttg tctttttcgc 4680cacatgtaat atctgtagta gatacctgat acattgtgga tgctgagtga aattttagtt 4740aataatggag gcgctcttaa taattttggg gatattggct ttttttttta aagtttacaa 4800atgaattttt tccgccagga taacgattct gaagttactc ttagcgttcc tatcggtaca 4860gccatcaaat catgcctata aatcatgcct atatttgcgt gcagtcagta tcatctacat 4920gaaaaaaact cccgcaattt cttatagaat acgttgaaaa ttaaatgtac gcgccaagat 4980aagataacat atatctagat gcagtaatat acacagattc cggccggccg cggccgc 5037354959DNAArtificial SequenceTDH3p-FRDg-TDH3t artificial sequence 35ggatccggcg cgccctattt tcgaggacct tgtcaccttg agcccaagag agccaagatt 60taaattttcc tatgacttga tgcaaattcc caaagctaat aacatgcaag acacgtacgg 120tcaagaagac atatttgacc tcttaacagg ttcagacgcg actgcctcat cagtaagacc 180cgttgaaaag aacttacctg aaaaaaacga atatatacta gcgttgaatg ttagcgtcaa 240caacaagaag tttaatgacg cggaggccaa ggcaaaaaga ttccttgatt acgtaaggga 300gttagaatca ttttgaataa aaaacacgct ttttcagttc gagtttatca ttatcaatac 360tgccatttca aagaatacgt aaataattaa tagtagtgat tttcctaact ttatttagtc 420aaaaaattag ccttttaatt ctgctgtaac ccgtacatgc ccaaaatagg gggcgggtta 480cacagaatat ataacatcgt aggtgtctgg gtgaacagtt tattcctggc atccactaaa 540tataatggag cccgcttttt aagctggcat ccagaaaaaa aaagaatccc agcaccaaaa 600tattgttttc ttcaccaacc atcagttcat aggtccattc tcttagcgca actacagaga 660acaggggcac aaacaggcaa aaaacgggca caacctcaat ggagtgatgc aacctgcctg 720gagtaaatga tgacacaagg caattgaccc acgcatgtat ctatctcatt ttcttacacc 780ttctattacc ttctgctctc tctgatttgg aaaaagctga aaaaaaaggt tgaaaccagt 840tccctgaaat tattccccta cttgactaat aagtatataa agacggtagg tattgattgt 900aattctgtaa atctatttct taaacttctt aaattctact tttatagtta gtcttttttt 960tagttttaaa acaccaagaa cttagtttcg aataaacaca cataaacaaa caaaatggtt 1020gatggtagat cttctgcttc cattgttgcc gttgacccag aaagagctgc cagagaaaga 1080gatgctgctg ccagagcttt gttgcaagac tctccattgc acaccaccat gcaatacgct 1140acctctggtt tggaattgac tgttccatac gctttgaagg ttgttgcttc tgctgacact 1200ttcgacagag ccaaggaagt tgctgatgaa gtcttgagat gtgcctggca attggctgac 1260accgttttga actctttcaa cccaaactct gaagtctctt tagtcggtag attaccagtc 1320ggtcaaaagc atcaaatgtc tgctccattg aaacgtgtca tggcttgttg tcaaagagtc 1380tacaactcct ctgctggttg tttcgaccca tccactgctc cagttgccaa ggctttgaga 1440gaaattgctt tgggtaagga aagaaacaat gcttgtttgg aagctttgac tcaagcttgt 1500accttgccaa actctttcgt cattgatttc gaagctggta ctatctccag aaagcacgaa 1560cacgcttctt tggatttggg tggtgtttcc aagggttaca tcgtcgatta cgtcattgac 1620aacatcaatg ctgctggttt ccaaaacgtt ttctttgact ggggtggtga ctgtcgtgcc 1680tccggtatga acgccagaaa cactccatgg gttgtcggta tcactagacc tccttccttg 1740gacatgttgc caaaccctcc aaaggaagct tcttacatct ccgtcatctc tttggacaat 1800gaagctttgg ctacctctgg tgattacgaa aacttgatct acactgctga cgataaacca 1860ttgacctgta cctacgattg gaaaggtaag gaattgatga agccatctca atccaatatc 1920gctcaagttt ccgtcaagtg ttactctgcc atgtacgctg acgctttggc taccgcttgt 1980ttcatcaagc gtgacccagc caaggtcaga caattgttgg atggttggag atacgttaga 2040gacaccgtca gagattaccg tgtctacgtc agagaaaacg aaagagttgc caagatgttc 2100gaaattgcca ctgaagatgc tgaaatgaga aagagaagaa tttccaacac tttaccagct 2160cgtgtcattg ttgttggtgg tggtttggct ggtttgtccg ctgccattga agctgctggt 2220tgtggtgctc aagttgtttt gatggaaaag gaagccaagt tgggtggtaa ctctgccaag 2280gctacctctg gtatcaacgg ttggggtact agagctcaag ctaaggcttc cattgtcgat 2340ggtggtaagt acttcgaaag agatacctac aagtctggta tcggtggtaa caccgatcca 2400gctttggtta agactttgtc catgaaatct gctgacgcta tcggttggtt gacttctcta 2460ggtgttccat tgactgtttt gtcccaatta ggtggtcact ccagaaagag aactcacaga 2520gctccagaca agaaggatgg tactccattg ccaattggtt tcaccatcat gaaaacttta 2580gaagatcatg ttagaggtaa cttgtccggt agaatcacca tcatggaaaa ctgttccgtt 2640acctctttgt tgtctgaaac caaggaaaga ccagacggta ccaagcaaat cagagttacc 2700ggtgtcgaat tcactcaagc tggttctggt aagaccacca ttttggctga tgctgttatc 2760ttggccaccg gtggtttctc caacgacaag actgctgatt ctttgttgag agaacatgcc 2820ccacacttgg ttaacttccc aaccaccaac ggtccatggg ctactggtga tggtgtcaag 2880ttggctcaaa gattaggtgc tcaattggtc gatatggaca aggttcaatt gcacccaact 2940ggtttgatca acccaaagga cccagccaac ccaaccaaat tcttgggtcc agaagctcta 3000agaggttctg gtggtgtttt gttgaacaaa caaggtaaga gatttgtcaa cgaattggat 3060ttgagatctg ttgtttccaa ggccatcatg gaacaaggtg ctgaataccc aggttctggt 3120ggttccatgt ttgcttactg tgtcttgaac gctgctgctc aaaaattgtt tggtgtttcc 3180tctcacgaat tctactggaa gaagatgggt ttgttcgtca aggctgacac catgagagac 3240ttggctgctt tgattggttg tccagttgaa tccgttcaac aaactttaga agaatacgaa 3300agattatcca tctctcaaag atcttgtcca attaccagaa aatctgttta cccatgtgtt 3360ttgggtacca aaggtccata ctatgtcgcc tttgtcactc catctatcca ctacaccatg 3420ggtggttgtt tgatttctcc atctgctgaa atccaaatga agaacacttc ttccagagct 3480ccattgtccc actccaaccc aatcttgggt ttattcggtg ctggtgaagt caccggtggt 3540gtccacggtg gtaacagatt aggtggtaac tctttgttgg aatgtgttgt tttcggtaga 3600attgccggtg acagagcttc taccattttg caaagaaagt cctctgcttt gtctttcaag 3660gtctggacca ctgttgtttt gagagaagtc agagaaggtg gtgtctacgg tgctggttcc 3720cgtgtcttga gattcaactt accaggtgct ctacaaagat ctggtctatc cttgggtcaa 3780ttcattgcca tcagaggtga ctgggacggt caacaattga ttggttacta ctctccaatc 3840actttgccag acgatttggg tatgattgac attttggcca gatctgacaa gggtacttta 3900cgtgaatgga tctctgcttt ggaaccaggt gacgctgtcg aaatgaaggc ttgtggtggt 3960ttggtcatcg aaagaagatt atctgacaag cacttcgttt tcatgggtca cattatcaac 4020aagctatgtt tgattgctgg tggtaccggt gttgctccaa tgttgcaaat catcaaggcc 4080gctttcatga agccattcat cgacactttg gaatccgtcc acttgatcta cgctgctgaa 4140gatgtcactg aattgactta cagagaagtt ttggaagaac gtcgtcgtga atccagaggt 4200aaattcaaga aaactttcgt tttgaacaga cctcctccat tatggactga cggtgtcggt 4260ttcatcgacc gtggtatctt gaccaaccac gttcaaccac catctgacaa cttattggtt 4320gccatctgtg gtccaccagt tatgcaaaga attgtcaagg ccactttaaa gactttaggt 4380tacaacatga acttggtcag aaccgttgac gaaactgaac catctggaag ttaaggcccg 4440ggcgtgaatt tactttaaat cttgcattta aataaatttt ctttttatag ctttatgact 4500tagtttcaat ttatatacta ttttaatgac attttcgatt cattgattga aagctttgtg 4560ttttttcttg atgcgctatt gcattgttct tgtctttttc gccacatgta atatctgtag 4620tagatacctg atacattgtg gatgctgagt gaaattttag ttaataatgg aggcgctctt 4680aataattttg gggatattgg cttttttttt taaagtttac aaatgaattt tttccgccag 4740gataacgatt ctgaagttac tcttagcgtt cctatcggta cagccatcaa atcatgccta 4800taaatcatgc ctatatttgc gtgcagtcag tatcatctac atgaaaaaaa ctcccgcaat 4860ttcttataga atacgttgaa aattaaatgt acgcgccaag ataagataac atatatctag 4920atgcagtaat atacacagat tccggccggc cgcggccgc 495936438PRTSchizosaccharomyces pombe 36Met Gly Glu Leu Lys Glu Ile Leu Lys Gln Arg Tyr His Glu Leu Leu1 5 10 15Asp Trp Asn Val Lys Ala Pro His Val Pro Leu Ser Gln Arg Leu Lys 20 25 30His Phe Thr Trp Ser Trp Phe Ala Cys Thr Met Ala Thr Gly Gly Val 35 40 45Gly Leu Ile Ile Gly Ser Phe Pro Phe Arg Phe Tyr Gly Leu Asn Thr 50 55 60Ile Gly Lys Ile Val Tyr Ile Leu Gln Ile Phe Leu Phe Ser Leu Phe65 70 75 80Gly Ser Cys Met Leu Phe Arg Phe Ile Lys Tyr Pro Ser Thr Ile Lys 85 90 95Asp Ser Trp Asn His His Leu Glu Lys Leu Phe Ile Ala Thr Cys Leu 100 105 110Leu Ser Ile Ser Thr Phe Ile Asp Met Leu Ala Ile Tyr Ala Tyr Pro 115 120 125Asp Thr Gly Glu Trp Met Val Trp Val Ile Arg Ile Leu Tyr Tyr Ile 130 135 140Tyr Val Ala Val Ser Phe Ile Tyr Cys Val Met Ala Phe Phe Thr Ile145 150 155 160Phe Asn Asn His Val Tyr Thr Ile Glu Thr Ala Ser Pro Ala Trp Ile 165 170 175Leu Pro Ile Phe Pro Pro Met Ile Cys Gly Val Ile Ala Gly Ala Val 180 185 190Asn Ser Thr Gln Pro Ala His Gln Leu Lys Asn Met Val Ile Phe Gly 195 200 205Ile Leu Phe Gln Gly Leu Gly Phe Trp Val Tyr Leu Leu Leu Phe Ala 210 215 220Val Asn Val Leu Arg Phe Phe Thr Val Gly Leu Ala Lys Pro Gln Asp225 230 235 240Arg Pro Gly Met Phe Met Phe Val Gly Pro Pro Ala Phe Ser Gly Leu 245 250 255Ala Leu Ile Asn Ile Ala Arg Gly Ala Met Gly Ser Arg Pro Tyr Ile 260 265 270Phe Val Gly Ala Asn Ser Ser Glu Tyr Leu Gly Phe Val Ser Thr Phe 275 280 285Met Ala Ile Phe Ile Trp Gly Leu Ala Ala Trp Cys Tyr Cys Leu Ala 290 295 300Met Val Ser Phe Leu Ala Gly Phe Phe Thr Arg Ala Pro Leu Lys Phe305 310 315 320Ala Cys Gly Trp Phe Ala Phe Ile Phe Pro Asn Val Gly Phe Val Asn 325 330 335Cys Thr Ile Glu Ile Gly Lys Met Ile Asp Ser Lys Ala Phe Gln Met 340 345 350Phe Gly His Ile Ile Gly Val Ile Leu Cys Ile Gln Trp Ile Leu Leu 355 360 365Met Tyr Leu Met Val Arg Ala Phe Leu Val Asn Asp Leu Cys Tyr Pro 370 375 380Gly Lys Asp Glu Asp Ala His Pro Pro Pro Lys Pro Asn Thr Gly Val385 390 395 400Leu Asn Pro Thr Phe Pro Pro Glu Lys Ala Pro Ala Ser Leu Glu Lys 405 410 415Val Asp Thr His Val Thr Ser Thr Gly Gly Glu Ser Asp Pro Pro Ser 420 425 430Ser Glu His Glu Ser Val

435371317DNAArtificial sequenceS. pombe malae permease cpo for S. cerevisiae 37atgggtgaat tgaaggaaat cttgaagcaa cgttaccatg aattgttgga ctggaacgtc 60aaggctccac acgttccatt gtctcaaaga ttgaagcatt tcacctggtc ctggtttgct 120tgtaccatgg ccactggtgg tgtcggtttg atcattggtt ctttcccatt cagattctac 180ggtttgaaca ccattggtaa gattgtctac atcttacaaa tcttcttatt ctctttgttt 240ggttcttgta tgttgttcag attcatcaaa tacccatcta ccatcaagga ctcctggaac 300caccacttgg aaaaattatt cattgctacc tgtttgctat ccatctccac tttcattgac 360atgttggcca tctacgctta cccagacact ggtgaatgga tggtctgggt tatcagaatc 420ttatactaca tctacgttgc tgtctctttc atctactgtg tcatggcttt cttcaccatt 480ttcaacaacc acgtttacac cattgaaact gcttctccag cttggatctt accaattttc 540ccaccaatga tctgtggtgt cattgctggt gctgtcaact ccactcaacc agctcaccaa 600ttgaagaaca tggttatctt cggtatctta ttccaaggtt tgggtttctg ggtttacttg 660ttgttgtttg ctgtcaacgt tttgagattc ttcaccgttg gtttggccaa gcctcaagac 720agaccaggta tgttcatgtt tgttggtcca ccagctttct ccggtttggc tttgatcaac 780attgcccgtg gtgctatggg ttccagacca tacattttcg tcggtgccaa ttcttctgaa 840tacttgggtt tcgtttccac tttcatggcc attttcatct ggggtttggc tgcttggtgt 900tactgtttgg ccatggtttc tttcttggct ggtttcttca ccagagctcc attgaaattt 960gcttgtggtt ggtttgcttt catcttccca aacgtcggtt tcgttaactg taccattgaa 1020attggtaaga tgattgactc caaggccttc caaatgttcg gtcacatcat cggtgtcatc 1080ctatgtatcc aatggatctt gttgatgtac ttgatggtca gagctttctt ggtcaacgat 1140ttgtgttacc caggtaagga tgaagatgct cacccacctc caaagccaaa cactggtgtt 1200ttgaacccaa ctttcccacc agaaaaggct ccagcttctt tggaaaaggt tgacacccac 1260gttacttcca ctggtggtga atctgatcct ccatcttctg aacacgaaag cgtttaa 131738600DNAArtificial sequenceENO1 promotor T at position -5 was changed to A in order to obtain a better Kozak sequence 38ccgcggaacc gccagatatt cattacttga cgcaaaagcg tttgaaataa tgacgaaaaa 60gaaggaagaa aaaaaaagaa aaataccgct tctaggcggg ttatctactg atccgagctt 120ccactaggat agcacccaaa cacctgcata tttggacgac ctttacttac accaccaaaa 180accactttcg cctctcccgc ccctgataac gtccactaat tgagcgatta cctgagcggt 240cctcttttgt ttgcagcatg agacttgcat actgcaaatc gtaagtagca acgtctcaag 300gtcaaaactg tatggaaacc ttgtcacctc acttaattct agctagccta ccctgcaagt 360caagaggtct ccgtgattcc tagccacctc aaggtatgcc tctccccgga aactgtggcc 420ttttctggca cacatgatct ccacgatttc aacatataaa tagcttttga taatggcaat 480attaatcaaa tttattttac ttctttcttg taacatctct cttgtaatcc cttattcctt 540ctagctattt ttcataaaaa accaagcaac tgcttatcaa cacacaaaca ctaaaacaaa 60039300DNAArtificial sequenceENO1 terminator 39agcttttgat taagccttct agtccaaaaa acacgttttt ttgtcattta tttcattttc 60ttagaatagt ttagtttatt cattttatag tcacgaatgt tttatgattc tatatagggt 120tgcaaacaag catttttcat tttatgttaa aacaatttca ggtttacctt ttattctgct 180tgtggtgacg cgggtatccg cccgctcttt tggtcaccca tgtatttaat tgcataaata 240attcttaaaa gtggagctag tctatttcta tttacatacc tctcatttct catttcctcc 300402240DNAArtificial sequenceENO1p-SpMAE-ENO1t synthetic construct 40ggatccggcg cgccccgcgg aaccgccaga tattcattac ttgacgcaaa agcgtttgaa 60ataatgacga aaaagaagga agaaaaaaaa agaaaaatac cgcttctagg cgggttatct 120actgatccga gcttccacta ggatagcacc caaacacctg catatttgga cgacctttac 180ttacaccacc aaaaaccact ttcgcctctc ccgcccctga taacgtccac taattgagcg 240attacctgag cggtcctctt ttgtttgcag catgagactt gcatactgca aatcgtaagt 300agcaacgtct caaggtcaaa actgtatgga aaccttgtca cctcacttaa ttctagctag 360cctaccctgc aagtcaagag gtctccgtga ttcctagcca cctcaaggta tgcctctccc 420cggaaactgt ggccttttct ggcacacatg atctccacga tttcaacata taaatagctt 480ttgataatgg caatattaat caaatttatt ttacttcttt cttgtaacat ctctcttgta 540atcccttatt ccttctagct atttttcata aaaaaccaag caactgctta tcaacacaca 600aacactaaaa caaaatgggt gaattgaagg aaatcttgaa gcaacgttac catgaattgt 660tggactggaa cgtcaaggct ccacacgttc cattgtctca aagattgaag catttcacct 720ggtcctggtt tgcttgtacc atggccactg gtggtgtcgg tttgatcatt ggttctttcc 780cattcagatt ctacggtttg aacaccattg gtaagattgt ctacatctta caaatcttct 840tattctcttt gtttggttct tgtatgttgt tcagattcat caaataccca tctaccatca 900aggactcctg gaaccaccac ttggaaaaat tattcattgc tacctgtttg ctatccatct 960ccactttcat tgacatgttg gccatctacg cttacccaga cactggtgaa tggatggtct 1020gggttatcag aatcttatac tacatctacg ttgctgtctc tttcatctac tgtgtcatgg 1080ctttcttcac cattttcaac aaccacgttt acaccattga aactgcttct ccagcttgga 1140tcttaccaat tttcccacca atgatctgtg gtgtcattgc tggtgctgtc aactccactc 1200aaccagctca ccaattgaag aacatggtta tcttcggtat cttattccaa ggtttgggtt 1260tctgggttta cttgttgttg tttgctgtca acgttttgag attcttcacc gttggtttgg 1320ccaagcctca agacagacca ggtatgttca tgtttgttgg tccaccagct ttctccggtt 1380tggctttgat caacattgcc cgtggtgcta tgggttccag accatacatt ttcgtcggtg 1440ccaattcttc tgaatacttg ggtttcgttt ccactttcat ggccattttc atctggggtt 1500tggctgcttg gtgttactgt ttggccatgg tttctttctt ggctggtttc ttcaccagag 1560ctccattgaa atttgcttgt ggttggtttg ctttcatctt cccaaacgtc ggtttcgtta 1620actgtaccat tgaaattggt aagatgattg actccaaggc cttccaaatg ttcggtcaca 1680tcatcggtgt catcctatgt atccaatgga tcttgttgat gtacttgatg gtcagagctt 1740tcttggtcaa cgatttgtgt tacccaggta aggatgaaga tgctcaccca cctccaaagc 1800caaacactgg tgttttgaac ccaactttcc caccagaaaa ggctccagct tctttggaaa 1860aggttgacac ccacgttact tccactggtg gtgaatctga tcctccatct tctgaacacg 1920aaagcgttta agagcttttg attaagcctt ctagtccaaa aaacacgttt ttttgtcatt 1980tatttcattt tcttagaata gtttagttta ttcattttat agtcacgaat gttttatgat 2040tctatatagg gttgcaaaca agcatttttc attttatgtt aaaacaattt caggtttacc 2100ttttattctg cttgtggtga cgcgggtatc cgcccgctct tttggtcacc catgtattta 2160attgcataaa taattcttaa aagtggagct agtctatttc tatttacata cctctcattt 2220ctcatttcct ccgcggccgc 2240411180PRTSaccharomyces cerevisiae 41Met Ser Ser Ser Lys Lys Leu Ala Gly Leu Arg Asp Asn Phe Ser Leu1 5 10 15Leu Gly Glu Lys Asn Lys Ile Leu Val Ala Asn Arg Gly Glu Ile Pro 20 25 30Ile Arg Ile Phe Arg Ser Ala His Glu Leu Ser Met Arg Thr Ile Ala 35 40 45Ile Tyr Ser His Glu Asp Arg Leu Ser Met His Arg Leu Lys Ala Asp 50 55 60Glu Ala Tyr Val Ile Gly Glu Glu Gly Gln Tyr Thr Pro Val Gly Ala65 70 75 80Tyr Leu Ala Met Asp Glu Ile Ile Glu Ile Ala Lys Lys His Lys Val 85 90 95Asp Phe Ile His Pro Gly Tyr Gly Phe Leu Ser Glu Asn Ser Glu Phe 100 105 110Ala Asp Lys Val Val Lys Ala Gly Ile Thr Trp Ile Gly Pro Pro Ala 115 120 125Glu Val Ile Asp Ser Val Gly Asp Lys Val Ser Ala Arg His Leu Ala 130 135 140Ala Arg Ala Asn Val Pro Thr Val Pro Gly Thr Pro Gly Pro Ile Glu145 150 155 160Thr Val Gln Glu Ala Leu Asp Phe Val Asn Glu Tyr Gly Tyr Pro Val 165 170 175Ile Ile Lys Ala Ala Phe Gly Gly Gly Gly Arg Gly Met Arg Val Val 180 185 190Arg Glu Gly Asp Asp Val Ala Asp Ala Phe Gln Arg Ala Thr Ser Glu 195 200 205Ala Arg Thr Ala Phe Gly Asn Gly Thr Cys Phe Val Glu Arg Phe Leu 210 215 220Asp Lys Pro Lys His Ile Glu Val Gln Leu Leu Ala Asp Asn His Gly225 230 235 240Asn Val Val His Leu Phe Glu Arg Asp Cys Ser Val Gln Arg Arg His 245 250 255Gln Lys Val Val Glu Val Ala Pro Ala Lys Thr Leu Pro Arg Glu Val 260 265 270Arg Asp Ala Ile Leu Thr Asp Ala Val Lys Leu Ala Lys Val Cys Gly 275 280 285Tyr Arg Asn Ala Gly Thr Ala Glu Phe Leu Val Asp Asn Gln Asn Arg 290 295 300His Tyr Phe Ile Glu Ile Asn Pro Arg Ile Gln Val Glu His Thr Ile305 310 315 320Thr Glu Glu Ile Thr Gly Ile Asp Ile Val Ser Ala Gln Ile Gln Ile 325 330 335Ala Ala Gly Ala Thr Leu Thr Gln Leu Gly Leu Leu Gln Asp Lys Ile 340 345 350Thr Thr Arg Gly Phe Ser Ile Gln Cys Arg Ile Thr Thr Glu Asp Pro 355 360 365Ser Lys Asn Phe Gln Pro Asp Thr Gly Arg Leu Glu Val Tyr Arg Ser 370 375 380Ala Gly Gly Asn Gly Val Arg Leu Asp Gly Gly Asn Ala Tyr Ala Gly385 390 395 400Ala Thr Ile Ser Pro His Tyr Asp Ser Met Leu Val Lys Cys Ser Cys 405 410 415Ser Gly Ser Thr Tyr Glu Ile Val Arg Arg Lys Met Ile Arg Ala Leu 420 425 430Ile Glu Phe Arg Ile Arg Gly Val Lys Thr Asn Ile Pro Phe Leu Leu 435 440 445Thr Leu Leu Thr Asn Pro Val Phe Ile Glu Gly Thr Tyr Trp Thr Thr 450 455 460Phe Ile Asp Asp Thr Pro Gln Leu Phe Gln Met Val Ser Ser Gln Asn465 470 475 480Arg Ala Gln Lys Leu Leu His Tyr Leu Ala Asp Leu Ala Val Asn Gly 485 490 495Ser Ser Ile Lys Gly Gln Ile Gly Leu Pro Lys Leu Lys Ser Asn Pro 500 505 510Ser Val Pro His Leu His Asp Ala Gln Gly Asn Val Ile Asn Val Thr 515 520 525Lys Ser Ala Pro Pro Ser Gly Trp Arg Gln Val Leu Leu Glu Lys Gly 530 535 540Pro Ser Glu Phe Ala Lys Gln Val Arg Gln Phe Asn Gly Thr Leu Leu545 550 555 560Met Asp Thr Thr Trp Arg Asp Ala His Gln Ser Leu Leu Ala Thr Arg 565 570 575Val Arg Thr His Asp Leu Ala Thr Ile Ala Pro Thr Thr Ala His Ala 580 585 590Leu Ala Gly Ala Phe Ala Leu Glu Cys Trp Gly Gly Ala Thr Phe Asp 595 600 605Val Ala Met Arg Phe Leu His Glu Asp Pro Trp Glu Arg Leu Arg Lys 610 615 620Leu Arg Ser Leu Val Pro Asn Ile Pro Phe Gln Met Leu Leu Arg Gly625 630 635 640Ala Asn Gly Val Ala Tyr Ser Ser Leu Pro Asp Asn Ala Ile Asp His 645 650 655Phe Val Lys Gln Ala Lys Asp Asn Gly Val Asp Ile Phe Arg Val Phe 660 665 670Asp Ala Leu Asn Asp Leu Glu Gln Leu Lys Val Gly Val Asn Ala Val 675 680 685Lys Lys Ala Gly Gly Val Val Glu Ala Thr Val Cys Tyr Ser Gly Asp 690 695 700Met Leu Gln Pro Gly Lys Lys Tyr Asn Leu Asp Tyr Tyr Leu Glu Val705 710 715 720Val Glu Lys Ile Val Gln Met Gly Thr His Ile Leu Gly Ile Lys Asp 725 730 735Met Ala Gly Thr Met Lys Pro Ala Ala Ala Lys Leu Leu Ile Gly Ser 740 745 750Leu Arg Thr Arg Tyr Pro Asp Leu Pro Ile His Val His Ser His Asp 755 760 765Ser Ala Gly Thr Ala Val Ala Ser Met Thr Ala Cys Ala Leu Ala Gly 770 775 780Ala Asp Val Val Asp Val Ala Ile Asn Ser Met Ser Gly Leu Thr Ser785 790 795 800Gln Pro Ser Ile Asn Ala Leu Leu Ala Ser Leu Glu Gly Asn Ile Asp 805 810 815Thr Gly Ile Asn Val Glu His Val Arg Glu Leu Asp Ala Tyr Trp Ala 820 825 830Glu Met Arg Leu Leu Tyr Ser Cys Phe Glu Ala Asp Leu Lys Gly Pro 835 840 845Asp Pro Glu Val Tyr Gln His Glu Ile Pro Gly Gly Gln Leu Thr Asn 850 855 860Leu Leu Phe Gln Ala Gln Gln Leu Gly Leu Gly Glu Gln Trp Ala Glu865 870 875 880Thr Lys Arg Ala Tyr Arg Glu Ala Asn Tyr Leu Leu Gly Asp Ile Val 885 890 895Lys Val Thr Pro Thr Ser Lys Val Val Gly Asp Leu Ala Gln Phe Met 900 905 910Val Ser Asn Lys Leu Thr Ser Asp Asp Ile Arg Arg Leu Ala Asn Ser 915 920 925Leu Asp Phe Pro Asp Ser Val Met Asp Phe Phe Glu Gly Leu Ile Gly 930 935 940Gln Pro Tyr Gly Gly Phe Pro Glu Pro Leu Arg Ser Asp Val Leu Arg945 950 955 960Asn Lys Arg Arg Lys Leu Thr Cys Arg Pro Gly Leu Glu Leu Glu Pro 965 970 975Phe Asp Leu Glu Lys Ile Arg Glu Asp Leu Gln Asn Arg Phe Gly Asp 980 985 990Ile Asp Glu Cys Asp Val Ala Ser Tyr Asn Met Tyr Pro Arg Val Tyr 995 1000 1005Glu Asp Phe Gln Lys Ile Arg Glu Thr Tyr Gly Asp Leu Ser Val 1010 1015 1020Leu Pro Thr Lys Asn Phe Leu Ala Pro Ala Glu Pro Asp Glu Glu 1025 1030 1035Ile Glu Val Thr Ile Glu Gln Gly Lys Thr Leu Ile Ile Lys Leu 1040 1045 1050Gln Ala Val Gly Asp Leu Asn Lys Lys Thr Gly Gln Arg Glu Val 1055 1060 1065Tyr Phe Glu Leu Asn Gly Glu Leu Arg Lys Ile Arg Val Ala Asp 1070 1075 1080Lys Ser Gln Asn Ile Gln Ser Val Ala Lys Pro Lys Ala Asp Val 1085 1090 1095His Asp Thr His Gln Ile Gly Ala Pro Met Ala Gly Val Ile Ile 1100 1105 1110Glu Val Lys Val His Lys Gly Ser Leu Val Lys Lys Gly Glu Ser 1115 1120 1125Ile Ala Val Leu Ser Ala Met Lys Met Glu Met Val Val Ser Ser 1130 1135 1140Pro Ala Asp Gly Gln Val Lys Asp Val Phe Ile Lys Asp Gly Glu 1145 1150 1155Ser Val Asp Ala Ser Asp Leu Leu Val Val Leu Glu Glu Glu Thr 1160 1165 1170Leu Pro Pro Ser Gln Lys Lys 1175 1180423543DNASaccharomyces cerevisiae 42atgagcagta gcaagaaatt ggccggtctt agggacaatt tcagtttgct cggcgaaaag 60aataagatct tggtcgccaa tagaggtgaa attccgatta gaatttttag atctgctcat 120gagctgtcta tgagaaccat cgccatatac tcccatgagg accgtctttc aatgcacagg 180ttgaaggcgg acgaagcgta tgttatcggg gaggagggcc agtatacacc tgtgggtgct 240tacttggcaa tggacgagat catcgaaatt gcaaagaagc ataaggtgga tttcatccat 300ccaggttatg ggttcttgtc tgaaaattcg gaatttgccg acaaagtagt gaaggccggt 360atcacttgga tcggccctcc agctgaagtt attgactctg tgggtgacaa agtctctgcc 420agacacttgg cagcaagagc taacgttcct accgttcccg gtactccagg acctatcgaa 480actgtgcaag aggcacttga cttcgttaat gaatacggct acccggtgat cattaaggcc 540gcctttggtg gtggtggtag aggtatgaga gtcgttagag aaggtgacga cgtggcagat 600gcctttcaac gtgctacctc cgaagcccgt actgccttcg gtaatggtac ctgctttgtg 660gaaagattct tggacaagcc aaagcatatt gaagttcaat tgttggctga taaccacgga 720aacgtggttc atcttttcga aagagactgt tctgtgcaaa gaagacacca aaaagttgtc 780gaagtcgctc cagcaaagac tttgccccgt gaagttcgtg acgctatttt gacagatgct 840gttaaattag ctaaggtatg tggttacaga aacgcaggta ccgccgaatt cttggttgac 900aaccaaaaca gacactattt cattgaaatt aatccaagaa ttcaagtgga gcataccatc 960actgaagaaa tcaccggtat tgacattgtt tctgcccaaa tccagattgc cgcaggtgcc 1020actttgactc aactaggtct attacaggat aaaatcacca cccgtgggtt ttccatccaa 1080tgtcgtatta ccactgaaga tccctctaag aatttccaac cggataccgg tcgcctggag 1140gtctatcgtt ctgccggtgg taatggtgtg agattggacg gtggtaacgc ttatgcaggt 1200gctactatct cgcctcacta cgactcaatg ctggtcaaat gttcatgctc tggttctact 1260tatgaaatcg tccgtaggaa gatgattcgt gccctgatcg aattcagaat cagaggtgtt 1320aagaccaaca ttcccttcct attgactctt ttgaccaatc cagtttttat tgagggtaca 1380tactggacga cttttattga cgacacccca caactgttcc aaatggtatc gtcacaaaac 1440agagcgcaaa aactgttaca ctatttggca gacttggcag ttaacggttc ttctattaag 1500ggtcaaattg gcttgccaaa actaaaatca aatccaagtg tcccccattt gcacgatgct 1560cagggcaatg tcatcaacgt tacaaagtct gcaccaccat ccggatggag acaagtgcta 1620ctggaaaagg gaccatctga atttgccaag caagtcagac agttcaatgg tactctactg 1680atggacacca cctggagaga cgctcatcaa tctctacttg caacaagagt cagaacccac 1740gatttggcta caatcgctcc aacaaccgca catgcccttg caggtgcttt cgctttagaa 1800tgttggggtg gtgctacatt cgacgttgca atgagattct tgcatgagga tccatgggaa 1860cgtctgagaa aattaagatc tctggtgcct aatattccat tccaaatgtt attacgtggt 1920gccaacggtg tggcttactc ttcattacct gacaatgcta ttgaccattt tgtcaagcaa 1980gccaaggata atggtgttga tatatttaga gtttttgatg ccttgaatga tttagaacaa 2040ttaaaagttg gtgtgaatgc tgtcaagaag gccggtggtg ttgtcgaagc tactgtttgt 2100tactctggtg acatgcttca gccaggtaag aaatacaact tagactacta cctagaagtt 2160gttgaaaaaa tagttcaaat gggtacacat atcttgggta ttaaggatat ggcaggtact 2220atgaaaccgg ccgctgccaa attattaatt ggctccctaa gaaccagata tccggattta 2280ccaattcatg ttcacagtca tgactccgca ggtactgctg ttgcgtctat gactgcatgt 2340gccctagcag gtgctgatgt tgtcgatgta gctatcaatt caatgtcggg cttaacttcc 2400caaccatcaa ttaatgcact gttggcttca ttagaaggta acattgatac tgggattaac 2460gttgagcatg ttcgtgaatt agatgcatac tgggccgaaa tgagactgtt gtattcttgt 2520ttcgaggccg acttgaaggg accagatcca gaagtttacc aacatgaaat cccaggtggt 2580caattgacta acttgttatt ccaagctcaa caactgggtc ttggtgaaca atgggctgaa 2640actaaaagag cttacagaga agccaattac ctactgggag atattgttaa agttacccca 2700acttctaagg ttgtcggtga tttagctcaa ttcatggttt ctaacaaact gacttccgac 2760gatattagac gtttagctaa ttctttggac tttcctgact ctgttatgga cttttttgaa 2820ggtttaattg gtcaaccata cggtgggttc ccagaaccat taagatctga tgtattgaga 2880aacaagagaa gaaagttgac gtgccgtcca ggtttagaat tagaaccatt tgatctcgaa 2940aaaattagag aagacttgca gaacagattc ggtgatattg atgaatgcga tgttgcttct 3000tacaatatgt

atccaagggt ctatgaagat ttccaaaaga tcagagaaac atacggtgat 3060ttatcagttc taccaaccaa aaatttccta gcaccagcag aacctgatga agaaatcgaa 3120gtcaccatcg aacaaggtaa gactttgatt atcaaattgc aagctgttgg tgacttaaat 3180aagaaaactg ggcaaagaga agtgtatttt gaattgaacg gtgaattaag aaagatcaga 3240gttgcagaca agtcacaaaa catacaatct gttgctaaac caaaggctga tgtccacgat 3300actcaccaaa tcggtgcacc aatggctggt gttatcatag aagttaaagt acataaaggg 3360tctttggtga aaaagggcga atcgattgct gttttgagtg ccatgaaaat ggaaatggtt 3420gtctcttcac cagcagatgg tcaagttaaa gacgttttca ttaaggatgg tgaaagtgtt 3480gacgcatcag atttgttggt tgtcctagaa gaagaaaccc tacccccatc ccaaaaaaag 3540taa 35434330DNAArtificial sequenceP1 primer 43ggactagtat gagcagtagc aagaaattgg 304431DNAArtificial sequenceP2 primer 44ccgctcgagt tacttttttt gggatggggg t 31

Claims

1. A recombinant eukaryotic cell selected from the group consisting of a yeast and a filamentous fungus comprising a nucleotide sequence encoding a NAD(H)-dependent fumarate reductase that catalyses the conversion of fumaric acid to succinic acid.

2. A cell according to claim 1, wherein the cell expresses a nucleotide sequence encoding an enzyme that catalyses the formation of succinic acid, wherein the nucleotide sequence encodes a NAD(H)-dependent fumarate reductase, comprising an amino acid sequence that has at least 40% 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;

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

4. A cell according to claim 1, wherein the NAD(H)-dependent fumarate reductase is active in the cytosol upon expression of the nucleotide sequence encoding NAD(H)-dependent fumarate reductase.

5. A cell according to claim 1, wherein the cell overexpresses a nucleotide sequence encoding a pyruvate carboxylase.

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

7. A 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.

8. A 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.

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

10. A cell according to claim 1, wherein at least one gene encoding alcohol dehydrogenase is not functional.

11. A cell according to claim 1, wherein at least one gene encoding glycerol-3-phosphate dehydrogenase is not functional.

12. A cell according to claim 1, wherein at least one gene encoding succinate dehydrogenase is not functional.

13. A cell according to claim 1, which is an Aspergillus, preferably an Aspergillus niger.

14. A cell according to claim 1, which is a Saccharomyces cerevisiae

15. A process for the preparation of succinic acid, comprising fermenting a eukaryotic cell according to claim 1, in a suitable fermentation medium, wherein succinic acid is prepared.

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

17. A fermentation broth comprising succinic acid obtainable by the process according to claim 16.