WO2014142647A1

WO2014142647A1 - Fungals strains with improved citric acid and itaconic acid production

Info

Publication number: WO2014142647A1
Application number: PCT/NL2013/050174
Authority: WO
Inventors: Laura VAN DER STRAAT; Tom SCHONEWILLE; Juan Antonio Tamayo Ramos; Leendert Hendrik De Graaff
Original assignee: Wageningen Universiteit
Priority date: 2013-03-14
Filing date: 2013-03-14
Publication date: 2014-09-18

Abstract

The present invention relates to cells with improved citric acid and itaconic acid production and to the use of these cells for the production of citric acid and itaconic acid.

Description

Fungal strains with improved citric acid and itaconic acid production

Field of the invention

Background of the invention

Itaconic acid is a C5 dicarboxylic acid, also known as methyl succinic acid. Itaconic acid has the potential to be a key building block for deriving both commodity and specialty chemicals. The basic chemistry of itaconic acid is similar to that of the petrochemicals derived from maleic acid/anhydride. Being able to do various kinds of addition-, esterification- and polymerization-reactions, it is an important compound for the chemical synthetic industry as well as for the production of chemical intermediates.

Currently, itaconic acid is used as a co-monomer in acrylic fibres and styrene materials to aid the dyeing and painting properties. Acrylic fibers, which have included itaconic acid as the third monomer, are much easier to dye. Itaconic acid is also used to improve the optical properties of plastics. Polymers, which contain itaconic acid, have special transparency and lustre qualities.

The problem of current itaconic acid manufacturing is the high production cost, thus limiting the use of this promising biological molecule as a building block for high value chemical intermediates and polymers. Should the price of itaconic acid be reduced then it is reasonable to expect more applications in the area of bio-based chemical building blocks.

Itaconic acid can be produced chemically by the pyrolysis of citric acid, resulting in water loss and conversion of citric acid in aconitate. Subsequent decarboxylation of aconitate gives two isomers itaconic acid and citraconic acid. This chemical synthesis route of itaconic acid has proven uneconomical for a number of reasons, including the relatively high substrate costs, the low yields and the co-production of various other acids such as succinic acid and tartaric acid (Brian Currell, R.C. et al).

A currently more promising production route is via fungal fermentation. Itaconic acid is commercially produced by Aspergillus terreus. The global production volume remains relatively low (estimated to be ca. 5000-10000 tonnes per annum) and the price relatively high (ca. €2500-4000 per tonne). Though fungal fermentation is economically a more viable route compared to chemical production, the cost price of also the fungal production is still a major hurdle for the development of itaconic acid as a building block for commodity chemicals.

It is thus an object of the present invention to provide for means and methods that allow for a more cost effective production of citric acid and/or itaconic acid.

Description of the invention

Definitions

The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or

RNA molecule in single or double stranded form, particularly a DNA having promoter activity according to the invention or a DNA encoding a protein or protein fragment. An "isolated nucleic acid" refers to a nucleic acid, which is no longer in the natural environment from which it was isolated, e.g. the nucleic acid sequence in a fungal host cell or in the plant nuclear or plastid genome.

The term peptide herein refers to any molecule comprising a chain of amino acids that are linked in peptide bonds. The term peptide thus includes oligopeptides, polypeptides and proteins, including multimeric proteins, without reference to a specific mode of action, size, 3 -dimensional structure or origin. The terms "protein" or "polypeptide" are used interchangeably. A "fragment" or "portion" of a protein may thus still be referred to as a "protein". An "isolated protein" is used to refer to a protein which is no longer in its natural environment, for example in vitro or in a recombinant (fungal or plant) host cell. The term peptide also includes post-expression modifications of peptides, e.g. glycosylations, acetylations, phosphorylations, and the like.

The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable transcription regulatory regions (e.g. a promoter). A gene may thus comprise several operably linked sequences, such as a promoter, a 5' non-translated leader sequence (also referred to as 5'UTR, which corresponds to the transcribed mRNA sequence upstream of the translation start codon) comprising e.g. sequences involved in translation initiation, a (protein) coding region (cDNA or genomic DNA) and a 3 'non-translated sequence (also referred to as 3' untranslated region, or 3'UTR) comprising e.g. transcription termination sites and polyadenylation site (such as e.g. AAUAAA or variants thereof).

A "chimeric gene" (or recombinant gene) refers to any gene, which is not normally found in nature in a species, in particular a gene in which one or more parts of the nucleic acid sequence are present that are not associated with each other in nature. For example the promoter is not associated in nature with part or all of the transcribed region or with another regulatory region. The term "chimeric gene" is understood to include expression constructs in which a promoter or transcription regulatory sequence is operably linked to one or more sense sequences (e.g. coding sequences) or to an antisense (reverse complement of the sense strand) or inverted repeat sequence (sense and antisense, whereby the RNA transcript forms double stranded RNA upon transcription).

A "3' UTR" or "3' non-translated sequence" (also often referred to as 3' untranslated region, or 3 'end) refers to the nucleic acid sequence found downstream of the coding sequence of a gene, which comprises, for example, a transcription termination site and (in most, but not all eukaryotic mRNAs) a polyadenylation signal (such as e.g. AAUAAA or variants thereof). After termination of transcription, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which is involved in the transport of the mRNA to the cytoplasm (where translation takes place).

"Expression of a gene" refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into a RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide (or active peptide fragment) or which is active itself (e.g. in posttranscriptional gene silencing or RNAi, or silencing through miRNAs). The coding sequence is preferably in sense-orientation and encodes a desired, biologically active protein or peptide, or an active peptide fragment.

"Ectopic expression" refers to expression in a tissue in which the gene is normally not expressed.

A "transcription regulatory sequence" is herein defined as a nucleic acid sequence that is capable of regulating the rate of transcription of a nucleic acid sequence operably linked to the transcription regulatory sequence. A transcription regulatory sequence as herein defined will thus comprise all of the sequence elements necessary for initiation of transcription (promoter elements), for maintaining and for regulating transcription, including e.g. attenuators or enhancers, but also silencers. Although mostly the upstream (5') transcription regulatory sequences of a coding sequence are referred to, regulatory sequences found downstream (3') of a coding sequence are also encompassed by this definition.

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 (5') with respect to the direction of transcription of the transcription initiation site of the gene (the transcription start is referred to as position +1 of the sequence and any upstream nucleotides relative thereto are referred to using negative numbers), and is structurally identified by the presence of a binding site for DNA-dependent RNA polymerase, transcription initiation sites and any other DNA domains (cis acting sequences), including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. Examples of eukaryotic cis acting sequences upstream of the transcription start (+1) include the TATA box (commonly at approximately position -20 to -30 of the transcription start), the CAAT box (commonly at approximately position -75 relative to the transcription start), 5 'enhancer or silencer elements, etc. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmentally regulated, e.g. by the application of a chemical inducer. A "tissue specific" promoter is only active in specific types of tissues or cells.

As used herein, the term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter, or a transcription regulatory sequence, is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame so as to produce a "chimeric protein". A "chimeric protein" or "hybrid protein" is a protein composed of various protein "domains" (or motifs) which is not found as such in nature but which are joined to form a functional protein, which displays the functionality of the joined domains (for example a DNA binding domain or a repression of function domain leading to a dominant negative function). A chimeric protein may also be a fusion protein of two or more proteins occurring in nature. The term "domain" as used herein means any part(s) or domain(s) of the protein with a specific structure or function that can be transferred to another protein for providing a new hybrid protein with at least the functional characteristic of the domain.

A "nucleic acid construct" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. A nucleic acid construct is a nucleic acid molecule, either single- or double-stranded, which has been modified to contain segments of nucleic acids, which are combined and juxtaposed in a manner, which would not otherwise exist in nature. A nucleic acid construct usually is a "vector", i.e. a nucleic acid molecule which is used to deliver exogenously created DNA into a host cell. Vectors usually comprise further genetic elements to facilitate their use in molecular cloning, such as e.g. selectable markers, multiple cloning sites and the like.

One type of nucleic acid construct is an "expression cassette" or "expression vector". These terms refers to nucleotide sequences that are capable of effecting expression of a gene in host cells or host organisms compatible with such sequences. Expression cassettes or expression vectors typically include at least suitable transcription regulatory sequences and optionally, 3' transcription termination signals. Additional factors necessary or helpful in effecting expression may also be present, such as expression enhancer elements. DNA encoding the polypeptides of the present invention will typically be incorporated into the expression vector. The expression vector will be introduced into a suitable host cell and be able to effect expression of the coding sequence in an in vitro cell culture of the host cell. The expression vector preferably is suitable for replication in a fungal, plant and/or in a prokaryotic host.

A "host cell" or a "recombinant host cell" or "transformed cell" are terms referring to a new individual cell (or organism), arising as a result of the introduction into said cell of at least one nucleic acid construct, especially comprising a chimeric gene encoding a desired protein. The host cell may be a plant cell, a bacterial cell, a fungal cell (including a yeast cell), etc. The host cell may contain the nucleic acid construct as an extra-chromosomally (episomal) replicating molecule, or more preferably, comprises the chimeric gene integrated in the nuclear or plastid genome of the host cell.

The term "selectable marker" is a term familiar to one of ordinary skill in the art and is used herein to describe any genetic entity which, when expressed, can be used to select for a cell or cells containing the selectable marker. Selectable markers may be dominant or recessive or bidirectional. The selectable marker may be a gene coding for a product which confers antibiotic or herbicide resistance to a cell expressing the gene or a non-antibiotic marker gene, such as a gene relieving other types of growth inhibition, i.e. a marker gene which allow cells containing the gene to grow under otherwise growth-inhibitory conditions. Examples of such genes include a gene which confers prototrophy to an auxotrophic strain. The term "reporter" is mainly used to refer to visible markers, such as green fluorescent protein (GFP), eGFP, luciferase, GUS and the like, as well as nptll markers and the like.

The term "ortholog" of a gene or protein refers herein to the homologous gene or protein found in another species, which has the same function as the gene or protein, but (usually) diverged in sequence from the time point on when the species harbouring the genes diverged (i.e. the genes evolved from a common ancestor by speciation). Orthologs of a gene from one species may thus be identified in other species based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and functional analysis.

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. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's). "Self-cloning" is defined herein as in European Directive 98/81/EC Annex II: Self-cloning consists in the removal of nucleic acid sequences from a cell of an organism which may or may not be followed by reinsertion of all or part of that nucleic acid (or a synthetic equivalent) with or without prior enzymic or mechanical steps, into cells of the same species or into cells of phylogenetically closely related species which can exchange genetic material by natural physiological processes where the resulting micro-organism is unlikely to cause disease to humans, animals or plants. Self-cloning may include the use of recombinant vectors with an extended history of safe use in the particular micro-organisms.

When used to indicate the relatedness of two nucleic acid sequences the term

"homologous" means that one single-stranded nucleic acid sequence may hybridise to a complementary single-stranded nucleic acid sequence. The degree of hybridisation may depend on a number of factors including the amount of identity between the sequences and the hybridisation conditions such as temperature and salt concentration as discussed later.

"Stringent hybridisation conditions" can be used to identify nucleotide sequences, which are substantially identical to a given nucleotide sequence. The stringency of the hybridization conditions are sequence dependent and will be different in different circumstances. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (T_m) for the specific sequences at a defined ionic strength and pH. The T_m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridises to a perfectly matched probe. Typically stringent conditions will be chosen in which the salt (NaCl) concentration is about 0.02 molar at pH 7 and the temperature is at least 60°C. Lowering the salt concentration and/or increasing the temperature increases stringency. Stringent conditions for RNA-DNA hybridisations (Northern blots using a probe of e.g. 100 nt) are for example those which include at least one wash in 0.2X SSC at 63°C for 20 min, or equivalent conditions. Stringent conditions for DNA-DNA hybridisation (Southern blots using a probe of e.g. lOOnt) are for example those which include at least one wash (usually 2) in 0.2X SSC at a temperature of at least 50°C, usually about 55°C, for 20 min, or equivalent conditions. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

"High stringency" conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCl, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (100X Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 μ§/ιη1 denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0.1 * SSC, 0.1% SDS. "Moderate stringency" refers to conditions equivalent to hybridization in the above described solution but at about 60-62° C. In that case the final wash is performed at the hybridization temperature in lx SSC, 0.1% SDS. "Low stringency" refers to conditions equivalent to hybridization in the above described solution at about 50-52° C. In that case, the final wash is performed at the hybridization temperature in 2x SSC, 0.1% SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).

"Sequence identity" and "sequence similarity" can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using a global alignment algorithms (e.g. Needleman Wunsch) which aligns the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using a local alignment algorithm (e.g. Smith Waterman). Part of a sequence may be used to calculate an sequence identity/similarity. In this context, part of a sequence means at least 50%, 60%, 70%, 80%), 90%) or 100%) of the length of said sequence. Sequences may then be referred to as "substantially identical" or "essentially similar" when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program "needle" (using the global Needleman Wunsch algorithm) or "water" (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for 'needle' and for 'water' and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall lengths, local alignments, such as using the Smith Waterman algorithm, are preferred. Alternatively percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc.

Optionally, in determining the degree of "amino acid similarity", the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur-containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine- valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

"Fungi" are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962 et al). The term fungus thus includes both filamentous fungi and yeast. "Filamentous fungi" are herein defined as eukaryotic microorganisms that include all filamentous forms of the subdivision Eumycotina and Oomycota (as defined by Hawksworth et al). The filamentous fungi are characterized by a mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan, and other complex polysaccharides. Vegetative growth is by hyphal elongation and carbon catabolism is obligately aerobic. Filamentous fungal strains include, but are not limited to, strains of Acremonium, Aspergillus, Aureobasidium, Candida, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Monascus, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, Yarrowia and Ustilago. Preferably the filamentous fungus is of a species selected from Aspergillus niger, Aspergillus terreus, Aspergillus itaconicus, Monascus ruber, Penicillium simplicissimum, Penicillium expansion, Penicillium digitatum, Penicillium italicum, Candida oleophila and Yarrowia lipolytica. "Yeasts" are herein defined as eukaryotic microorganisms and include all species of the subdivision Eumycotina that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism.

The term "fungal", when referring to a protein or nucleic acid molecule thus means a protein or nucleic acid whose amino acid or nucleotide sequence, respectively, naturally occurs in a fungus.

In principle, any "plant" may be a suitable host for the nucleic acid constructs of the invention, such as monocotyledonous plants or dicotyledonous plants, for example sugar beet, sugar cane, maize/corn (Zea species), wheat (Triticum species), barley (e.g. Hordeum vulgare), oat (e.g. Avena sativa), sorghum (Sorghum bicolor), rye (Secale cereale), soybean (Glycine spp, e.g. G. max), cotton (Gossypium species, e.g. G hirsutum, G barbadense), Brassica spp. (e.g. B. napus, B. juncea, B. oleracea, B. rapa, etc), sunflower (Helianthus annus), safflower, yam, cassava, tobacco (Nicotiana species), alfalfa (Medicago sativa), rice (Oryza species, e.g. O. sativa indica cultivar- group or japonica cultivar-group), forage grasses, pearl millet (Pennisetum spp. e.g. P. glaucum), tree species (Pinus, poplar, fir, plantain, etc), tea, coffea, oil palm, coconut, vegetable species, such as tomato (Lycopersicon ssp e.g. Lycopersicon esculentum), potato (Solanum tuberosum, other Solanum species), eggplant (Solanum melongena), peppers (Capsicum annuum, Capsicum frutescens), pea, zucchini, beans (e.g. Phaseolus species), cucumber, artichoke, asparagus, broccoli, garlic, leek, lettuce, onion, radish, turnip, Brussels sprouts, carrot, cauliflower, chicory, celery, spinach, endive, fennel, beet, fleshy fruit bearing plants (grapes, peaches, plums, strawberry, mango, apple, plum, cherry, apricot, banana, blackberry, blueberry, citrus, kiwi, figs, lemon, lime, nectarines, raspberry, watermelon, orange, grapefruit, etc.), ornamental species (e.g. Rose, Petunia, Chrysanthemum, Lily, Gerbera species), herbs (mint, parsley, basil, thyme, etc.), woody trees (e.g. species of Populus, Salix, Quercus, Eucalyptus), fibre species e.g. flax (Linum usitatissimum) hemp {Cannabis sativa) and grasses, e.g. Miscanthus and switchgrass (Panicum species).

Typical host plants for use in the method according to the invention are plants which can easily be grown, which give a high yield of plant material per hectare and which can be easily harvested and processed. Typical host plants suitable for use in the method according to the invention include corn, wheat, rice, barley, sorghum, millets, sunflower, cassava, canola, soybean, oil palm, groundnut, cotton, sugar cane, chicory, bean, pea, cawpea, banana, tomato, beet, sugar beet, Jerusalem artichoke, tobacco, potato, sweet potato, coffee, cocoa and tea. In addition, said plants should preferably after transformation be able to produce large amounts of itaconic acid, give a high content of produced itaconic acid based on fresh plant material and preferably be able to deposit said itaconic acid in a concentrated manner in parts of the plant, preferably in tap roots or tubers, which can be easily harvested, stored and processed.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

Detailed description of the invention

The commercial production of itaconic acid (i.e. methyl succinic acid) is reminiscent to the production of citric acid. Citric acid is commercially produced on a very large scale by Aspergillus niger, a close relative of the itaconic acid producing Aspergillus terreus. The citric acid production rate in A. niger is much more cost effective and efficient than itaconic acid production in A. terreus. The relatively high citric acid production rate of A. niger is the result of 65 years of work examining the biochemistry, molecular biology and industrial biotechnology of citric acid production in A. niger. This has resulted is a relatively highly efficient industrial production platform, which is highly optimized with respect to directing the metabolic flux towards citric acid. In contrast, the itaconic acid producing A. terreus is a rather underdeveloped industrial platform in comparison to A. niger. In WO 2009/102205, fungal strains were designed that are able to produce higher amounts of itaconic acid thanks to the expression of a czs-aconitate decarboxylase that confers the capacity to convert citric acid into itaconic acid. However, there is still room for improvement of a process for producing citric acid or itaconic acid.

The inventors surprisingly found that a cell expressing or capable of expressing at least one of:

a polypeptide having pyruvate carboxylase activity (defined as b) later on) and/or a polypeptide having phosphofructokinase activity (defined as a) later on) (i.e. defined as a first cell) is highly attractive to be used for producing citric acid. Optionally such a cell also expresses or is capable of expressing a polypeptide having malate dehydrogenase activity (defined as d) later on) and a polypeptide having fumarase activity (defined as c) later on). A first cell (capable of expressing b) and/or a), and optionally c) and d)) surprisingly produces higher amounts of citric acid and has a higher productivity of citric acid by comparison to the citric acid production and productivity of a corresponding cell not expressing a polypeptide having pyruvate carobxylase activity and/or not expressing a polypeptide having phosphofructokinase activity. This increase of production and of productivity of citric acid is surprising at least since citrate is produced in mitochondria and each of the four enzymes identified above is a cytosolic enzyme.

The inventors further surprisingly found that a cell expressing or capable of expressing a polypeptide having fumarase activity and/or a polypeptide having pyruvate carboxylase activity and/or a polypeptide having phosphofructokinase activity and/or a polypeptide having malate dehydrogenase activity (i.e. (a) and/or b) and/or c) and/or d) andthat further expresses or is capable of expressing the following three polypeptides: one having cis-aconitate decarboxylase activity (defined e) later on), one being a mitochondrial transporter (defined as f) later on) and one being a plasma membrane transporter (defined as g) later on) (i.e. defined as a second cell herein or a cell capable of expressing a) and/or b) and/or c) and/or d) in combination with e) and f) and g)) is highly attractive to be used for producing itaconic acid. Such a cell produces higher amount of itaconic acid and has a higher productivity of itaconic acid by comparison to the itaconic acid production and productivity of a corresponding cell not expressing these three last polypeptides.

Below we first describe each of the polypeptides, encoding nucleotide sequences and nucleic acid construct that could be used in the present invention. Subsequently, we describe cells and methods of the invention. (a) Phosphofructokinase

A polypeptide with phosphofructokinase activity is part of this disclosure. A polypeptide with phosphofructokinase activity (EC 2.7.1.11) is herein defined as an enzyme that catalyzes the irreversible phosphorylation of fructose-6-phosphate into fructose- 1,6-bisphosphate. A preferred phosphofructokinase polypeptide has been described in Mlakar et al. One phosphofructokinase unit (U) is defined as the production of 1 μπιοΐ of fructose- 1,6-bisphosphate per minute under standard reaction conditions. An assay for quantifying phosphofructokinase activity is described in Mlakar et al.

Polypeptides of the invention with phosphofructokinase activity may be further defined by their amino acid sequence as herein described below. A preferred phosphofructokinase polypeptide comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 10. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100% identity with SEQ ID NO:9. The length of a preferred phosphofructokinase polypeptide as defined above may be at least 400, 430, 450, 480, 485, 500 amino acids, preferably 485 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred phosphofructokinase polypeptide may be at least 1200, 1300, 1400, 1455, 1500 nucleotides. Likewise phosphofructokinase may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a phosphofructokinase as herein described below. Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with phosphofructokinase activity is also part of this disclosure. A nucleotide sequence encoding a polypeptide with phosphofructokinase activity preferably is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 10;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO. 9;

iii) a nucleotide sequence the complementary strand of which hybridises to a nucleotide sequence of ii); and,

iiii) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code.

The length of said nucleotide sequence is preferably as defined above.

A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptides having phosphofructokinase activity are represented by SEQ ID NO: 10 which is a phosphofructokinase derived from the phosphofructokinase of Aspergillus niger. The phosphofructokinase represented by SEQ ID NO: 10 has been modified in such a way that the citrate inhibitory part of the enzyme has been removed. Examples of amino acid sequences that have at least 40% sequence identity with the amino acid sequence of SEQ ID NO: 10 are a phosphofructokinase of a plant species such as Fumaria officinalis, Arabidopsis thalia, or of a fungal species such as Rhizopus species, Rhizopus oryzae, Rhizopus delamares, Aspergillus species such as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus terreus, or of a Monascus species such as Monascus albidulus, Monascus argentinensis, Monascus aurantiacus, Monascus barken, Monascus bisporus, Monascus eremophilus, Monascus floridanus, Monascus fuliginosus, Monascus fumeus, Monascus kaoliang, Monascus lunisporas, Monascus mucoroides, Monascus olei, Monascus pollens, Monascus paxii, Monascus pilosus, Monascus pubigerus, Monascus purpureus, Monascus ruber, Monascus rubropunctatus, Monascus rutilus, Monascus sanguineus, Monascus serorubescens, Monascus vitreus, Monascus ruber, or of a Pseudomonas species such as Alcaligenes faecalis , Pseudomonas fluorescens, Pseudomonas alcaligenes XD-1 (S. I. Ichikawa et al) or of a Bacillus species such as Bacillus stearothermophilus, Bacillus brevis, Bacillus sp. MI- 105 (Roa Engel et al).

(b) Pyruvate carboxylase

A polypeptide with pyruvate carboxylase activity is part of this disclosure. A polypeptide with pyruvate carboxylase activity (EC 6.4.1.1) is herein defined as an enzyme that catalyzes the irreversible carboxylation of pyruvate into oxaloacetate. A preferred pyruvate carboxylase polypeptide has been described in Bloom & Johnsonl962. One pyruvate carboxylase unit (U) is defined as the production of 1 μπιοΐ of oxaloacetate per minute under standard reaction conditions. An assay for quantifying pyruvate carboxylase activity is described in Osmani et al.

Polypeptides used in the invention with pyruvate carboxylase activity may be further defined by their amino acid sequence as herein described below. A preferred pyruvate carboxylase polypeptide comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 6 or 8. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100% identity with SEQ ID NO:6 or 8. The length of a preferred pyruvate carboxylase polypeptide as defined above may be at least 1100, 1150, 190, 1196, 1200 amino acids, preferably 1196 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred pyruvate carboxylase polypeptide may be at least 3300, 3400, 3500, 3588, 3600 nucleotides. Likewise pyruvate carboxylase may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a pyruvate carboxylase as herein described below.

Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with pyruvate carboxylase activity is also part of this disclosure. A nucleotide sequence encoding a polypeptide with pyruvate carboxylase activity preferably is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 6 or 8;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO. 5 or 7;

The length of said nucleotide sequence is preferably as defined above.

A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptides having pyruvate carboxylase activity are represented by SEQ ID NO:6 which is a pyruvate carboxylase of Aspergillus niger or SEQ ID NO: 8 which is a pyruvate carboxylase polypeptide modified from SEQ ID NO:6 and optimised for higher expression in Aspergillus niger. Examples of amino acid sequences that have at least 40% sequence identity with the amino acid sequence of SEQ ID NO 6 or 8 are a pyruvate carboxylase of a plant species such as Fumaria officinalis, Arabidopsis thalia, or of a fungal species such as Rhizopus species, Rhizopus oryzae, Rhizopus delamares, Aspergillus species such as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus terreus, or of a Monascus species such as Monascus albidulus, Monascus argentinensis, Monascus aurantiacus, Monascus barken, Monascus bisporus, Monascus eremophilus, Monascus floridanus, Monascus fuliginosus, Monascus fumeus, Monascus kaoliang, Monascus lunisporas, Monascus mucoroides, Monascus olei, Monascus pollens, Monascus paxii, Monascus pilosus, Monascus pubigerus, Monascus purpureus, Monascus ruber, Monascus rubropunctatus, Monascus rutilus, Monascus sanguineus, Monascus serorubescens, Monascus vitreus, Monascus ruber, or of a Pseudomonas species such as Alcaligenes faecalis , Pseudomonas fluorescens, Pseudomonas alcaligenes XD-1 (S. I. Ichikawa et al) or of a Bacillus species such as Bacillus stear other mophilus, Bacillus brevis, Bacillus sp. MI- 105 (Roa Engel et al).

(c) Fumarase A polypeptide with fumarase activity is part of this disclosure. A polypeptide with fumarase activity (EC 4.2.1.2) is herein defined as an enzyme that catalyzes the reversible hydratation of fumarate to malate. A preferred fumarase polypeptide has been described in De Jongh & Nielsen. One fumarase unit (U) is defined as the production of 1 μπιοΐ of fumarate or L-malate per minute under standard reaction conditions. An assay for quantifying fumarase activity is described in Song et al.

Polypeptides used in the invention with fumarase activity may be further defined by their amino acid sequence as herein described below. A preferred fumarase polypeptide comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 2 or 4. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100%) identity with SEQ ID NO:2 or 4. The length of a preferred fumarase polypeptide as defined above may be at least 450, 460, 470, 479, 480, 490, 500 amino acids, preferably 479 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred fumarase polypeptide may be at least 1350, 1400, 1437, 1450, 1500 nucleotides.

Likewise fumarase may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a fumarase as herein described below.

Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with fumarase activity is also part of this disclosure. A nucleotide sequence encoding a polypeptide with fumarase activity preferably is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,

90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 2 or 4;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO. 1 or 3;

iii) a nucleotide sequence the complementary strand of which hybridises to a nucleotide sequence of ii); and, iiii) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code. The length of said nucleotide sequence is preferably as defined above.

A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptides having fumarase activity are represented by SEQ ID NO:2 which is a fumarase of Rhizopus oryzae or SEQ ID NO:4 which is a fumarase polypeptide modified from SEQ ID NO:2 and optimised for higher expression in Aspergillus niger. Examples of amino acid sequences that have at least 40% sequence identity with the amino acid sequence of SEQ ID NO 2 or 4 are fumarase of a plant species such as Fumaria officinalis, Arabidopsis thalia, or of a fungal species such as Rhizopus species, Rhizopus oryzae, Rhizopus delamares, Aspergillus species such as Aspergillus niger, Aspergillus terreus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus clavatus, or of a Monascus species such as Monascus albidulus, Monascus argentinensis, Monascus aurantiacus, Monascus barken, Monascus bisporus,

Monascus eremophilus, Monascus floridanus, Monascus fuliginosus, Monascus fumeus, Monascus kaoliang, Monascus lunisporas, Monascus mucoroides, Monascus olei, Monascus pollens, Monascus paxii, Monascus pilosus, Monascus pubigerus, Monascus purpureus, Monascus ruber, Monascus rubropunctatus, Monascus rutilus, Monascus sanguineus, Monascus serorubescens, Monascus vitreus, Monascus ruber, or of a Pseudomonas species such as Alcaligenes faecalis , Pseudomonas fluorescens, Pseudomonas alcaligenes XD-1 (S. I. Ichikawa et al) or of a Bacillus species such as Bacillus stearothermophilus, Bacillus brevis, Bacillus sp. MI- 105 (Roa Engel et al).

(d) Malate dehydrogenase

A polypeptide with malate dehydrogenase activity is part of this disclosure. A polypeptide with malate dehydrogenase activity (EC 1.1.1.37) is herein defined as an enzyme that reversible catalyzes the oxidation of malate to oxaloacetate. A preferred malate dehydrogenase polypeptide has been described in Xu Guoqiang et al. One malate dehydrogenase unit (U) is defined as the production of 1 μπιοΐ of malate or oxaloacetate per minute under standard reaction conditions. An assay for quantifying malate dehydrogenase activity is described in Osmani et al.

Polypeptides of the invention with malate dehydrogenase activity may be further defined by their amino acid sequence as herein described below. A preferred malate dehydrogenase polypeptide comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 12 or 14. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100% identity with SEQ ID NO: 12 or 14. The length of a preferred malate dehydrogenase polypeptide as defined above may be at least 300, 310, 330, 338, 350, 360 amino acids, preferably 338 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred malate dehydrogenase polypeptide may be at least 900, 1014, 1080 nucleotides. Likewise malate dehydrogenase may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a malate dehydrogenase as herein described below.

Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with malate dehydrogenase activity is also part of this disclosure. A nucleotide sequence encoding a polypeptide with malate dehydrogenase activity preferably is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 12 or 14;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO. 11 or 13;

The length of said nucleotide sequence is preferably as defined above.

A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptides having malate dehydrogenase activity are represented by SEQ ID NO: 12 which is a malate dehydrogenase oiRhizopus oryzae or SEQ ID NO: 14 which is a malate dehydrogenase optimised for expression in Aspergillus niger. Examples of amino acid sequences that have at least 40% sequence identity with the amino acid sequence of SEQ ID NO 12 or 14 are a malate dehydrogenase of a plant species such as Fumaria officinalis, Arabidopsis thalia, or of a fungal species such as Rhizopus species, Rhizopus oryzae, Rhizopus delamares, Aspergillus species such as, Aspergillus niger, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus terreus, or of a Monascus species such as Monascus albidulus, Monascus argentinensis, Monascus aurantiacus, Monascus barken, Monascus bisporus, Monascus eremophilus, Monascus floridanus, Monascus fuliginosus, Monascus fumeus, Monascus kaoliang, Monascus lunisporas, Monascus mucoroides, Monascus olei, Monascus pollens, Monascus paxii, Monascus pilosus, Monascus pubigerus, Monascus purpureus, Monascus ruber, Monascus rubropunctatus, Monascus rutilus, Monascus sanguineus, Monascus serorubescens, Monascus vitreus, Monascus ruber, or of a Pseudomonas species such as Alcaligenes faecalis , Pseudomonas fluorescens, Pseudomonas alcaligenes XD-1 (S. I. Ichikawa et al) or of a Bacillus species such as Bacillus stearothermophilus, Bacillus brevis, Bacillus sp. MI- 105 (Roa Engel et al).

(e) c/ -aconitate decarboxylase

A polypeptide with cz^'s-aconitate decarboxylase activity is part of this disclosure. A polypeptide with cz^'s-aconitate decarboxylase activity (EC 4.1.1.6.) is herein defined as an enzyme that catalyses the decarboxylation of cis-aconitate to itaconate and C0₂ and vice versa, czs-aconitate decarboxylase (CAD) is also known as cz^'s-aconitic decarboxylase, czs-aconitate carboxy-lyase or cz^'s-aconitate carboxy-lyase (itaconate- forming). CAD enzyme activity determination is essentially performed as described by Bentley and Thiessen and Dwiarti et al. or in the experimental part of the application. A preferred czs-aconitate decarboxylase polypeptide has been described in Dwiarti et al. One czs-aconitate decarboxylase unit (U) is defined as the production of 1 μπιοΐ of itaconate per minute under standard reaction conditions.

Polypeptides of the invention with cz^'s-aconitate decarboxylase activity may be further defined by their amino acid sequence as herein described below. A preferred cz^'s-aconitate decarboxylase polypeptide comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 16 or 33. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100% identity with SEQ ID NO: 16, or 33. The length of a preferred czs-aconitate decarboxylase polypeptide as defined above may be at least 400, 430, 450, 470, 490, 492, 500, 520 amino acids, preferably 492 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred czs-aconitate decarboxylase polypeptide may be at least 1200, 1300, 1400, 1476, 1500, 1560 nucleotides.

Likewise czs-aconitate decarboxylase may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a czs-aconitate decarboxylase as herein described below. Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide with cz^'s-aconitate decarboxylase activity is also part of this disclosure. A nucleotide sequence encoding a polypeptide with cz^'s-aconitate decarboxylase activity preferably is selected from the group consisting of:

90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 16 or 33;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO.15 or 32;

The length of said nucleotide sequence is preferably as defined above.

A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptide having cz^'s-aconitate decarboxylase activity are represented by SEQ ID NO: 16 which is a czs-aconitate decarboxylase of Aspergillus terreus.

(f) Mitochondrial transporter (MtaA) A polypeptide being a mitochondrial transporter is part of this disclosure. A preferred polypeptide being a mitochondrial transporter has been described in Li An et al.

Polypeptides of the invention being mitochondrial transporter may be further defined by their amino acid sequence as herein described below. A preferred mitochondrial transporter comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 18 or 35. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100% identity with SEQ ID NO: 18 or 35. The length of a preferred mitochondrial transporter polypeptide as defined above may be at least 250, 270, 290, 300, 303, 320 amino acids, preferably 303 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred mitochondrial transporter polypeptide may be at least 750, 830, 860, 900, 909, 960 nucleotides. Examples of such preferred homologues having at least 40% identity with SEQ ID NO: 18 or 35 are identified as SEQ ID NO: 38-46.

Likewise a mitochondrial transporter may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a mitochondrial transporter as herein described below. Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide being a mitochondrial transporter is also part of this disclosure. A nucleotide sequence encoding a polypeptide being a mitochondrial transporter preferably is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 18 or 35;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO.17 or 34;

iiii) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code. The length of said nucleotide sequence is preferably as defined above.

A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptide being a mitochondrial transporter is represented by SEQ ID NO: 18 which is a mitochondrial transporter of Aspergillus terreus.

(g) plasma membrane transporter (PmtA)

A polypeptide being a plasma membrane transporter has been described in Li An et al.

Polypeptides of the invention being plasma membrane transporter may be further defined by their amino acid sequence as herein described below. A preferred plasma membrane transporter comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 20 or 37. However, said polypeptide may have at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99%, 100% identity with SEQ ID NO:20 or 37. The length of a preferred plasma membrane transporter polypeptide as defined above may be at least 400, 420, 450, 470, 480, 489, 500 amino acids, preferably 489 amino acids. Accordingly, the length of a preferred nucleotide sequence encoding said preferred plasma membrane transporter polypeptide may be at least 1200, 1300, 1400, 1467, 1500 nucleotides. Examples of such preferred homologues having at least 40% identity with SEQ ID NO: 20 or 37 are identified as SEQ ID NO: 51- 58.

Likewise a plasma membrane transporter may be defined by the nucleotide sequences encoding the polypeptide as well as by nucleotide sequences hybridising to a reference nucleotide sequence encoding a plasma membrane transporter as herein described below.

Therefore a nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide being a plasma membrane transporter is also part of this disclosure. A nucleotide sequence encoding a polypeptide being a plasma membrane transporter preferably is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 100% sequence identity with the amino acid sequence of SEQ ID NO 20 or 37; ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%,99% sequence identity with SEQ ID NO.19 or 36;

The length of said nucleotide sequence is preferably as defined above. A nucleic acid molecule used in the invention preferably is an isolated nucleic acid molecule. Examples of polypeptide being a plasma membrane transporter is represented by SEQ ID NO:20. Examples of amino acid sequences that have at least 40% sequence identity with the amino acid sequence of SEQ ID NO 20 are given in Li An et al.

In coming paragraph, we explained how each of the defined polypeptides and/or corresponding encoding nucleotide sequences (as identified in a), b), c), d), e), f) and/or g)) could be introduced into a cell of the invention.

Each of the nucleotide sequences as identified in a), b), c), d), e), f) and/or g) may be functionally expressed in suitable host cells (see below). Each of these nucleotide sequences preferably encode a polypeptide that naturally occurs in certain fungi and bacteria. A preferred nucleotide sequence as disclosed herein thus encodes a polypeptide with an amino acid sequence that is identical to that of a polypeptide that is obtainable from (or naturally occurs in) Basidiomycota or Ascomycota (formerly referred to as "Basidiomycetes" or "Ascomycetes" resp.). More preferably, the nucleotide sequence encodes a polypeptide that is obtainable from (or naturally occurs in) a fungus that belongs to a genus selected from Aspergillus, Gibberella (Fusarium), Monascus, Pichia, Ustilago, Candida and Rhodotorula. Most preferred are nucleotide sequences from Aspergillus terreus, Aspergillus itaconicus, Aspergillus oryzae, Aspergillus niger, Monascus ruber, Ustilago zeae, Ustilago maydis, Rhodotorula rubra or a Candida species. Alternatively, the nucleotide sequences used in the invention preferably encode a polypeptide with an amino acid sequence that is identical to that of a polypeptide that is obtainable from (or naturally occurs in) a bacterium that belongs to the genera of Pseudozyma antarctica NRRL Y-7808. It is however understood that nucleotide sequences encoding engineered forms of the fungal and bacterial polypeptides defined above in a), b), c), d), e), f) and/or g) and that comprise one or more amino acid substitutions, insertions and/or deletions as compared to the corresponding naturally occurring fungal and bacterial polypeptides but that are within the ranges of identity or similarity as defined herein are expressly included in the invention. Nucleotide sequences encoding polypeptides may e.g. be engineered in such way that the expressed protein is less susceptible to proteolytic degradation, has an improved oxygen stability or has an altered pH optimum, e.g. to a lower pH.

Each of the nucleotide sequences used in the invention (as defined in a), b), c), d), e), f) and/or g)), encoding polypeptides as herein defined, are obtainable from genomic and/or cDNA of a fungus, yeast or bacterium that belongs to a phylum, class or genus as described above, using method for isolation of nucleotide sequences that are well known in the art per se (see e.g. Sambrook and Russell (2001)). The nucleotide sequences used in the invention are e.g. obtainable in a process wherein:

A) degenerate PCR primers are used on genomic and/or cDNA of a suitable fungus, yeast or bacterium (as indicated above) to generate a PCR fragment comprising part of a nucleotide sequence encoding the polypeptides as earlier defined herein in a), b), c), d), e), f) and/or g);

B) the PCR fragment obtained in A) is used as probe to screen a cDNA and/or genomic library of the fungus, yeast or bacterium; and

C) producing a cDNA or genomic DNA comprising the nucleotide sequence encoding a polypeptide as earlier defined herein in a), b), c), d), e), f) and/or g).

Preferred fungal strains for source of cDNA or genomic DNA in a process for obtaining a nucleotide sequence used in the invention are e.g. A. terreus NRRL 1960, A. terreus NIH 2624 and A. terreus ATCC 20542.

To increase the likelihood that a polypeptide for used in the invention and as earlier defined herein in a), b), c), d), e), f) and/or g) is expressed at sufficient levels and in active form in the transformed host cells of the invention, the nucleotide sequence encoding these enzymes, are preferably adapted to optimise their codon usage to that of the host cell in question. The adaptiveness of a nucleotide sequence encoding an enzyme to the codon usage of a host cell may be expressed as codon adaptation index (CAI). The codon adaptation index is herein defined as a measurement of the relative adaptiveness of the codon usage of a gene towards the codon usage of highly expressed genes in a particular host cell or organism. The relative adaptiveness (w) of each codon is the ratio of the usage of each codon, to that of the most abundant codon for the same amino acid. The CAI index is defined as the geometric mean of these relative adaptiveness values. Non-synonymous codons and termination codons (dependent on genetic code) are excluded. CAI values range from 0 to 1, with higher values indicating a higher proportion of the most abundant codons (see Sharp and Li , 1987; also see: Jansen et al, 2003). An adapted nucleotide sequence preferably has a CAI of at least 0.2, 0.3, 0.4, 0.5, 0.6 or 0.7. Most preferred is the sequences as listed in SEQ ID NO 3, 7, 13, 32, 34, 36 which have been codon optimised for expression in niger cells. For expression in plants the sequences listed in SEQ ID NO's: are more preferred, which have been codon optimised for expression, in particular for expression in potato and sugarbeet. SEQ ID NO's: are most preferred for expression in plants because these sequences have been designed to have a higher GC content than SEQ ID NO: to avoid deletion/truncation of the sequence during cloning. In one embodiment the invention therefore relates to codon optimised coding sequence having a GC content higher than that of SEQ ID NO: or higher than 25, 30, 35, 40 or 45%. For changing GC content of a coding sequence while maintaining a CAI for a plant host cell that is higher than the wild type coding sequnece, preferably RSCU (Relative Synonymous Codon Usage) values present in plant genes found to have high transcript levels are used as described by Wang and Roossinck.

Further example of methods adaptation of codon usage in a coding nucleotide sequence are described in WO 2006/077258 and WO2008/000632.

Nucleotide sequence encoding polypeptides used in the invention and earlier defined herein as a), b), c), d), e), f) and/or g) may also be optimised for mRNA instability, mRNA secondary structure, self homology, RNAi effects. The disclosure provides nucleic acid constructs and several regulatory nucleic acid molecules (i.e. promoter, selectable marker (gene), leader sequence, terminator sequence, enhancer, integration factors, reporter gene, intron sequence, centromers, telomers, matrix attachment sequence ...) that could be used in such nucleic acid constructs. Since a cell of the invention has been engineered to be able to express a nucleotide sequence encoding a polypeptide as defined herein (i.e. a), b), c), d), e), f) and/or g)), it is understood that each of these nucleic acid constructs may comprise identical or distinct regulatory nucleic acid molecules as identified herein.

The disclosure provides a nucleic acid construct comprising a nucleotide sequence encoding a polypeptide as herein defined in a), b), c), d), e), f) and/or g), wherein the nucleotide sequence is operably linked to a promoter. Preferably, the promoter may be derived from a gene, which is highly expressed (defined herein as the mRNA concentration with at least 0.5% (w/w) of the total cellular mRNA). In another preferred embodiment, the promoter may be derived from a gene, which is medium expressed (defined herein as the mRNA concentration with at least 0.01% until 0.5% (w/w) of the total cellular mRNA). In a further preferred embodiment, the promoter may be a promoter that is insensitive to catabolite (glucose) repression. More preferably, micro array data is used to select genes, and thus promoters of those genes, that have a certain transcriptional level and regulation. In this way one can optimally adapt the gene expression cassettes to the conditions under which it should function. These promoter fragments can be derived from many sources, i.e. different species, PCR amplified, synthetically and the like.

In the nucleic acid construct used in the invention the promoter preferably is a promoter that regulates transcription in a fungal cell or a plant cell. The nucleic acid construct used in the invention is thus preferably an expression vector for a plant cell or a fungal cell.

Cell

A first cell

In a first aspect therefore, the present invention relates to a cell capable of expressing at least one of the following nucleic acid molecules: b) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with pyruvate carboxylase activity, wherein the nucleotide sequence is selected from the group consisting of: i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 6 or 8;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 5 or 7;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code and a) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with phosphofructokinase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ

ID NO 10;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 9;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code.

This cell capable of expressing b) and/or a) is called a first cell of the invention. Said first cell is attractive for producing citric acid.

Optionally this first cell is also capable of expressing the two following nucleic acid molecules:

c) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with fumarase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 2 or 4; ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 1 or 3;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code and d) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with malate dehydrogenase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 12 or 14;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 11 or 13;

This cell capable of expressing b) and/or a) and optionally c) and d) is preferred a first cell of the invention and is also attractive for producing citric acid.

Surprisingly, a first cell (i.e. capable of expressing b) and/or a) and optionally c) and d)) is also preferred when it is further also capable of expressing g) as defined below: g) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide which is a plasma membrane transporter, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 20 or 37;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 19 or 36; iii) a nucleotide sequence the complementary strand of which hybridises to a nucleotide sequence of ii); and,

An even more preferred first preferred cell is capable of expressing the following nucleotide sequence(s):

b),

a) ,

a) and b) and c) and d),

b) and a),

c) and b) and d),

a) and c) and d),

b) and g)

a) and g)

a) and b) and c) and d) and g)

b) and a) and g)

c) and b) and d) and g)

a) and c) and d) and g).

A second cell

In another embodiment, a cell capable of expressing a nucleotide sequence as defined in a), b), c) and/or d), is also disclosed herein. Such a cell is further capable of expressing the following nucleic acid molecules:

e) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with cis-aconitate decarboxylase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 16, or 33;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 15 or 32; iii) a nucleotide sequence the complementary strand of which hybridises to a nucleotide sequence of ii); and,

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code; f) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide which is a mitochondrial transporter, wherein the nucleotide sequence is selected from the group consisting of:

ID NO 18 or 35;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 17 or 34;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code; and g) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide which is a plasma membrane transporter, wherein the nucleotide sequence is selected from the group consisting of:

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 19 or 36;

As earlier explained herein, this cell or second cell is attractive for producing itaconic acid. This cell is also named herein a cell capable of expressing a nucleotide sequence as defined in a), b), c) and/or d) and all of e), f and g) or a second cell. Below several preferred second cells are provided.

A second preferred cell is capable of expressing a nucleotide sequence as defined in: c), b) and/or d) and all of e), f) and g). In an embodiment, such a second cell is optionally capable of expressing a nucleotide sequence as defined in a).

Another second preferred cell is capable of expressing a nucleotide sequence as defined in: b) and all of e), f) and g). In an embodiment, such a second cell is optionally capable of expressing a nucleotide sequence as defined in a) and c) and d).

Another second preferred cell is capable of expressing a nucleotide sequence as defined in: a) and all of e), f) and g). In an embodiment, such a second cell is optionally capable of expressing a nucleotide sequence as defined in c) and b) and d).

Another second preferred cell is capable of expressing a nucleotide sequence as defined in: a) and b) and c) and d) and all of e), f) and g).

Another second preferred cell is capable of expressing a nucleotide sequence as defined in: b) and a) and all of e), f) and g). In an embodiment, such a second cell is optionally capable of expressing a nucleotide sequence as defined in c) and d).

Another second preferred cell is capable of expressing a nucleotide sequence as defined in: c) and b) and d) and all of e), f) and g). In an embodiment, such a second cell is optionally capable of expressing a nucleotide sequence as defined in a).

Another second preferred cell is capable of expressing a nucleotide sequence as defined in: a) and c) and d) and all of e), f) and g). In an embodiment, such a second cell is optionally capable of expressing a nucleotide sequence as defined in b).

A cell of the invention has been preferably transformed with a nucleic acid molecule or construct comprising a nucleotide sequence encoding a polypeptide as herein defined above (i.e. b) and/or a) and optionally c) and d) for producing citric acid, or a), b), c) and/or d) and all of e), f) and g) for producing itaconic acid). The transformed first or second cell (or host cell) may be any cell that produces citric acid and that comprises aconitate dehydratase (E.C. 4.2.1.3). The recipient cell for the nucleic acid molecule or construct comprising a nucleotide sequence encoding a polypeptide as herein defined above may be a bacterial, fungal or plant cell.

Preferred fungal cells for transformation with the nucleic acid molecules or constructs used in the invention include fungal cells of a genus selected from Aspergillus, Monascus, Penicillium, Candida and Yarrowia. More preferably, the fungal cell is of a species selected from Aspergillus niger, Aspergillus terreus, Aspergillus itaconicus, Monascus ruber, Penicillium simplicissimum, Penicillium expansum, Penicillium digitaium, Penicillium italicum, Candida oleophila and Yarrowia lipolytica. Preferred strains are Aspergillus niger CBS 120.49 and derived strains lacking oxaloacetate hydrolase (EC 3.7.1.1) and glucose oxidase (EC 1.1.3.4) activity like NW 185 and Candida oleophila ATCC 20177.

Preferred cells for transformation with the nucleic acid molecules or constructs used in the invention are cells of an (micro)organisms (in particular filamentous fungi such as Aspergillus) that are able to produce citric acid and/or itaconic acid at high yield and high rate from a suitable source of carbohydrate like e.g. glucose, fructose, sucrose, molasses, cassava, starch or corn. Measurement of citric acid is done by simple acid-base titration with NaOH keeping in mind that all acids are measured in this way. To measure citric acid in the presence of other acids, HPLC is used (e.g. with lonPac AS-1 1 anion exchange column of Dionex, as described in their publicly available application note No. 123 of Dec. 1998 "The determination of inorganic anions and organic acids in fermentation broths", Dionex Corp., Sunnyvale, California). When measured for instance by HPLC or titration, preferred (micro)organisms for transformation with the nucleic acid molecules or constructs of the invention are able to produce citric acid from sucrose at a level of at least 10, 20, 50, 100, or 200 g/1 respectively. Modified microorganisms capable of producing citric acid in even higher quantities of at least 300 g/1 when produced by submerged fermentation starting from sucrose are disclosed in WO2007/063133, and these may also suitably be used as recipient cells for transformation with the nucleic acid constructs of the invention for the production of itaconic acid.

Measurement of itaconic acid is done by simple acid-base titration with NaOH keeping in mind that all acids are measured in this way. To measure itaconic acid in the presence of other acids, HPLC is used (e.g. with lonPac AS-1 1 anion exchange column of Dionex, as described above). When measured for instance by HPLC or titration, preferred (micro)organisms for transformation with the nucleic acid molecules or constructs of the invention are able to produce itaconic acid from sucrose at a level of at least 0.5, 1, 5, 10 g/1 respectively.

Nucleic acid constructs for expression of coding nucleotide sequences in fungi are well known in the art. In such constructs the nucleotide sequence encoding a polypeptide as earlier defined herein (i.e. b) and/or a) and optionally c) and d)) is preferably operably linked to a promoter that causes sufficient expression of the nucleotide sequences in the cell (i.e. first cell) to confer to the cell the ability to produce citric acid.

In such constructs the nucleotide sequence encoding a polypeptide as earlier defined herein (i.e. a), b), c) and/or d) and all of e), f) and g)) is preferably operably linked to a promoter that causes sufficient expression of the nucleotide sequences in the cell (i.e. second cell) to confer to the cell the ability to convert cis-aconitate to itaconate and C0₂.

Suitable promoters for expression of the nucleotide sequence as defined above include promoters that are insensitive to catabolite (glucose) repression and/or that do require induction. Promoters having these characteristics are widely available and known to the skilled person. Suitable examples of such promoters include e.g. promoters from glycolytic genes such as the phosphofructokinase, triose phosphate isomerase, glyceraldehyde-3 -phosphate dehydrogenase, pyruvate kinase, phosphogly cerate kinase, glucose-6-phosphate isomerase from yeasts or filamentous fungi. Other useful promoters are ribosomal protein encoding gene promoters, alcohol dehydrogenase promoters, the enolase promoter, the cytochrome cl promoter, promoters from genes encoding amylo- or cellulolytic enzymes (glucoamylase, TAKA- amylase and cellobiohydrolase), promoters from a beta-xylosidase gene. A preferred promoter is a sugar-inducible promoter. Such sugar-inducible promoter may be from plants. In this context, a sugar may be a cell wall hydrolysate, xylose, arabinose, galactose. A preferred promoter is a XlnR-dependent promoter (Van Peij N.N. et al). More preferably such XlnR promoter is used in a cell as defined herein that comprises additional copies of the XlnR gene or that over-express said gene. Even more preferably such cell has a dysfunctional creA gene. Other promoters, both constitutive and inducible and enhancers or upstream activating sequences will be known to those of skill in the art. The promoters used in the nucleic acid constructs of the present invention may be modified, if desired, to affect their control characteristics. Preferably, the promoter used in the nucleic acid construct for expression of a polypeptide as identified herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) is homologous to the host cell in which said polypeptide is expressed. In the nucleic acid construct used in the invention for fungal expression, the 3'- end of the nucleotide acid sequence encoding the polypeptide as defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) preferably is operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice. In any case the choice of the terminator is not critical; it may e.g. be from any fungal gene. Preferred terminators for filamentous fungal cells are obtained from the genes encoding A. oryzae TAKA amylase, the Penicillium chrysogenum pcbAB, pcbC and penDE terminators A. niger glucoamylase (glaA), A. nidulans anthranilate synthase, A. niger beta-xylosidase, A. niger alpha-glucosidase, Aspergillus nidulans trpC gene and Fusarium oxysporum trypsin-like protease.

In the nucleic acid construct used in the invention for fungal expression may further comprise a suitable leader sequence, a non-translated region of an mRNA that is important for translation by the cell. The leader sequence is operably linked to the 5'- terminus of the nucleic acid sequence encoding a polypeptide as defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)). Any leader sequence, which is functional in the cell, may be used in the present invention. Preferred leaders for filamentous fungal cells are obtained from the genes encoding A. niger beta-xylosidase, Aspergillus oryzae TAKA amylase and Aspergillus nidulans triose phosphate isomerase and Aspergillus niger glaA.

Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term "marker" refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. The marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Examples of suitable antibiotic resistance markers include e.g. dihydrofolate reductase, hygromycin-B-phosphotransf erase, 3'-0-phosphotransferase II (kanamycin, neomycin and G418 resistance). Although the use of antibiotic resistance markers may be most convenient for the transformation of polyploid host cells, preferably however, non- antibiotic resistance markers are used, such as auxotrophic markers (URA3, TRP1, LEU2) or the S. pombe TPI gene (Russell P R, 1985). Alternatively, a screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, or beta-glucuronidase may be incorporated into the nucleic acid constructs of the invention allowing screening for transformed cells.

A variety of selectable marker genes are available for use in the transformation of fungi. Suitable markers include auxotrophic marker genes involved in amino acid or nucleotide metabolism, such as e.g. genes encoding ornithine-transcarbamylases (argB), orotidine-5'-decaboxylases (pyrG, URA3) or glutamine-amido-transferase indoleglycerol-phosphate-synthase phosphoribosyl-anthranilate isomerases (trpC), or involved in carbon or nitrogen metabolism, such as e.g. nitrate reductase (niaD) or facA, and antibiotic resistance markers such as genes providing resistance against phleomycin, bleomycin or neomycin (G418). Preferably, bidirectional selection markers are used for which both a positive and a negative genetic selection is possible. Examples of such bidirectional markers are the pyrG (URA3), facA and amdS genes. Due to their bidirectionality these markers can be deleted from transformed filamentous fungus while leaving the introduced recombinant DNA molecule in place, in order to obtain fungi that do not contain selectable markers, as is disclosed in EP-A-0 635 574, which is herein incorporated by reference. Of these selectable markers the use of dominant and bidirectional selectable markers such as acetamidase genes like the amdS genes of A. nidulans, A. niger and P. chrysogenum is most preferred, the amdS genes of A. niger and P. chrysogenum are disclosed in US6,548,285. In addition to their bidirectionality these markers provide the advantage that they are dominant selectable markers that, the use of which does not require mutant (auxotrophic) strains, but which can be used directly in wild type strains.

Optional further elements that may be present in the nucleic acid constructs used in the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences. The nucleic acid constructs used in the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKDl (Fleer et al, 1991) plasmids. An autonomously maintained nucleic acid construct suitable for filamentous fungi may comprise the AMAl-sequence ( Aleksenko and Clutterbuck (1997)). Alternatively the nucleic acid construct may comprise sequences for integration, preferably by homologous recombination (see e.g. W098/46772), or gene replacement (see e.g. EPO 357 127). Such sequences may thus be sequences homologous to the target site for integration in the host cell's genome. In order to promote targeted integration, the cloning vector is preferably linearised prior to transformation of the host cell. Linearization is preferably performed such that at least one but preferably either end of the cloning vector is flanked by sequences homologous to the target locus. The length of the homologous sequences flanking the target locus is preferably at least 30bp, preferably at least 50 bp, preferably at least O. lkb, even preferably at least 0.2kb, more preferably at least 0.5 kb, even more preferably at least 1 kb, most preferably at least 2 kb. Preferably, the efficiency of targeted integration into the genome of the host cell, i.e. integration in a predetermined target locus, is increased by augmented homologous recombination abilities of the host cell. Such phenotype of the cell preferably involves a deficient ku70 gene as described in WO2005/095624. WO2005/095624 discloses a preferred method to obtain a filamentous fungal cell comprising increased efficiency of targeted integration. Preferably, the DNA sequence in the cloning vector, which is homologous to the target locus is derived from a highly expressed locus meaning that it is derived from a gene, which is capable of high expression level in the filamentous fungal host cell. A gene capable of high expression level, i.e. a highly expressed gene, is herein defined as a gene whose mRNA can make up at least 0.5% (w/w) of the total cellular mRNA, e.g. under induced conditions, or alternatively, a gene whose gene product can make up at least 1% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.1 g/1 (as described in EP 357 127 Bl). A number of preferred highly expressed fungal genes are given by way of example: the amylase, glucoamylase, alcohol dehydrogenase, xylanase, glyceraldehyde-phosphate dehydrogenase or cellobiohydrolase (cbh) genes from Aspergilli or Trichoderma. Most preferred highly expressed genes for these purposes are a glucoamylase gene, preferably an A. niger glucoamylase gene, an A. oryzae TAKA-amylase gene, an A. nidulans gpdA gene, a Trichoderma reesei cbh gene, preferably cbhX .

More than one copy of a nucleic acid sequence encoding a polypeptide as defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) may be inserted into the host cell to increase production of the gene product. This can be done, preferably by integrating into its genome copies of the DNA sequence, more preferably by targeting the integration of the DNA sequence at one of the highly expressed locus defined in the former paragraph. Alternatively, this can be done by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent. To increase the copy number of the integrated nucleic acid constructs used in the invention even more, the technique of gene conversion as described in W098/46772 may be used.

The nucleic acid constructs used in the invention can be provided in a manner known per se, which generally involves techniques such as restricting, linking, amplifying, and the like nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and Russel (2001) or F. Ausubel et al, (1987). Transformation methods for filamentous fungi, such as Aspergilli, are well- known to the skilled person (Biotechnology of Filamentous fungi, (1992)). The skilled person will recognize that successful transformation of fungi is not limited to the use of vectors, selection marker systems, promoters and transformation protocols specifically exemplified herein. Specific transformation protocols for niger are described in e.g. WO 99/32617 or WO 98/46772.

Another preferred recipient cell for transformation with the nucleic acid molecules or constructs used in the invention is a plant cell. Expressly included invention are thus transgenic plants, plant cells or plant tissues or organs comprising a nucleic acid molecule or construct comprising a nucleotide sequence encoding a polypeptide as defined herein above (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)).

The construction of chimeric genes and nucleic acid constructs (vectors) for, preferably stable, introduction of a nucleotide sequence encoding a polypeptide as defined earlier herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) into the genome of plant host cells is generally known in the art. To generate a chimeric gene the nucleic acid sequence encoding said polypeptide is operably linked to a promoter sequence, suitable for expression in the host cells, using standard molecular biology techniques. The promoter sequence may already be present in a vector so that the encoding nucleic sequence as earlier defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) is simply inserted into the vector downstream of the promoter sequence. The vector is then used to transform the host cells and the chimeric gene is inserted in the nuclear genome or into the plastid, mitochondrial or chloroplast genome and expressed there using a suitable promoter (e.g. Mc Bride et al, 1995; US 5,693, 507). In one embodiment a chimeric gene comprises a suitable promoter for expression in plant cells, operably linked thereto a nucleic acid sequence encoding a polypeptide as earlier defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)), optionally followed by a 3'nontranslated nucleic acid sequence.

Each of the nucleic acid sequences as earlier identified herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)), preferably each of the corresponding chimeric gene, encoding a polypeptide, can be stably inserted in a conventional manner into the nuclear genome of a single plant cell, and the so- transformed plant cell can be used in a conventional manner to produce a transformed plant that has an altered phenotype due to the presence of said polypeptide ((i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) in certain cells at a certain time. In this regard, a T-DNA vector, comprising a nucleic acid sequence encoding said polypeptide (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) in Agrobacterium tumefaciens can be used to transform the plant cell, and thereafter, a transformed plant can be regenerated from the transformed plant cell using the procedures described, for example, in EP 0 116 718, EP 0 270 822, PCT publication WO84/02913 and published European Patent application EP 0 242 246 and in Gould et al, 1991. The construction of a T-DNA vector for Agrobacterium mediated plant transformation is well known in the art. The T-DNA vector may be either a binary vector as described in EP 0 120 561 and EP 0 120 515 or a co-integrate vector which can integrate into the Agrobacterium Ti-plasmid by homologous recombination, as described in EP O 116 718. Preferred T-DNA vectors each contain a promoter operably linked to nucleic acid sequence encoding said polypeptide (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) between T-DNA border sequences, or at least located to the left of the right border sequence. Border sequences are described in Gielen et al, 1984. Of course, other types of vectors can be used to transform the plant cell, using procedures such as direct gene transfer (as described, for example in EP 0 223 247), pollen mediated transformation (as described, for example in EP 0 270 356 and WO85/01856), protoplast transformation as, for example, described in US 4,684, 611, plant RNA virus- mediated transformation (as described, for example in EP 0 067 553 and US 4,407, 956), liposome-mediated transformation (as described, for example in US 4,536, 475), and other methods such as those described methods for transforming certain lines of corn (e. g., US 6, 140, 553; Fromm et al, 1990; Gordon-Kamm et al, 1990) and rice (Shimamoto et al, 1989; Datta et al. 1990) and the method for transforming monocots generally (PCT publication WO92/09696). The most widely used transformation method for dicot species is Agrobacterium mediated transformation. For cotton transformation see also WO 00/71733. Brassica species (e.g. cabbage species, broccoli, cauliflower, rapeseed etc.) can for example be transformed as described in US5750871 and legume species as described in US 5565346. Musa species (e.g. banana) may be transformed as described in US5792935. Agrobacterium-mediated transformation of strawberry is described in Plant Science, (1990). Likewise, selection and regeneration of transformed plants from transformed cells is well known in the art. Obviously, for different species and even for different varieties or cultivars of a single species, protocols are specifically adapted for regenerating transformants at high frequency.

Besides transformation of the nuclear genome, also transformation of the plastid genome, preferably chloroplast genome, is included in the invention. One advantage of plastid genome transformation is that the risk of spread of the transgene(s) can be reduced. Plastid genome transformation can be carried out as known in the art, see e.g. Sidorov VA et al. 1999, or Lutz KA et al. 2004, US 6541682, US6515206, US6512162 or US6492578.

Each of the nucleic acid sequences earlier described herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) is inserted in a plant cell genome so that the inserted coding sequence is downstream (i.e. 3') of, and under the control of, a promoter which can direct the expression in the plant cell. This is preferably accomplished by inserting the chimeric gene in the plant cell genome, particularly in the nuclear or plastid (e. g. chloroplast) genome.

Preferred promoters include: the strong constitutive 35S promoters or (double) enhanced 35S promoters (the "35S promoters") of the cauliflower mosaic virus (CaMV) of isolates CM 1841 (Gardner et al, 1981), CabbB-S (Franck et al, 1980) and CabbB-JI (Hull and Howell, 1987); the 35S promoter described by Odell et al. (1985) or in US5164316, promoters from the ubiquitin family (e.g. the maize ubiquitin promoter of Christensen et al, 1992, EP 0 342 926, see also Cornejo et al. 1993), the gos2 promoter (de Pater et al, 1992), the emu promoter (Last et al, 1990), Arabidopsis actin promoters such as the promoter described by An et al. 1996, rice actin promoters such as the promoter described by Zhang et al.1991, and the promoter described in US 5,641,876 or the rice actin 2 promoter as described in WO070067; promoters of the Cassava vein mosaic virus (WO 97/48819, Verdaguer et al. 1998), the pPLEX series of promoters from Subterranean Clover Stunt Virus (WO 96/06932, particularly the S7 promoter), a alcohol dehydrogenase promoter, e.g., pAdhl S (GenBank accession numbers X04049, X00581), and the TR1' promoter and the TR2' promoter (the "TR1 'promoter" and "TR2'promoter", respectively) which drive the expression of the Γ and 2' genes, respectively, of the T-DNA (Velten et al, 1984), the Figwort Mosaic Virus promoter described in US6051753 and in EP426641, histone gene promoters, such as the Ph4a748 promoter from Arabidopsis (PMB 8: 179-191), or others.

Alternatively, a promoter can be utilized which is not constitutive but rather is specific for one or more tissues or organs of the plant (tissue preferred / tissue specific, including developmentally regulated promoters), for example tap root preferred, fruit (or fruit development or ripening) preferred, leaf preferred, epidermis preferred, root preferred, flower tissue preferred, seed preferred, pod preferred, stem preferred, whereby each nucleic acid as earlier identified herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) is expressed only in cells of the specific tissue(s) or organ(s) and/or only during a certain developmental stage, for example during stem, leave or tap root development. For example, each of these nucleic acid can be selectively expressed in green tissue / aerial parts of a plant by placing the coding sequence under the control of a light-inducible promoter such as the promoter of the ribulose-1, 5-bisphosphate carboxylase small subunit gene of the plant itself or of another plant, such as pea, as disclosed in US 5,254, 799 or Arabidopsis as disclosed in US5034322. The choice of the promoter is obviously determined by the phenotype one aims to achieve, as described above.

The production of itaconic acid is particularly advantageous in plant organs able to store large amounts of water-soluble compounds, such as the tap roots of sugar beet or the stems of sugar cane, cereals or grasses, the tubers of cassava or potato, or the fruits of citrus, or the leaves of for example sugar beet, potato, grasses or tobacco. Therefore, a highly preferred promoter is a promoter, which is active in organs and cell types which normally are capable of accumulating water soluble compounds. An organ-specific promoter can for example be the tuber-specific potato proteinase inhibitor II or GBSS promoter, a tap root-specific promoter such as a sucrose synthase or a fructan:fructan fructosyltransferase promoter or any other inducible or tissue- specific promoter.

To achieve expression in seeds, a seed specific promoter, as described in EP723019, EP255378 or W09845461 can be used. For tuber specific expression (e.g. potatoes) a tuber or peel specific promoter is the most suitable such as the class II patatin promoter (Nap et al, 1992) that specifies expression in the outer layer of the tuber, or a promoter with leaf and tuber peel expression such as the potato UBI7 promoter (Garbarino et al., 1995). For root specific expression a promoter preferentially active in roots is described in WO00/29566. Another promoter for root preferential expression is the ZRP promoter (and modifications thereof) as described in US 5,633, 363.

Another alternative is to use a promoter whose expression is inducible, thus effecting induction of the expression of each of the nucleic acid as earlier described herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)), for example upon a change in temperature, wounding, microbial or insect attack, chemical treatment (e.g. substrate-inducible) etc. Examples of inducible promoters are wound-inducible promoters, such as the MPI promoter described by Cordera et al. 1994, which is induced by wounding (such as caused by insect or physical wounding), or the COMPTII promoter (WO0056897) or the promoter described in US6031151. Alternatively the promoter may be inducible by a chemical, such as dexamethasone as described by Aoyama and Chua 1997 and in US6063985 or by tetracycline (TOPFREE or TOP 10 promoter, see Gatz, 1997 and Love et al. 2000). Other inducible promoters are for example inducible by a change in temperature, such as the heat shock promoter described in US 5,447, 858, by anaerobic conditions (e.g. the maize ADH1S promoter), by light (US6455760), by pathogens (e.g. EP759085 or EP309862) or by senescence (SAG12 and SAG13, see US5689042). Obviously, there are a range of other promoters available. A podwall specific promoter from Arabidopsis is the FUL promoter (also referred to as AGL8 promoter, WO9900502; WO9900503; Liljegren et al. 2004), the Arabidopsis IND1 promoter (Lijegren et al. 2004; WO9900502; WO9900503) or the dehiscence zone specific promoter of a Brassica polygalacturonase gene (W09713856). Each of the nucleic acid sequences as earlier described herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) is inserted into the plant genome so that the coding sequence is upstream (i.e. 5') of suitable 3'end transcription regulation signals ("3'end") (i.e. transcript formation and polyadenylation signals). Polyadenylation and transcript formation signals include those of the CaMV 35S gene ("3' 35S"), the nopaline synthase gene ("3' nos") (Depicker et al), the octopine synthase gene ("3'ocs") (Gielen et al, 1984) and the T-DNA gene 7 ("3' gene 7") (Velten and Schell, 1985), which act as 3 '-untranslated DNA sequences in transformed plant cells, and others.

Introduction of the T-DNA vector into Agrobacterium can be carried out using known methods, such as electroporation or triparental mating.

Each of the nucleic acid sequences earlier defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) can optionally be inserted in the plant genome as a hybrid gene sequence whereby each of said nucleic acid sequence is linked in-frame to a (US 5,254, 799; Vaeck et al., 1987) gene encoding a selectable or scorable marker, such as for example the neo (or nptll) gene (EP 0 242 236) encoding kanamycin resistance, so that the plant expresses a fusion protein which is easily detectable.

Preferably, for selection purposes but also for weed control options, the transgenic plants of the invention are also transformed with a DNA encoding a protein conferring resistance to herbicide, such as a broad-spectrum herbicide, for example herbicides based on glufosinate ammonium as active ingredient (e.g. Liberty® or BASTA; resistance is conferred by the PAT or bar gene; see EP 0 242 236 and EP 0 242 246) or glyphosate (e.g. RoundUp®; resistance is conferred by EPSPS genes, see e.g. EPO 508 909 and EP 0 507 698). Using herbicide resistance genes (or other genes conferring a desired phenotype) as selectable marker further has the advantage that the introduction of antibiotic resistance genes can be avoided. Alternatively, other selectable marker genes may be used, such as antibiotic resistance genes. As it is generally not accepted to retain antibiotic resistance genes in the transformed host plants, these genes can be removed again following selection of the transformants. Different technologies exist for removal of transgenes. One method to achieve removal is by flanking the chimeric gene with lox sites and, following selection, crossing the transformed plant with a CRE recombinase-expressing plant (see e.g. EP506763B 1). Site specific recombination results in excision of the marker gene. Another site specific recombination systems is the FLP/FRT system described in EP686191 and US5527695. Site specific recombination systems such as CRE/LOX and FLP/FRT may also be used for gene stacking purposes. Further, one-component excision systems have been described, see e.g. WO9737012 or WO9500555).

When reference to "a transgenic plant cell" or "a recombinant plant cell" is made anywhere herein, this refers to a plant cell (or also a plant protoplast) as such in isolation or in tissue/cell culture, or to a plant cell (or protoplast) contained in a plant or in a differentiated organ or tissue, and these possibilities are specifically included herein. Hence, a reference to a plant cell in the description or claims is not meant to refer only to isolated cells in culture, but refers to any plant cell, wherever it may be located or in whatever type of plant tissue or organ it may be present. Also, parts removed from the recombinant plant, such as harvested fruit, tap roots, stems, tubers, seeds, cut flowers, pollen, etc. as well as cells derived from the recombinant cells, as well as seeds derived from traditional breeding (crossing, selfing, etc.) which retain each of the chimeric nucleic acid sequences as defined earlier herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) are specifically included.

In a preferred embodiment the production of itaconic acid is advantageously located in cell organelles containing intermediates of the Krebs cycle, such as the mitochondria, the plastids (or plastid like organelles, such as the chloroplast or leucoplast), the cytosol or the vacuole, Accordingly, each of the nucleic acid as earlier identified herein (i.e. or a), b), c) and/or d) and all of e), f) and g)) and used in the present invention, is preferably linked to a sequence encoding a transit peptide or targeting sequence which directs each of said encoded polypeptide to a subcellular compartment, such as for example said the mitochondrion, plastid, cytosol of vacuole. For this purpose each of the polypeptides as defined herein (i.e. a), b), c) and/or d) and all of e), f) and g)) may be endowed with target peptides The terms "target peptide" refers to amino acid sequences which target a protein to intracellular organelles such as vacuoles, plastids, preferably chloroplasts, mitochondria, leucoplasts or chromoplasts, the endoplasmic reticulum, or to the extracellular space (secretion signal peptide). A nucleic acid sequence encoding a target peptide may be fused (in frame) to the nucleic acid sequence encoding the amino terminal end (N-terminal end) of each of said polypeptide or may replace part of the amino terminal end of each of said polypeptide. In a further preferred embodiment, each of said polypeptides (i.e. a), b), c) and/or d) and all of e), f) and g)) is targeted to a (subcellular) compartment or organelle in the cell. This allows to create a metabolic sink which draws in the citric acid to be efficiently converted to itaconic acid.

In another preferred embodiment the cell transformed of the invention comprises one or more further genetic modifications that allow cheaper and/or more efficient production of citric acid and/or itaconic acid. Such further genetic modification may include any modification that increases the flux of carbohydrates to citric acid including e.g. modifications as described in WO2007/063133.

Another preferred further genetic modification is a modification that increases the aconitate dehydratase (E.C. 4.2.1.3) activity in the cell. An increase in aconitate dehydratase activity may e.g. be achieved by increasing the copy number of endogenous copies of the aconitate dehydratase in the cell and/or introducing additional exogenous aconitate dehydratase genes. Nucleic acid constructs for (over)expression of aconitate dehydratase genes may in principle be similar or identical to the constructs described above herein where the nucleotide sequence coding for a polypeptide as defined herein (i.e. b) and/or a) and optionally c) and d) or a), b), c) and/or d) and all of e), f) and g)) is replaced by a sequence coding for the aconitate dehydratase.

Yet another preferred further genetic modification may include modifications that allow the host cell to use pentoses such as xylose and/or arabinose as carbon- and energy source. For this purpose genes coding for xylose isomerases, xylulose kinases (as described e.g. in WO 03/062340 and WO 06/009434) and/or arabinose isomerases, a ribulokinases and ribulose-5-P-4-epimerases (as described in Wisselink et al, 2007; and in EP 1 499 708) are respectively introduced into the host cell.

Again another preferred further genetic modification may include transformation of the host cell with one or more expression constructs for (over)expression of the transporters encoded by ORF 14 and/or 16 of A. terreus ATCC 20542 (as defined by Kennedy et al, 1999, supra) or corresponding ORFs (orthologs) from other Aspergillus species or terreus strains.

Use/process In a further aspect the present invention relates to the use of a cell (i.e. first cell as identified herein) comprising a molecule or construct comprising a nucleotide sequence encoding a polypeptide as defined herein above (i.e. b) and/or a) and optionally c) and d) ), in the production of citric acid.

In a further aspect the present invention relates to a process for producing citric acid, whereby the process comprises the steps of (A) fermenting a medium comprising a source of carbon and energy with a cell as defined herein above (i.e. first cell), whereby the cell ferments the source of carbon and energy to citric acid, and optionally, (B) recovery of the citric acid.

The use of a first cell as defined herein capable of expressing at least one of a polypeptide having fumarase, pyruvate carboxylase, phosphofructokinase and malate dehydrogenase activity (i.e. as defined in b) and/or a) and optionally c) and d)) is highly attractive to be used for producing citric acid. Such a cell produces higher amount of citric acid and has a higher productivity of citric acid by comparison to the citric acid production and productivity of a corresponding cell not expressing at least one of these four polypeptides. It is expected that the increase of citric acid produced by a first cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to the citric acid produced by a cell not expressing at least one of a polypeptide as identified above, both cells being fermented in the same conditions. The production of citric acid in a process of the invention is expected to be at least 50 g/1, 70 g/1, 100 g/1, 150g/l, 200 g/1, 300 g/1, 350 g/1, 400 g/1. The presence of citric acid is assessed as earlier defined herein or as done in the experimental part. It is further expected that the increase of citric acid productivity of a first cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to the citric acid productivity of a cell not expressing at least one of a polypeptide as identified above, both cells being fermented in the same conditions.

In a further aspect the present invention relates to the use of a cell (i.e. second cell as identified herein) comprising a molecule or construct comprising a nucleotide sequence encoding a polypeptide as defined herein above (i.e. a), b), c) and/or d) and all of e), f) and g)) in the production of itaconic acid. In a further aspect the present invention relates to a process for producing itaconic acid, whereby the process comprises the steps of (A) fermenting a medium comprising a source of carbon and energy with a cell as defined herein above (i.e. second cell), whereby the cell ferments the source of carbon and energy to itaconic acid, and optionally, (B) recovery of the itaconic acid.

The use of a second cell as defined herein capable of expressing at least one of the four polypeptides of the first cell and the three following polypeptides: one having cis-aconitate decarboxylase activity, one being a mitochondrial transporter and one being a plasma membrane transporter (i.e. as defined as a), b), c) and/or d) and all of e), f) and g)) is highly attractive to be used for producing itaconic acid. Such a cell produces higher amount of itaconic acid and has a higher productivity of itaconic acid by comparison to the itaconic acid production and productivity of a corresponding cell not expressing these three polypeptides. Such a cell is also expected to produce higher amount of itaconic acid and has a higher productivity of itaconic acid by comparison to the itaconic acid production and productivity of a corresponding cell expressing none of the a), b), c), d), e), f) and g) polypeptides.

It is expected that the increase of itaconic acid produced by a first cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%), 85%), 90%), 95%), 100% or more compared to the itaconic acid produced by a cell not expressing the three polypeptides as identified above, both cells being fermented in the same conditions. The production of itaconic acid in a process of the invention is expected to be at least 1 g/1, 5 g/1, 7 g/1, lOg/1, 15 g/1, 20 g/1, 30 g/1, 40g/l, 50g/l, 60g/l, 70g/l, 80g/l, 90g/l, 100 g/1, 110 g/1, 120 g/1, 130 g/1, 140 g/1, 150 g/1, 160 g/1, 170 g/1, 180 g/1 , 190 g/1, 200 g/1, 210 g/1 ,220 g/1, 230 g/1, 240 g/1 ,250 g/1, 260 g/1. The production of itaconic acid is assessed as earlier defined herein or as done in the experimental part. It is further expected that the increase of itaconic acid productivity of a first cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% or more compared to the itaconic acid productivity of a cell not expressing the three polypeptides as identified above, both cells being fermented in the same conditions.

A preferred fermentation process is an aerobic fermentation process. An aerobic fermentation process of the invention may be run under aerobic oxygen-limited conditions. Preferably, in an aerobic process under oxygen-limited conditions, the rate of oxygen consumption is at least 5.5, more preferably at least 6 and even more preferably at least 7 mmol/L/h.

The fermentation process may either be a submerged or a solid state fermentation process. Itaconic acid may be produced via submerged fermentation starting from a carbohydrate raw material such as for instance cassava and/or corn, which may be milled and mixed with water. A seed fermentation may be prepared in a separate fermenter. The liquefaction of the starch may be performed in the presence of an amylolytic enzyme such as for instance amylases, cellulases, lactases or maltases and additives and nutrients such as antifoam may be added before or during fermentation. For the main fermentation, the concentration of carbohydrate, e.g. starch, in the mix may be in the range of 150 to 200 g/1, preferably about 180 g/1. Alternatively, itaconic acid may be produced via surface fermentation starting from a carbohydrate raw material such as for instance a mix of beet and cane molasses or sucrose.

The fermentation process is preferably run at a temperature that is optimal for the cells of the invention. Thus, for most fungal cells, the fermentation process is performed at a temperature which is less than 42°C, preferably less than 38°C. For filamentous fungal cells, the fermentation process is preferably performed at a temperature which is lower than 35, 33, 30 or 28°C and at a temperature which is higher than 20, 22, or 25°C.

Preferably in the fermentation processes of the invention, the cells stably maintain the nucleic acid constructs that confer to the cell the ability to produce itaconic acid. Preferably in the process at least 10, 20, 50 or 75% of the cells retain the ability to produce itaconic acid after 50 generations of growth, preferably under industrial fermentation conditions.

In a solid state fermentation process (sometimes referred to as semi-solid state fermentation) the transformed host cells are fermenting on a solid medium that provides anchorage points for the fungus in the absence of any freely flowing substance. The amount of water in the solid medium can be any amount of water. For example, the solid medium could be almost dry, or it could be slushy. A person skilled in the art knows that the terms "solid state fermentation" and "semi-solid state fermentation" are interchangeable. A wide variety of solid state fermentation devices have previously been described (for review see, Larroche et al, (1997); Roussos et al, "Zymotis: (1993); Smits et al. 1998). These devices fall within two categories, those categories being static systems and agitated systems. In static systems, the solid media is stationary throughout the fermentation process. Examples of static systems used for solid state fermentation include flasks, petri dishes, trays, fixed bed columns, and ovens. Agitated systems provide a means for mixing the solid media during the fermentation process. One example of an agitated system is a rotating drum (Larroche et al., supra). In a submerged fermentation process on the other hand, the transformed fungal host cells are fermenting while being submerged in a liquid medium, usually in a stirred tank ferm enter as are well known in the art, although also other types of fermenters such as e.g. airlift-type fermenters may also be applied (see e.g. US 6,746,862). Conditions described herein for a fermentation process for itaconic acid production may also be used for citric acid production.

In a further aspect the invention relates to a process for producing citric acid or itaconic acid, whereby the process comprises the steps of (a) growing a transgenic plant as herein defined above; (b) harvesting plant material comprising citric acid or itaconic acid from the transgenic plant obtained in (a); and optionally, (c) recovery of the citric acid or itaconic acid. In one embodiment the plant material comprising citric acid or itaconic acid in (b) comprises at least 9, 12, 15, 20, 30, 50 or 100 mg itaconic acid per gram dry weight of the plant material. Preferably the plant material is a tuber, more preferably a tuber of a potato.

Description of the figures Figure 1 Citric acid production of transformants expressing cadA and fumarase Results HPLC analysis Erlenmeyer cultures

Figure 2 Itaconic acid production of transformants expressing cadA and fumarase Results HPLC analysis Erlenmeyer cultures Examples

Example 1 Expression of fumarase gene in A. niger

Based on the sequence of fumarase from Rhizopus oryzae (SEQ ID NO: 1) excluding the mitochondrial targeting signal as it was published by de Jongh (de Jongh & Nielsen 2008), a gene was codon optimized for A. niger and synthesised by DNA 2.0 (SEQ ID NO:3). The synthesized gene was cloned into proter {an Aspergillus niger expression plasmid wherein the coding sequence can be cloned as a Nsil-Notl fragment 3' of the modified xlnD promoter and 5' of the xlnD terminator) by DNA 2.0 with the restriction sites Nsil and Notl. The construct was transformed in electrocompetent E. coli DH5a cells and plated on LB agar plates with 100 g/mL ampicillin. Colonies were grown in 5 mL LB broth with 100 g/mL ampicillin and plasmids were isolated with the GeneJET™ plasmid miniprep kit from Fermentas.

Transformation of Aspergillus niger 872.11 and 872.1 l+cadA

Protoplasts of Aspergillus niger 872.11, that is a pyrA, org mutant of NW185 described by Ruijter et al, (Ruijter & de Vondervoort 1999), and 872.11 + cadA, that is an 872.11 strain harbouring the cadA gene from A. terreus (SEQ ID NO: 32), were co- transformed according to L.H. de Graaff (1989, "The structure and expression of the pyruvate kinase gene of Aspergillus nidulans and Aspergillus niger", PhD thesis Agricultural University Wageningen) with the expression plasmid containing the fumarase gene (SEQ ID NO: 3) and with plasmid pAL69 containing the selection marker argB and plated on MMS 1% glucose or MMS 1% glucose and 0,02% arginine plates. Protoplasts of both A. niger strains were also co-transformed with the fumarase containing expression plasmid, the expression plasmid containing both mtaA and pmtA (SEQ ID NO: 34, 36) and with the selection plasmid pAL69.

Spores from developed colonies were harvested and plated on CM glucose or CM glucose arginine plates. Genomic DNA was isolated from the transformants and a colony PCR was done to confirm the presence of the fumarase gene using a forward primer (5 ' -TC AT AACCGATGTGGGGATT-3 ' , SEQ ID NO: 21) and a reverse primer (5'-ACGTTTTCAAGCCCGTTATG

-3', SEQ ID NO:22) located in the fumarase gene. From several developed colonies for each construct spores were harvested and used to inoculate PM medium (1.2 g NaN0₃, 0.5 g KH₂P0₄, 0.2 g MgS0₄ ·7Η₂0, 0.5 g Yeast extract and 40 uL Vishniac solution per L) containing 100 mM sorbitol and 0,02% arginine. After 18 hours induction took place by the addition of 50 mM xylose. Acid formation was followed by HPLC analysis. The most promising strains were selected to repeat the growth experiments in 1 L fermenters (Sartorius) (figures 1, 2, tables 1 and 2).

872.11 CAD Tl T2 T3 T4

6 h 0,00 0,34 4,49 1,32 1,09 1,13

30 h 4,98 4,32 4,42 8,39 10,14 3,31

54 h 6,76 7,87 12,48 2,43

78 h 11,03 8,26 0,19 0,38 18,86 0,33

872.11 CAD Tl T2 T3 T4

6 h 0,00 0,12 1,24 1,42 1,33 1,68

30 h 0,23 0,76 16,08 18,83 14,45 21,10

54 h 0,60 1,47 37,90 35,11 19,55 41,41

78 h 1,20 3,06 51,36 55,27 25,76 66,75

Example 2 Expression of pyruvate carboxylase gene in A. niger

To obtain the pyruvate carboxylase gene a proofreading PCR was performed using genomic DNA of strain N593 with forward primer 5'-

GAGAATGCATATGGCTGCTCCCCGCCAGCC-3 SEQ ID NO: 23 and the reverse primer 5'- GAGAGCGGCCGCCTAGGCCTTGACGATCTTGC-3', SEQ ID NO: 24 (restriction sites underlined). PCR was performed using phusion DNA polymerase and the following cycling conditions: predenaturation for 30 seconds at 98°C, followed by 35 cycles of amplification, denaturation 10 seconds at 98°C, hybridisation 20 seconds at 70 °C, extension 1 minute at 72°C and a final incubation for 7 minutes at 72°C. The PYC amplicon was visible on gel at approximately 3500 bp (3579 is size to expect). The band was cut from the gel and isolated with the Gene JET™ Gel Extraction Kit eluted in MQ water and ligated in pJET according to CloneJET™ PCR Cloning Kit and transformed in electrocompetent E. coli DH5a cells and plated on LB agar plates with 100 μg/mL ampicillin. Colonies were grown in 5 mL LB broth with 100 μg/mL ampicillin and plasmids were isolated with the GeneJET™ plasmid miniprep kit from Fermentas. Isolated plasmids were screened by Nsil and Notl digestion (Fermentas). A plasmid with the correct sized insert was sequenced (SEQ ID NO:5).

Based on the sequence of pyruvate carboxylase from A. niger (Pel et al. 2007) (SEQ ID NO:5), a gene was codon optimized for A. niger and synthesised by DNA 2.0. The synthesized gene was cloned into proter (SEQ ID NO: 7) (an Aspergillus niger expression plasmid wherein the coding sequence can be cloned as a Nsil-NotI fragment 3' of the modified xlnD promoter and 5' of the xlnD terminator) by DNA 2.0 with the restriction sites Nsil and Notl. The construct was transformed in electrocompetent E. coli DH5a cells and plated on LB agar plates with 100 μg/mL ampicillin. Colonies were grown in 5 mL LB broth with 100 μg/mL ampicillin and plasmids were isolated with the GeneJET™ plasmid miniprep kit from Fermentas.

Transformation of Aspergillus niger 872.11 and 872.1 l+cadA

Protoplasts of Aspergillus niger 872.11, that is a pyrA, argB mutant of NW185 described by Ruijter et al, (Ruijter & de Vondervoort 1999), and 872.11 + cadA, that is an 872.11 strain harbouring the cadA gene from terreus, were co-transformed according to L.H. de Graaff (1989, "The structure and expression of the pyruvate kinase gene of Aspergillus nidulans and Aspergillus niger", PhD thesis Agricultural University Wageningen) with the expression plasmid containing the pyruvate carboxylase gene. One strain is prepared with the endogenous and one with the optimised pyruvate carboxylase gene (SEQ ID NO:5 or 7) and with plasmid pAL69 containing the selection marker argB and plated on MMS 1% glucose or MMS 1% glucose and 0,02% arginine plates. Protoplasts of both niger strains were also co- transformed with the pyruvate carboxylase containing expression plasmid, the expression plasmid containing both mtaA and pmtA (SEQ ID NO: 34 and 36) and with the selection plasmid pAL69.

Spores from developed colonies were harvested and plated on CM glucose or CM glucose arginine plates. Genomic DNA was isolated from the transformants and a colony PCR was done to confirm the presence of the pyruvate carboxylase gene using a forward primer located in the xlnD promoter (5'-CCCACAGACTGGCTCAACC-3', SEQ ID NO: 25) and a reverse primer located in the synthetic pyruvate carboxylase gene (5 ' - AAGCCTC ATCTGCCTTTTGG-3 ' , SEQ ID NO: 26) or endogenous pyruvate carboxylase gene (5'- ACGCTCGAAGGAGTCACG-3', SEQ ID NO: 27). From 5 developed colonies for each construct spores were harvested and used to inoculate PM medium (1.2 g NaN0₃, 0.5 g KH₂P0₄, 0.2 g MgS0₄ ·7Η₂0, 0.5 g Yeast extract and 40 μΙ_, Vishniac solution per L) containing 100 mM sorbitol and 0,02% arginine. After 18 hours induction took place by the addition of 50 mM xylose. Acid formation was followed by HPLC analysis. The most promising strains were selected to repeat the growth experiments in 1 L fermenters (Sartorius) (see tables 3- 12).

Results HPLC analysis Erlenmeyer cultures:

872.11 CAD Tl T2 Τ3 Τ4 Τ5

6 h 0 0 0 0 0 0 0

30 h 0 3,74 2,85 1,33 5,35 4,89 10,76

54 h 3,88 4,59 3,24 3,06 11,65 11,39 21,21

78 h 5,37 6,29 5,2 8,46 19,67 16,96 37,54

872.11 CAD T6 T7 Τ8 Τ9 T10

6 h 0 0 0 0 0 0 0

30 h 0 3,74 4,4 3,67 7,4 9,22 7,32

54 h 3,88 4,59 8,45 4,34 23,7 19,65 19,18

78 h 5,37 6,29 17,05 7,56 41,61 31,74 30,85

ϊ' . >ΐ . w.._\\..-._f-^■ .

872.11 CAD Tl Τ2 Τ3 Τ4 Τ5

6 h 1,64 2,04 2,08 3,1 2,41 1,92 1,75

30 h 0,1 5,78 2,83 20 12,61 10,69 11,31

54 h 2,83 4,96 4,41 40,46 23,17 19,6 22,38

78 h 3,88 6,21 5,88 40,51 29,42 33,72 42,27

872.11 CAD Tl Τ2 Τ3 Τ4 Τ5

6 h 0 0 0 0 0 0 0

30 h 0 0,41 0,54 0,71 0,74 0,36 0,43

54 h 0 0,87 1,49 1,56 2 1,02 1,25

1,69 2,36 1,98 3,11 1,8 2,03

872.11 CAD T6 T7 T8 T9 T10

6 h 1,64 2,04 0,19 1,76 0,25 0,22 2,16

30 h 0,1 5,78 16,09 8,76 13,63 15,35 9,22 54 h jilll 30,24 15,94 23,11 26,51 14,78

78 h 3,88 49,11 25,38 35,27 39,93 20,78

872.11 CAD T6 T7 T8 Τ9 T10

6 h 0 o 0 0 0 0 0

30 h 0 0,41 0,77 0,29 0,6 0,85 0,43

54 h o 0,87 1,67 0,9 1,28 1,83 1,3

78 h 0 1,69 2,19 1,41 1,89 2,85 1,99

* issg CAii

872.11 CAD Tl T2 Τ3 Τ4 Τ5 Τ6 Τ7 Τ8

6 h 1,19 2,28 3,73 1,94 3,18 0,00 0,00 0,00 0,00 0,00

30 h 0,83 3,01 0,17 0,81 13,4 5,71 1,10 0,50 0,78 4,45

54 h 0,69 2,78 0,82 0,92 24,23 10,70 0,95 0,54 0,78 7,67

78 h 0,88 2,52 1,37 0,88 6,54 22,97 1,28 0,71 2,14 12,71

_:> CAB, Μ' ΓΑ, Ρ Χ .

e iiirfciisy!

872.11 CAD Tl T2 Τ3 Τ4 Τ5 Τ6 Τ7 Τ8

6 h 0 0,06 2,16 1,03 1,23 0,31 0,17 0,33 0,56 0,58

30 h 0 0,48 16,9 6,14 12,34 6,07 1,78 10,07 7,49 6,22

54 h 0 0,88 28,8 6,03 27,18 15,94 2,76 13,25 11,83 19,20

78 h 0 2,19 32,7 11,5 30,27 22,88 5,05 21,88 15,23 32,26

.siss¾ CAB, , ΤΑ, ΡΑ

pyrttvat : carfmyi

872.11 CAD Tl T2 Τ3 Τ4 Τ5

6 h 0,00 0,00 0,00 0,00 0,00 0,00 ο,οο

30 h 1,61 3,46 3,55 3,80 5,46 4,74 3,83

54 h 1,03 4,13 6,57 9,29 5,64 5,64

78 h 1,76 5,00 1,42 12,33 18,22 11,47 8,70

CAB, A ¾ A .,

872.11 CAD Tl T2 Τ3 Τ4 T5

6 h o 0,00 0,51 0,37 0,33 0,41 0,37

30 h 0 0,20 7,77 5,18 6,76 7,75 6,12

54 h 0 0,72 24,93 17,69 18,65 17,61

78 h 0 1,51 44,01 35,48 32,87 38,83 32,87

Example 3 Expression of the modified phosphofructokinase gene in A. niser

To obtain the modified phosphofructokinase gene a proofreading PCR was performed using genomic DNA of strain N593 with forward primer 5'-

GAGAATGCAT ATGGCTCCCCCCC AAGC-3 ' (SEQ ID NO: 28) and the reverse primer 5'- GAGAGCGGCCGCATCATAGTGCCGGCACAGACC-3 YSEQ ID NO: 29) (restriction sites underlined). PCR was performed using phusion DNA polymerase and the following cycling conditions: predenaturation for 30 seconds at 98°C, followed by 35 cycles of amplification, denaturation 10 seconds at 98°C, hybridisation 30 seconds at 57 °C, extension 45 seconds at 72°C and a final incubation for 5 minutes at 72°C. The PYC amplicon was visible on gel at approximately 1500 bp (1475 is size to expect). The band was cut from the gel and isolated with the GeneJET™ Gel

Extraction Kit eluted in MQ water and ligated in pJET according to CloneJET™ PCR Cloning Kit and transformed in electrocompetent E. coli DH5a cells and plated on LB agar plates with 100 μg/mL ampicillin. Colonies were grown in 5 mL LB broth with 100 μg/mL ampicillin and plasmids were isolated with the GeneJET™ plasmid miniprep kit from Fermentas. Isolated plasmids were screened by Nsil and Notl digestion (Fermentas). A plasmid with the correct sized insert was sequenced (SEQ ID NO: 9).

Transformation of Aspergillus niger 872.11 and 872.1 l+cadA

Protoplasts of Aspergillus niger 872.11, that is a pyrA, org mutant of NW185 described by Ruijter et al, (Ruijter & de Vondervoort 1999), and 872.11 + cadA, that is an 872.11 strain harbouring the cadA gene (SEQ ID NO: 32) from terreus, were co- transformed according to L.H. de Graaff (1989, "The structure and expression of the pyruvate kinase gene of Aspergillus nidulans and Aspergillus niger", PhD thesis Agricultural University Wageningen) with the expression plasmid containing the modified phosphofructokinase gene (SEQ ID NO: 9) and with plasmid pAL69 containing the selection marker argB and plated on MMS 1% glucose or MMS 1% glucose and 0,02% arginine plates. Protoplasts of both A. niger strains were also co- transformed with the modified phosphofructokinase containing expression plasmid, the expression plasmid containing both mtaA and pmtA and with the selection plasmid pAL69.

Spores from developed colonies were harvested and plated on CM glucose or CM glucose arginine plates. Genomic DNA was isolated from the transformants and a colony PCR was done to confirm the presence of the modified phosphofructokinase gene using a forward primer (5'-CCCACAGACTGGCTCAACC-3 \ SEQ ID NO: 30 ) located in the xlnD promoter and a reverse primer located in the modified phosphofructokinase gene (5'-ACCGCTCCGTCAAAATATCC-3\ SEQ ID NO: 31). From several developed colonies for each construct spores were harvested and used to inoculate PM medium (1.2 g NaN0₃, 0.5 g KH₂P0₄, 0.2 g MgS0₄ ·7Η₂0, 0.5 g Yeast extract and 40 μΙ_, Vishniac solution per L) containing 100 mM sorbitol and 0,02% arginine. After 18 hours induction took place by the addition of 50 mM xylose. Acid formation was followed by HPLC analysis. The most promising strains were selected to repeat the growth experiments in 1 L fermenters (Sartorius) (see tables 13-17).

Results HPLC analysis Erlenmeyer cultures

872.11 CAD Tl T2 T3

6 h 1,74 1,89 2,12 1,94 1,86

30 h 1,36 1,24 1,67 1,62 1,60

54 h 1,12 1,04 1,64 1,34 1,38

78 h 1,10 1,25 1,57 1,26 0,88

872.11 CAD Tl T2 T3 T4 T5 T6 T7

6 h 0,09 0,26 1,89 2,01 2,15 0,87 1,24 1,45 1,33

30 h 0,90 0,94 18,68 15,33 19,06 17,94 23,99 19,57 19,44

54 h 1,39 1,88 35,62 25,69 32,62 21,39 27,92 24,87 32,83

78 h 3,88 0,45 42,33 26,32 47,88 24,02 32,56 29,92 39,45

872.11 CAD Tl T2 T3 T4 T5 T6 T7

6 h 0,00 0,09 0,10 0,15 0,14 0,07 0,08 0,08 0,09

30 h 0,00 0,68 0,69 1,01 0,83 0,87 0,88 0,88 1,14

54 h 0,00 1,24 0,00 1,84 1,58 1,22 0,98 1,14 1,96

78 h 0,00 1,88 2,14 2,24 2,61 1,62 1,22 1,51 2,67

5 asTiiKk press €AS>

872.11 CAD Tl T2 T3 T4 T5 T6 T7 T8

6 h 0,09 0,26 1,18 1,38 0,74 0,86 0,50 0,56 0,83 0,50

30 h 0,90 0,94 4,76 4,09 7,10 8,28 0,59 8,43 5,42 6,02

54 h 1,39 1,88 5,48 8,77 10,81 9,38 0,71 13,31 6,69 6,00

78 h 3,88 0,45 7,45 17,22 13,28 11,04 0,00 12,63 10,41 10,94

872.11 CAD Tl T2 T3 T4 T5 T6 T7 T8

6 h 0,00 0,09 1,42 2,12 1,24 1,24 1,39 0,91 1,89 1,07

30 h 0,00 0,68 14,44 18,64 23,59 16,61 23,00 19,50 24,45 21,06

54 h 0,00 1,24 32,15 35,40 40,01 19,85 34,84 30,82 38,17 31,41 78 h 0,00 1,88 42,72 43,58 58,25 25,83 62,04 52,75 57,98 54,63

Example 4 Expression of malate dehydrogenase gene in A. niger

Based on the sequence of malate dehydrogenase from Rhizopus oryzae (SEQ ID

NO: 11) as it was published by Xu et al 2012 , a gene was codon optimized for A. niger and synthesised by DNA 2.0 (SEQ ID NO: 11). The synthesized gene was cloned into proter (an Aspergillus niger expression plasmid wherein the coding sequence can be cloned as a Nsil-Notl fragment 3' of the modified xlnD promoter and 5' of the xlnD terminator) by DNA 2.0 with the restriction sites Nsil and Notl. The construct was transformed in electrocompetent E. coli DH5a cells and plated on LB agar plates with 100 μg/mL ampicillin. Colonies were grown in 5 mL LB broth with 100 μg/mL ampicillin and plasmids were isolated with the GeneJET™ plasmid miniprep kit from Fermentas.

Transformation of Aspergillus niger 872.11 and 872.1 l+PYC

Protoplasts of Aspergillus niger 872.11, that is a pyrA, org mutant of NW185 described by Ruijter et al, (Ruijter & de Vondervoort 1999), and 872.11 + PYC, that is an 872.11 strain harbouring the optimized pyruvate carboxylase gene from niger (SEQ ID NO: 07), were co-transformed according to L.H. de Graaff (1989, "The structure and expression of the pyruvate kinase gene of Aspergillus nidulans and Aspergillus niger", PhD thesis Agricultural University Wageningen) with the expression plasmid containing the malate dehydrogenase gene (SEQ ID NO: 11) and with plasmid pGW635 containing the selection marker pyrA and plated on MMS 1% glucose or MMS 1% glucose and 0,12% uridine plates.

Spores from developed colonies were harvested and plated on CM glucose or CM glucose uridine plates. Genomic DNA was isolated from the transformants and a colony PCR was done to confirm the presence of the malate dehydrogenase gene using a forward primer (5'- AGTCAATCGGTCATTCTCCG -3', SEQ ID NO: 23) located in the xlnD promoter and a reverse primer (5'- CGCCATACAATCTCTTAGGG-3', SEQ ID NO:24) located in the malate dehydrogenase gene. From several developed colonies for each transformation spores were harvested and used to inoculate PM medium (1.2 g NaN0₃, 0.5 g KH₂P0₄, 0.2 g MgS0₄ ·7Η₂0, 0.5 g Yeast extract and 40 Vishniac solution per L) containing 100 mM sorbitol and 0,02% arginine. After 18 hours induction took place by the addition of 50 mM xylose. Acid formation was followed by HPLC analysis (tables 18-19).

5 Results HPLC analysis Erlenmeyer cultures:

872.11 Tl T2 T3 T4 T5 T6 T7 T8

h 4.88 5.77 4.47 5.54 3.22 4.75 4.61 3.91 4.78

7.09 9.38 6.67 9.33 5.00 6.50 6.15 6.93 6,39 h 9.19 7.91 5.36 7.95 4.09 6.17 4.94 5.66 5.32

T&bfe 18 Cime add HK¾? ¾>ss Is sss of A mge JUl sverapressiag ttse cts&iate dds «!g«si;;so gase

872.1 872.1 Tl T2 T3 T4 T5 T6 T7 T8 1 1+PYC h 0.97 2.41 3.35 2.62 2.31 2.45 2.39 2.32 2.55 2.37

h 4.16 16.31 19.45 15.26 20.85 15.39 18.45 19.16 16.03 18.96 h 5.43 21.31 29.94 21.99 27.10 30.44 24.32 23.41 25.18 29.12 h 6.73 33.16 35.05 34.50 37.82 39.94 39.10 33.06 40.72 34.92

0 Example 5 Expression of cadA, mtaA and pmtA in A. niger

Based on the sequence of mtaA and pmtA from Aspergillus terreus (SEQ ID

NO: 17, 19) the genes were codon optimized for niger and synthesised by DNA 2.0 (SEQ ID NO: 34,36). The synthesized genes were cloned into proter (an Aspergillus niger expression plasmid wherein the coding sequence can be cloned as a Nsil-Notl fragment 3' of the modified xlnD promoter and 5' of the xlnD terminator) by DNA 2.0 with the restriction sites Nsil and Notl. The p xlnD-mtaA-t xlnD fragment was cloned into the expression vector containing the pmtA gene for the expression of both transporter genes into strain 872.11 + cadA. The construct was transformed in electrocompetent E. coli DH5a cells and plated on LB agar plates with 100 μg/mL ampicillin. Colonies were grown in 5 mL LB broth with 100 μg/mL ampicillin and plasmids were isolated with the GeneJET™ plasmid miniprep kit from Fermentas. Transformation of Aspergillus niger 872.1 l+cadA

Protoplasts of Aspergillus niger 872.11 + cadA, that is an 872.11 strain harbouring the cadA gene from A. terreus (SEQ ID NO: 32), was co-transformed according to L.H. de Graaff (1989, "The structure and expression of the pyruvate kinase gene of Aspergillus nidulans and Aspergillus niger", PhD thesis Agricultural University Wageningen) with the expression plasmid containing both mtaA and pmtA (SEQ ID NO: 34, 36) and with plasmid pAL69 containing the selection marker argB and plated on MMS 1% glucose or MMS 1% glucose and 0,02% arginine plates.

Spores from developed colonies were harvested and plated on CM glucose or CM glucose arginine plates. Genomic DNA was isolated from the transformants and a colony PCR was done to confirm the presence of the mtaA and pmtA genes using a forward primer LS_pMTA_for (5'- ATTAAGACCCGCATGCAATC, SEQ ID NO: 47) and LS_pMTA_rev (5'- CTTCTCGTAGACGGGGAACA SEQ ID NO: 48) to check for the presence of the MTA gene. Primers LS_pPMT_for (5'- ACCTTCACTAGCTGGCGTGT, SEQ ID NO: 49) and LS_pPMT_rev (5'- GACATCCGTGGGACTGAACT, SEQ ID NO: 50) are used to check for the presence of the PMT gene. From several developed colonies for each construct spores were harvested and used to inoculate PM medium (1.2 g NaN0₃, 0.5 g KH₂P0₄, 0.2 g MgS0₄ ·7Η₂0, 0.5 g Yeast extract and 40 μΕ Vishniac solution per L) containing 100 mM sorbitol and 0,02% arginine. After 18 hours induction took place by the addition of 50 mM xylose. Acid formation was followed by HPLC analysis. All strains were selected to repeat the growth experiments in 1 L fermenters (Sartorius).

The transformants containing cadA, mtaA and pmtA and the control strains CAD, CAD +MTA, CAD+PMT were inoculated (10⁶ spores/mL) in 1 L fermentors (Sartorius) containing 0.75 L of PM medium with 100 mM sorbitol. After 18 hours the strains were induced by the addition of 50 mM xylose. The strains were grown for 5 days at 30°C with a stirrer speed of 1000 rpm. The pH in the culture broth was not controlled. Samples were taken 6, 30, 54 and 78 hours after induction for HPLC measurements and for dry weight determination (table 20).

CAD + PMT + MTA 3 17

Table 21 list of most used cDNA and amino acid sequences

Name of the enzyme SEQ ID NO: Type of sequence

a) Fumarase 1 cDNA of Rhizopus oryzae fumarase

2 Amino acid sequence of

Rhizopus oryzae fumarase

3 cDNA of fumarase optimised for niger

4 Amino acid sequence of

fumarase optimised for A. niger

b) Pyruvate 5 cDNA pyruvate carboxylase decarboxylase of niger

6 Amino acid sequence of

pyruvate carboxylase of A niger

7 cDNA pyruvate carboxylase optimised for niger

8 Amino acid sequence of

pyruvate carboxylase optimised for _^4 niger c) Phosphofructokinase 9 cDNA phosphofructokinase modified from A. niger

10 Amino acid sequenceof

phosphofructokinase modified from A. niger d) Malate dehydrogenase 11 cDNA of Rhizopus oryzae malate dehydrogenase

12 Amino acid sequence of

Rhizopus oryzae malate dehydrogenase

13 cDNA of malate

dehydrogenase optimised for A niger

14 Amino acid sequence of

malate dehydrogenase optimised for _^4 niger e) c/5-aconitate 15 cDNA of c/5-aconitate dehydrogenase dehydrogenase of A. terreus (CAD) 16 Amino acid sequence of cis- aconitate dehydrogenase of A. terreus

f) Mitochondrial 17 cDNA of mitochondrial transporter (MTA) transporter of A. terreus

18 Amino acid sequence of

mitochondrial transporter of A. terreus g) Plasma membrane 19 cDNA of plasma membrane transporter (PMT) transporter of A. terreus

20 Amino acid sequence of

plasma membrane transporter of terreus

CAD 32 cDNA of cis-aconitate

dehydrogenase of A. terreus optimised for A niger.

33 Amino acid sequence of cis- aconitate dehydrogenase of A. terreus optimised for A niger.

MTA 34 cDNA of mitochondrial

transporter of A. terreus optimised for A niger.

35 Amino acid sequence of

mitochondrial transporter of A. terreus optimised for _^4 niger.

38 Amino acid sequence of

mitochondrial transporter of A. clavatus

39 Amino acid sequence of

mitochondrial transporter of A. fumigatus

40 Amino acid sequence of

mitochondrial transporter of Neosartorya fischeri

41 Amino acid sequence of

mitochondrial transporter of Neurospora crassa

42 Amino acid sequence of

mitochondrial transporter of Talaromyces stipitatus

43 Amino acid sequence of

mitochondrial transporter of Paracoccidioides brasiliensis

44 Amino acid sequence of

mitochondrial transporter of Sordaria macrospora

45 Amino acid sequence of

mitochondrial transporter of A. oryzae

46 Amino acid sequence of

mitochondrial transporter of A. niger

PMT 36 cDNA of plasma membrane transporter of A. terreus optimised for A niger.

37 Amino acid sequence of plasma membrane transporter of A. terreus optimised for A niger.

51 Amino acid sequence of plasma membrane transporter of terreus

52 Amino acid sequence of plasma membrane transporter of oryzae

53 Amino acid sequence of plasma membrane transporter of A. flavus

54 Amino acid sequence of plasma membrane transporter of Neosartorya fischeri

55 Amino acid sequence of plasma membrane transporter οΐΡ. chrysogenum

56 Amino acid sequence of plasma membrane transporter of A. fumigatus

57 Amino acid sequence of plasma membrane transporter of nidulans

58 Amino acid sequence of plasma membrane transporter of Talaromyces stipitatus

References

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Claims

1. A cell capable of expressing at least one of the following nucleic acid molecules: a) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with phosphofructokinase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 10;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code and b) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with pyruvate carboxylase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 6 or 8;

2. A cell according to claim 1 further being capable of expressing the two following nucleic acid molecules: c) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with fumarase activity, wherein the nucleotide sequence is selected from the group consisting of:

ID NO 2 or 4;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 1 or 3;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code; and d) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with malate dehydrogenase activity, wherein the nucleotide sequence is selected from the group consisting of:

3. A cell, which is capable of expressing at least one of the following nucleic acid molecules: a) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with phosphofructokinase activity, wherein the nucleotide sequence is selected from the group consisting of: i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 10;

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code, b) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with pyruvate carboxylase activity, wherein the nucleotide sequence is selected from the group consisting of:

ID NO 6 or 8;

iii) a nucleotide sequence the complementary strand of which hybridises to a nucleotide sequence of ii); and

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code; c) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with fumarase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 2 or 4;

iii) a nucleotide sequence the complementary strand of which hybridises to a nucleotide sequence of ii); and, iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code; and d) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with malate dehydrogenase activity, wherein the nucleotide sequence is selected from the group consisting of:

iv) a nucleotide sequence the sequence of which differs from the sequence of a nucleotide sequence of ii) or iii) due to the degeneracy of the genetic code, said cell being further capable of expressing the following nucleic acid molecules: e) a nucleic acid molecule represented by a nucleotide sequence encoding a polypeptide with cis-aconitate decarboxylase activity, wherein the nucleotide sequence is selected from the group consisting of:

i) a nucleotide sequence encoding a polypeptide which comprises an amino acid sequence that has at least 40% sequence identity with the amino acid sequence of SEQ ID NO 16 or 33;

ii) a nucleotide sequence comprising a nucleotide sequence that has at least 40% sequence identity with the nucleotide sequence SEQ ID NO. 15 or 32;

ID NO 18 or 35;

4. A cell according to claim 1 or 2, wherein the cell is a plant cell or a fungal cell including a yeast cell.

5. A cell according to claim 3, wherein the cell is a plant cell or a fungal cell including a yeast cell.

6. A cell according to claim 4, wherein the fungal cell is of a genus selected from Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Monascus, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, and Ustilago, preferably from Aspergillus, Penicillium, Candida and Yarrowia, more preferably the fungal cell is of a species selected from Aspergillus niger, Aspergillus terreus, Aspergillus itaconicus, Monascus ruber, Penicillium simplicissimum, Penicillium expansum, Penicillium digitatum, Penicillium italicum, Candida oleophila and Yarrowia lipolytica.

7. A cell according to claim 5, wherein the fungal cell is of a genus selected from Acremonium, Aspergillus, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Monascus, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Piromyces, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Tolypocladium, Trichoderma, and Ustilago, preferably from Aspergillus, Penicillium, Candida and Yarrowia, more preferably the fungal cell is of a species selected from Aspergillus niger, Aspergillus terreus, Aspergillus itaconicus, Monascus ruber, Penicillium simplicissimum, Penicillium expansum, Penicillium digitatum, Penicillium italicum, Candida oleophila and Yarrowia lipolytica.

8. Use of a cell as defined in any one of claims 1, 2, 4 or 6 in the production of citric acid.

9. Use of a cell as defined in any one of claims 3, 5 or 7 in the production of itaconic acid.

10. A process for producing citric acid, whereby the process comprises the steps of: a) fermenting a medium comprising a source of carbon and energy with a cell as defined in any one of claims 1, 2, 4 or 6, whereby the cell ferments the source of carbon and energy to citric acid, and optionally,

b) recovery of the citric acid.

11. A process for producing itaconic acid, whereby the process comprises the steps of:

a) fermenting a medium comprising a source of carbon and energy with a cell as defined in any one of claims 3, 5 or 7, whereby the cell ferments the source of carbon and energy to itaconic acid, and optionally,

b) recovery of the itaconic acid.