The invention relates to the field of microbial production, specifically production of itaconic acid (itaconate), more specifically production of itaconate in fungi.

Production and metabolism of itaconic acid in microbial cells has been studied extensively for several decades (Calam, C.T. et al., 1939, Thorn. J. Biochem., 33:1488-1495; Bentley, R. and Thiessen, CP., 1956, J. Biol. Chem. 226:673-720; Cooper, R.A. and Romberg, H.L., 1964, Biochem. J., 91:82-91; Bonnarme, P. et al., 1995, J. Bacteriol. 117:3573-3578; Dwiarti, L. et al., 2002, J. Biosci. Bioeng. 1:29-33), but the metabolic pathway for itaconic acid has not been unequivocally established (Wilke, Th. and Vorlop, K.-D., 2001, Appl. Microbiol. Biotechnol. 56:289-295; Bonnarme, P. et al., 1995, J. Bacteriol. 177:3573-3578). Two complicating factors in this respect are that the biosynthesis route for itaconic acid is thought to occur both in the cytosol and the mitochondria (Jaklitsch, W.M. et al., 1991, J. Gen.

Microbiol. Appl. 6:51-61) and that aconitase, the enzyme that interconverts citric acid into cis-aconitate, and vice versa, and other enzymes in the metabolic pathway have been found to be present in many isoforms in microbial cells.

Production of itaconic acid is now commercially achieved in

Aspergillus terreus, which has physiological similarity to A. niger and A. oryzae. However, these latter two accumulate citric acid, due to the absence of cis-aconic acid decarboxylase (CAD) activity. Substrates used by these fungi include mono- and disaccharides, such as glucose, sucrose and fructose and starches, as they exist in forms that are degradable by the microorganism, and molasses. Recently, it has been discovered that also glycerol is a useful substrate in itaconic acid production by A. terreus (US 5,637,485). The general scheme currently envisioned for itaconic acid biosynthesis is given in Fig. 21, wherein clearly the existence of the biosynthetic route both in the cytosol and the mitochondria is depicted and the connection between these two compartments. At several points of this scheme possibilities exist to try to improve the existing commercial production of itaconic acid in micro-organisms.

SUMMARY OF THE INVENTION

The present inventors now have found mutant strains selected for increased itaconic acid resistance and reduced itaconic acid degradation and/or biochemical conversion, thus producing improved levels of itaconic acid. Moreover the present inventors have found that inhibition of the activity of itaconyl-CoA transferase (EC 2.8.3.-), itaconyl-CoA hydratase / citramalyl-CoA hydro-lyase (EC 4.2.1.56) or citramalyl-CoA lyase (EC 4.1.3.25) and/or inhibition of the expression of the genes encoding these enzymes from the organism in which itaconic acid is produced, e.g.

Aspergillus are able to overcome the toxic effects caused by itaconic acid and concomitantly to boost the production of itaconic acid. Accordingly, the invention comprises methods to increase production of itaconic acid in a micro-organism by increasing resistance to the toxic effects of itaconic acid and decreasing itaconic acid degradation and/or biochemical conversion. One approach to obtain increased itaconic acid production is by selection of mutant strains of itaconic acid producing organisms which are resistant to the toxic effects of itaconic acid. Another approach is by inhibiting the functioning of the enzyme itaconyl-CoA transferase (EC 2.8.3.-) , and/or itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase, EC 4.2.1.56) and/or citramalyl-CoA lyase (EC 4.1.3.25)or inhibiting the expression of the genes encoding these enzymes . Said inhibition can be effected by mutation of the gene coding for said itaconyl-CoA transferase and/or itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase (EC 4.1.3.25), wherein said mutation is chosen from:

a) mutation of the promoter or insertion of an inducible promoter; b) mutation of the coding sequence, chosen from insertion, deletion or change of one or more nucleotides;

c) insertion of a protein binding site; and

d) combinations thereof.

In an alternative embodiment, the expression of said enzyme is silenced, which can be effected by:

a) antisense silencing;

b) sense co-suppression; or

c) RNA interference.

Further comprised in the invention is a method according to any of the above, wherein said micro-organism is a micro-organism which naturally produces itaconic acid, wherein optionally said micro-organism is further provided with a gene coding for the enzyme aconitase and/or a gene coding for the enzyme citrate synthase, 2-methylcitrate dehydratase, and/or cis-aconitate decarboxylase. Also genes coding for transporter proteins that can transport aconitase(-metabolites) such as Aspergillus terreus

ATEG_09970.1 or ATEG_09972.1 can add to the production of itaconic acid.

Alternatively, said micro-organism is genetically constructed to produce itaconic acid, preferably by introducing a gene coding for the enzyme cis-aconitic acid decarboxylase, wherein optionally said microorganism is further provided with a gene coding for the enzyme aconitase and/or a gene coding for the enzyme citrate synthase, and/or 2-methylcitrate dehydratase and/or aconitase transporters such as Aspergillus terreus

ATEG_09970.1 or ATEG_09972.1. Preferably, said micro-organism is an Aspergillus, preferably A. terreus or A. niger.

Another embodiment of the invention is a micro-organism, preferably Aspergillus, more preferably A. terreus or A. niger, in which expression of the gene coding for the enzyme itaconyl-CoA transferase and/or itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase (EC 4.1.3.25) is inhibited.

LEGENDS TO THE FIGURES

Fig. 1: Controlled fermentation of Aspergillus terreus NRRL 1960 showing itaconic acid production and degradation. Glucose concentration (g/1), itaconic acid titer (g/1) and biomass (g DWT/kg) are shown in relation to fermentation time (hours).

Fig. 2: Controlled fermentation of Aspergillus niger strains CitB #77 and CitB #101 producing IA and CA, showing itaconic acid production and degradation. A. itaconic acid (IA) titer and citric acid (CA) titer (g/1) and B. glucose concentration (g/1), and biomass (g/kg) are shown in relation to fermentation time EFT (h).

Fig. 3: Controlled fermentation of an IA producing Aspergillus niger strain Q199 (CBS 143051), derived from an industrial citric acid production strain, showing citric acid production and itaconic acid

production and degradation. Glucose concentration (g/1), itaconate titer (g/1), citrate titer (g/L) and biomass (g DWT/kg) are shown in relation to fermentation time EFT (hours).

Fig. 4: Growth of AB1.13 CAD 4.1 (CBS 141653) strain in M12+Cu supplemented with several concentrations of itaconic acid. Medium devoid of C-source is used as negative control. An extracellular

concentration of 10 g/1 IA in the production medium results in an 75% decrease in biomass development. At 20 g/1 IA growth and even higher concentrations of IA further deteriorated growth is observed after 7 days of incubation at 33°C.

Fig. 5: Organic acid production and biomass formation in controlled fermentation of A. niger IA producing strains CitB #99 (CBS 141659) and #113 (CBS 141660) showing reduced biomass formation compared to AB1.13 #49B (CBS 141657). A. itaconic acid (IA) titer and citric acid (CA) titer (g/1) and B. glucose concentration (g/1), and biomass (g/kg) are shown in relation to fermentation time EFT (h).

Fig. 6: Biomass generation of shakeflask experiment with evolution mutants EE #3, #7, #9, #10, #13, #25 (CBS 141661), and #26 (CBS 141662) and CitB #99 (CBS141659). Shakeflasks were filled with production medium both with and without IA. Evolution experiment mutant strains and their parental strain CitB #99 were used in this experiment. This experiment was performed in duplicate. Biomass (g/kg) is shown in relation to fermentation condition (+/- IA) after 5 days of incubation at 33°C.

Fig. 7: Average growth inhibition of shakeflask experiment with evolution mutants EE #3, #7, #9, #10, #13, #25 (CBS 141661), and #26 (CBS 141662) and CitB #99 (CBS 141659). Shakeflasks were filled with

production medium with and without IA. Evolution experiment mutant strains and their parental strain CitB #99 were used in this experiment. This experiment was performed in duplicate. Reduction of growth by IA is calculated by dividing biomass values with IA by these without IA after 5 days of incubation at 33°C.

Fig. 8: Degradation of IA in shakeflask experiment. Shakeflasks were filled with production medium supplemented with 20 g/1 IA and glucose (1 g/1 and 5 g/1). Production medium only supplemented with 1 g/1 glucose was added as positive control for this experiment. Evolution experiment mutant strains EE #25 (CBS 141661), EE #26 (CBS 141662) and their parental strain CitB #99 (CBS 141659) and AB1.13 CAD 4.1 (CBS 141653) were used in this experiment. This experiment was performed in duplicate. Degradation of IA is expressed in IA (g/1) decrease in the culture fluid after 5 days of incubation at 33°C.

Fig. 9: IA and CA production in IA resistant strains. Shakeflasks were filled with production medium. Evolution experiment mutant strains EE #25 (CBS 141661), EE #26 (CBS 141662) and their parental strain CitB #99 (CBS 141659) were used in this experiment. Itaconic acid and citric acid levels obtained in these cultivations are given in g/1 after 5 days of incubation at 33°C.

Fig. 10: Specific itaconic acid production in IA resistant A. niger strains. Shakeflasks were filled with production medium. Evolution experiment mutant strains EE #25 (CBS 141661), EE #26 (CBS 141662) and their parental strain CitB #99 (CBS 141659) were used in this experiment. Specific itaconic acid production expressed in g/g biomass after 5 days of incubation at 33°C is given.

Fig. 11: Blast results for itaconyl-CoA transferase.

Fig. 12: Blast results for itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase).

Fig. 13: Blast results with citramalyl-CoA lyase.

Fig. 14: Blast results with trans-aconitate 2-methyltransferase.

Fig. 15: Blast results with alternative itaconic acid pathway genes trans-aconitase decarboxylase (A) and aconitate isomerase (B).

Fig. 16: (A) Splitmarker design for gene An07g00760 (itaconyl- CoA transferase) using the A. oryzae pyrE selection marker flanked by direct repeat sequences. (B) Splitmarker design for gene An07g00760 (itaconyl-CoA transferase) using the E. coli hygromycin B

phosphotransferase (hph) selection marker. (C) Splitmarker design for gene An07g09220 (itaconyl-CoA hydratase) using the A. oryzae pyrE selection marker flanked by direct repeat sequences. (D) Splitmarker design for gene Anl6g06510 (trans-aconitate 2-methyltransferase) using the A. oryzae pyrE selection marker flanked by direct repeat sequences.

Fig. 17: Improved itaconic acid production in modified IA producing A. niger strains grown in flasks. Erlenmeyer flasks were filled with production medium. Deletion mutants CitB #99 AICT #RD1 (CBS 143055), CitB #99 AICH #RB2 (CBS 143056) and their parental strain CitB #99 (CBS 141659) were used in this experiment. Flasks were incubated at 33°C and (A) put to shake (250 RPM) or (B) left static. Itaconic acid levels (g/1) were obtained in time.

Fig. 18: Improved itaconic acid production in a modified IA producing A. niger strain, derived from an industrial citric acid production strain, grown in flasks. Erlenmeyer flasks were filled with production medium. Deletion mutants Q199 AICT #4 (CBS 143050) and its parental strain Q199 (CBS 143051) were used in this experiment. Shakeflasks were incubated at 35°C at 250 RPM and itaconic acid levels (g/1) were obtained in time.

Fig. 19: Controlled fermentation of modified IA producing A. niger strains CitB #99 AICT #RD1 (CBS 143055) and CitB #99 AICH #RB2 (CBS 143056) showing improved production and no degradation of itaconic acid. A. itaconic acid (IA) titer (g/1) and B. glucose concentration (g/1), and biomass (g DWT /kg) are shown in relation to fermentation time EFT (h).

Fig. 20: Controlled fermentation of Q199 AICT #4 (CBS 143050), a modified IA producing A. niger strain, derived from an industrial citric acid production strain, showing production and no degradation of itaconic acid. Itaconate titer (g/1), citrate titer (g/1), glucose concentration (g/1), and biomass (g DWT /kg) are shown in relation to fermentation time EFT

(hours).

Fig. 21: Postulated biosynthesis and catabolism route(s) for itaconic acid in A. terreus. 1, citrate synthase A; 2, aconitase; 3, cis-aconitic acid decarboxylase (itaconate-forming); 4, cis-aconitic acid decarboxylase (citraconate-forming); 5, citraconate isomerase; 6, mitochondrial

dicarboxylate-tricarboxylate antiporter; 7, mitochondrial tricarboxylate transporter; 8, dicarboxylate transporter; 9, 2-methylcitrate dehydratase; 10, citrate synthase B; 11, itaconyl-CoA transferase; 12, itaconyl-CoA hydratase; 13, citramalyl-CoA lyase; 14, trans-aconitate 2- methyltransferase. Fig. 22. Itaconic acid degradation pathway (adapted from

Sasikaran et al., 2014, Nat. Chem. Biol. 10:371-377).

DETAILED DESCRIPTION OF THE INVENTION

"Fungi" are herein defined as eukaryotic micro-organisms and include all species of the subdivision Eumycotina (Alexopoulos, C. J., 1962, In: Introductory Mycology, John Wiley & Sons, Inc., New York). The term fungus thus includes both filamentous fungi and yeast. "Filamentous fungi" are herein defined as eukaryotic micro-organisms that include all filamentous forms of the subdivision Eumycotina. These fungi are characterized by a vegetative mycelium composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi used in the present invention are morphologically, physiologically, and genetically distinct from yeasts. Vegetative growth by filamentous fungi is by hyphal elongation and carbon catabolism of most filamentous fungi are obligately aerobic. "Yeasts" are herein defined as eukaryotic micro-organisms 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.

The term "gene", as used herein, refers to a nucleic acid sequence containing a template for a nucleic acid polymerase, in eukaryotes, RNA polymerase II. Genes are transcribed into mRNAs that are then translated into protein. "Expression" refers to the transcription of a gene into structural RNA (rRNA, tRNA) or messenger RNA (mRNA) with subsequent

translation into a protein.

The term "inhibition" can be both used for inhibition of expression of a protein or for inhibition of function of a protein. When used in

conjunction with protein expression the term "inhibition" refers to a measurable reduction in expression of mRNA encoding said protein or in the concentration of the protein in the cell. The reduction can be anything from less than normal to zero (i.e. no mRNA or protein measurable). Further, with respect to protein function, the term "inhibition" refers to any action and/or treatment which operates against the full activity of a protein thus reducing and/or completely suppressing protein function.

The term "vector" as used herein, includes reference to an autosomal expression vector and to an integration vector used for

integration into the chromosome.

The term "expression vector" refers to a DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. In particular an expression vector comprises a nucleotide sequence that comprises in the 5' to 3' direction and operably linked: (a) a fungal- recognized transcription and translation initiation region, (b) a coding sequence for a polypeptide of interest, and (c) a fungal-recognized

transcription and translation termination region. "Plasmid" refers to autonomously replicating extrachromosomal DNA which is not integrated into a microorganism's genome and is usually circular in nature. An "integration vector" refers to a DNA molecule, linear or circular, that can be incorporated in a microorganism's genome and provides for stable inheritance of a gene encoding a polypeptide of interest. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e., operably linked to) additional nucleic acid segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the

incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the host cell, but which has a replicon that is non-functional in that organism. Integration of the segment comprising the gene of interest may be selected if an

appropriate marker is included within that segment.

"Transformation" and "transforming", as used herein, refer to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion, for example, direct uptake, transduction, f- mating or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host cell genome.

By "host cell" is meant a cell that contains a vector or

recombinant nucleic acid molecule and supports the replication and/or expression of the vector or recombinant nucleic acid molecule. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, fungus, plant, insect, amphibian, or mammalian cells. Preferably, host cells are fungal cells.

Key in the biosynthetic pathway for itaconic acid is the localisation of the various substrates. It is thought that production of itaconic acid mainly occurs in the cytosol, but part of the production takes place in the mitochondrion. There is also an active transport of itaconic acid (or other compounds in the metabolic route in which itaconic acid is formed) in between the mitochondrion and the cytoplasma. In many biochemical pathways, the end-product is inhibiting its own production to prevent excess end-product in the biological system. Excess end-product will not only lead to loss of energy in an economical sense, it can also give rise to unwanted side effects such as toxicity. It is contemplated that by depleting the cell of itaconic acid the formation of new itaconic acid will continue without end- product inhibition and without any inhibitions caused by the toxicity of the itaconate, thus giving—in total- an increase yield of itaconic acid. Inhibition by IA toxicity can also be relieved by selection of IA resistant mutants.

The enzymes itaconyl-CoA transferase, itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and citramalyl-CoA lyase (EC 4.1.3.25)are the most important enzymes in the catabolism of itaconic acid (see Fig. 21), and they degrade itaconic acid via itaconyl-CoA into citramalyl-CoA, which is further degraded by citramalyl-CoA pyruvate lyase into acetyl- CoA and pyruvate.

The present invention discloses that inhibition of one or more of these enzymes prevents growth inhibition by toxic effects of itaconic acid and inhibits degradation or biochemical conversion of itaconic acid or its precursors and would be advantageously used for overproduction of itaconic acid in micro-organisms which are capable of producing itaconic acid.

Inhibition of the enzyme(s) can take place by various mechanisms, which are shortly discussed below.

In principle, inhibition of the first step in the degradation of itaconic acid (i.e. the enzyme itaconyl-CoA transferase) would already be sufficient to block the degradation of itaconic acid. It is however

advantageous to also block the second step, the conversion by the enzyme itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase), if only as a

precautious safety measure. Blocking this second step has additional benefits as the absence of this second step may downregulate the entire degradation pathway and/or it may avoid the production of toxic

intermediates originating from the second step.

Next to these two enzymes, it would also be possible to (additionally) block the enzyme citramalyl-CoA pyruvate lyase.

Additional inhibition of this enzyme in organisms in which such an enzyme is active would further increase the production of itaconic acid.

It is believed that any method which results in an inhibition of the degradation or biochemical conversion of itaconic acid by inhibiting the above-mentioned enzymes,, whether said inhibition is on the translational, transcriptional, post-transcriptional or functional level, would give the desired effect.

The enzyme(s) preferably is/are inhibited by inhibition on transcriptional or translational level, e.g. by mutation, anti-sense inhibition or by RNA interference (RNAi). Mutation of the gene coding for the enzyme can be accomplished by site-directed mutagenesis with a mutated nucleotide sequence, which causes aberrant expression of the enzyme or expression of an aberrant enzyme. Such a mutation can comprise, but is not limited to, the following examples:

1) a change in the promoter sequence of the gene, thereby decreasing promoter function. The promoter is usually situated upstream (5') of the coding sequence. In its broader scope, the term "promoter" includes the RNA polymerase binding site as well as regulatory sequence elements located within several hundreds of base pairs, occasionally even further away, from the transcription start site. Such regulatory sequences are, e.g., sequences that are involved in the binding of protein factors that control the

effectiveness of transcription initiation in response to physiological conditions. A change in the promoter sequence can be accomplished by, for instance, deletion of the ribosome binding site or by deletion of the TATA- box, which causes loss of recognition or binding of the polymerase enzyme and thus an inhibited formation of mRNA. Alternatively, the promoter can be shortened, or even deleted totally, or replaced by a promoter which is inducible. In the latter case, enzyme product will only be formed after induction of the promoter. Inducible promoters are known to the person skilled in the art. Typically, the factor that binds specifically to an inducible promoter to activate transcription is present in an inactive form which is then directly or indirectly converted to the active form by the inducer. The inducer may be a chemical agent such as a protein, metabolite (sugar, alcohol, etc.), a growth regulator, herbicide, or a phenolic compound or a physiological stress imposed directly by heat, salt, wounding, toxic elements etc., or indirectly through the action of a pathogen or disease agent such as a virus. A cell containing an inducible promoter may be exposed to an inducer by externally applying the inducer to the cell such as by spraying, heating, or similar methods. Inducible promoters are known to those familiar with the art and several exist that could conceivably be used to inhibit expression of the enzyme. Inducible promoters suitable for use in accordance with the present invention include, but are not limited to, the heat shock promoter, promoters inducible by the mammalian steroid receptor system and any chemically inducible promoter. Examples of inducible promoters include the inducible 70 kD heat shock promoter of Drosophila melanogaster (Wing et al 1989, Mol Gen Genet, 219: 9-16) and the alcohol dehydrogenase promoter which is induced by ethanol. A promoter that is inducible by a simple chemical is particularly useful. Such simple or common chemicals are used in the induction of so-called gene switch promoters. Examples of gene switch promoters include the alcA/alcR gene switch promoter as described in published International Patent Application No. WO 93/21334; the GST promoter, as described in published International Patent Application Nos. WO 90/08826 and WO 93/031294; and the ecdysone switch system as described in published International Patent Application No. WO 96/37609. In such switch systems, the timing of gene expression is controlled by application of an external chemical. The switch chemical may be applied as a spray or vapor to all or part of the transgenic plant or as a root drench. Examples of suitable switch chemicals are provided in the above references describing switch promoter systems. The external chemical stimulus is preferably an chemical, the use of which is not detrimental to the microbial cells. Inducible switch promoter systems preferably include one or two component systems; nevertheless, systems comprising more than two components are encompassed by the present invention. The alcA/alcR switch promoter system is particularly preferred. In the alcA/alcR promoter switch system, the preferred chemical inducer is ethanol, in either liquid or vapour form. One of the main advantages of the use of ethanol is that small quantities of ethanol generate high levels of expression. The alcA/alcR inducible promoter system is a two-component system involving DNA sequences coding for the alcA promoter and the alcR protein, the expression of which is placed under the control of desired promoters. The alcR protein activates the alcA promoter in the presence of an inducer and any gene under the control of the alcA promoter (in this case the gene coding for the enzyme), will therefore be expressed only in the presence of that inducer. With such a system the activity of the gene construct can be limited both by place and by time. Other gene-switch systems and/or inducible promoters are known in the art and would also be equally applicable. An advantage in using such an inducible promoter or gene-switch system is that it may be possible that expression of the enzyme is desired during certain moments of growth of the culture. In that case, during these times, the inducer can be introduced into the culture, resulting in expression of the enzyme. At other moments, no inducer is present and accumulation of itaconic acid will take place.

2) a change in the coding sequence. Such a change can be effected by the insertion, deletion or change of one of more nucleotides in the open reading frame of the gene coding for the itaconyl-CoA transferase and/or itaconyl- CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase enzyme . Such a change should be able to cause a change in the amino acid sequence of the transcribed enzyme. A preferred change-type is causing a frame-shift mutation by inserting or deleting one or two nucleotides. Such a mutation would distort the three-nucleotide codon based information and would cause construction of a completely different sequence of amino acids from the mutation point until a stop codon would be encountered. Generally, such a frame-shift mutation (especially where the mutation is near the 5' end of the gene) yields proteins which do no longer have the biological function of the enzyme encoded by the original gene. Alternatively, a stop codon can be inserted in the gene, which causes termination of the

production of the amino acid sequence at that point, which thus results in the production of N-terminally truncated proteins. Also in this case, when the mutation is located near the 5' end of the gene, the resulting truncated protein will no longer have any biologic functionality.

3) introduction of a protein binding site. The insertion of a protein binding site will cause attachment of the corresponding protein (if present), thereby introducing steric hindrance for transcription of the gene. Preferably such a binding site is introduced in front of or in the neighborhood of the start codon (either in the promoter sequence or in the coding sequence) and the presence of an attached protein will hinder the polymerase to start or continue transcription. This again enables a regulatable system, whereby the amount of transcription can be regulated by the amount of protein that is available for binding. Preferred binding sites for such a system are those specific for the transcriptional repressor protein CreA relevant for carbon- catabolite-repression in Aspergillus species (Mathieu, M. et al., 2005, Mol. Microbiol. 56(2):535-548; Felenbok, B. et al., 2001, Prog. Nucleic Acid Res. Mol. Biol. 69:149-204; Mathieu, M. and Felenbok, B., 1994, EMBO J.

13(17):4022-4027). 4) a change in gene splicing. A eukaryotic gene typically is present in a structure in which parts having a coding sequence (called 'exons') are interspersed with parts having a non-coding sequence ('introns'). For a correct expression of the eukaryotic gene the whole gene is transcribed into (pre-)mRNA, but then the introns are spliced out of the RNA to result in a final mRNA only having coding sequences (and some regulatory sequences, such as a poly- A tail).

It is also known in especially in various diseases alternative RNA

transcripts can be produced from the same genomic region of DNA. These alternative transcripts are generally known as "variants." More specifically, "pre-mRNA variants" are transcripts produced from the same genomic DNA that differ from other transcripts produced from the same genomic DNA in either their start or stop position and contain both intronic and exonic sequence. Upon excision of one or more exon or intron regions, or portions thereof during splicing, pre-mRNA variants produce smaller "mRNA variants." Consequently, mRNA variants are processed pre-mRNA variants and each unique pre-mRNA variant must always produce a unique mRNA variant as a result of splicing. These mRNA variants are also known as "alternative splice variants." If no splicing of the pre-mRNA variant occurs then the pre-mRNA variant is identical to the mRNA variant.

It is also known in nature such variants can be produced through the use of alternative signals to start or stop transcription and that pre- mRNAs and mRNAs can possess more than one start codon or stop codon. Variants that originate from a pre-mRNA or mRNA that use alternative start codons are known as "alternative start variants" of that pre-mRNA or mRNA. Those transcripts that use an alternative stop codon are known as "alternative stop variants" of that pre-mRNA or mRNA. One specific type of alternative stop variant is the "polyA variant" in which the multiple transcripts produced result from the alternative selection of one of the "polyA stop signals" by the transcription machinery, thereby producing transcripts that terminate at unique polyA sites.

As used herein, "antisense mechanisms" are all those involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.

As used herein, the terms "precursor mRNA" or "pre-mRNA" refer to an immature single strand of messenger ribonucleic acid (mRNA) that contains one or more intervening sequence(s) (introns). Pre-mRNA is transcribed by an RNA polymerase from a DNA template in the cell nucleus and is comprised of alternating sequences of introns and coding regions (exons). Once a pre-mRNA has been completely processed by the splicing out of introns and joining of exons, it is referred to as "messenger RNA" or "mRNA," which is an RNA that is completely devoid of intron sequences. Eukaryotic pre-mRNAs exist only transiently before being fully processed into mRNA. When a pre-mRNA has been properly processed to an mRNA sequence, it is exported out of the nucleus and eventually translated into a protein by ribosomes in the cytoplasm.

As used herein, the terms "splicing" and "(pre-)mRNA processing" refer to the modification of a pre-mRNA following transcription, in which introns are removed and exons are joined. Pre-mRNA splicing involves two sequential biochemical reactions. Both reactions involve the spliceosomal transesterification between RNA nucleotides. In a first reaction, the 2'-OH of a specific branch-point nucleotide within an intron, which is defined during spliceosome assembly, performs a nucleophilic attack on the first nucleotide of the intron at the 5' splice site forming a lariat intermediate. In a second reaction, the 3'-OH of the released 5' exon performs a nucleophilic attack at the last nucleotide of the intron at the 3' splice site thus joining the exons and releasing the intron lariat. Pre-mRNA splicing is regulated by intronic silencer sequence (ISS), exonic silencer sequences (ESS) and terminal stem loop (TSL) sequences.

As used herein, "modulation of splicing" refers to altering the processing of a pre-mRNA transcript such that there is an increase or decrease of one or more splice products, or a change in the ratio of two or more splice products. Modulation of splicing can also refer to altering the processing of a pre-mRNA transcript such that a spliced mRNA molecule contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or additional sequence not normally found in the spliced mRNA (e.g., intron sequence).

As used herein, "splice site" refers to the junction between an exon and an intron in a pre-mRNA (unspliced RNA) molecule (also known as a "splice junction"). A "cryptic splice site" is a splice site that is not typically used but may be used when the usual splice site is blocked or unavailable or when a mutation causes a normally dormant site to become an active splice site. An "aberrant splice site" is a splice site that results from a mutation in the native DNA and pre-mRNA.

Changing the splicing of a gene and thereby producing expression products that are not or only partial functional may be achieved by blocking the splice sites that are needed for a proper expression of the gene.

Changing of the splicing may be effected by introducing antisense oligomeric compounds, generally oligonucleotides or oligonucleotide analogs or mimetics, that are capable of interacting with and/or hybridizing to a pre- mRNA thereby modifying gene expression and/or splicing. Oligomeric compounds can be introduced in the form of single-stranded, double- stranded, circular, branched or hairpins and can contain structural elements such as internal or terminal bulges or loops. Oligomeric double- stranded compounds can be two strands hybridized to form double- stranded compounds or a single strand with sufficient self complementarity to allow for hybridization and formation of a fully or partially double-stranded compound. Enzyme- dependent antisense oligonucleotides include forms that are dependent on RNase H activity to degrade target mRNA, and include single-stranded DNA, RNA, and phosphorothioate antisense. Steric blocking antisense oligonucleotides (RNase-H independent antisense) interfere with gene expression or other mRNA- dependent cellular processes by binding to a target sequence of mRNA. Steric blocking antisense includes 2'-0 alkyl antisense oligonucleotides, morpholino antisense oligonucleotides, and tricyclo-DNA antisense oligonucleotides.

In the current invention blocking of splicing sites that cause splicing of intron 1 with the help of oligomeric compounds as defined above would lead to the expression of a non-functional protein.

Another embodiment for providing inhibition of the expression of the enzyme itaconyl-CoA transferase and/or citramalyl-CoA hydro-lyase

(itaconyl-CoA hydratase) and/or citramalyl-CoA lyase is formed by silencing of the expression of the gene. Basically, three methods for silencing are known at this moment and are contemplated in this application: antisense expression, sense co-suppression and RNA-inhibition. However, the invention is not limited to these methods and any other method which causes silencing of the genes coding for the enzyme itaconyl-CoA

transferase and/or citramalyl-CoA hydro-lyase (itaconyl-CoA hydratase) and/or citramalyl-CoA lyase is included.

For antisense expression, a nucleotide sequence coding for said gene, its homologue or variant, or at least a part thereof of 40 nucleotides or more, is put behind a suitable promoter in anti- sense direction. After transcription of this nucleotide sequence an mRNA is produced which is complementary to the mRNA formed through transcription of the endogenous female suppressor gene. It is well proven by now that production of such an anti- sense mRNA is capable of inhibition of the endogenous expression of the gene for which it is complementary. Furthermore, it has been proven that to achieve this effect even sequences with a less than 100% homology are useful. Also antisense mRNA's which are shorter than the endogenous mRNA which they should inhibit can be used. Generally, it is accepted that mRNA sequences of 23 nucleotides or more which have an identity of 70% or more will be capable of generating an inhibitory effect. The principal patent reference is EP 240,208 of Calgene Inc. There is no reason to doubt the operability of antisense technology. It is well-established, used routinely in laboratories around the world and products in which it is used are on the market.

The second approach is commonly called sense co- suppression. This phenomenon occurs when the gene or part of said gene is expressed in its sense direction. Although this kind of expression when full length genes are used most often results in overexpression of the gene, it has been found that in some cases and especially in cases when a sequence shorter than the full length sequence is used, expression of this gene or fragment causes inhibition of the endogenous gene. The principal patent reference on sense co- suppression is EP 465,572 in the name of DNA Plant Technology Inc.

Sense and antisense gene regulation is reviewed by Bird and Ray (Gen. Eng. Reviews 9: 207-221, 1991). Gene silencing can thus be obtained by inserting into the genome of a target organism an extra copy of the target female suppressor gene coding sequence which may comprise either the whole or part or be a truncated sequence and may be in sense or in antisense orientation. Additionally, intron sequences which are obtainable from the genomic gene sequence may be used in the construction of suppression vectors. There have also been reports of gene silencing being achieved within organisms of both the transgene and the endogenous gene where the only sequence identity is within the promoter regions.

The third possible way to silence genes is by using the so-called RNAi technology, which covers all applications in which double-stranded RNAs are used to achieve silencing of an endogenous gene. As has been demonstrated by Fire et al. (Nature, 391: 806-811, 1998) application of a dsRNA of which one strand is at least partly complementary to the endogenously produced mRNA whether produced intracellularly or added extracellularly is extremely capable of inhibiting translation of the mRNA into a protein. It is believed that this phenomenon works through the intermediate production of short stretches of dsRNA (with a length of 23 nucleotides). To achieve production of dsRNA a construct is made harboring both a sense and an antisense nucleotide sequence (together also called an inverted repeat) of at least 19, usually 23 nucleotides or more, of which one is complementary to the endogenous gene which needs to be silenced. The sense and antisense nucleotide sequences can be connected through a spacer nucleotide sequence of any length which allows for a fold back of the formed RNA so that a double stranded RNA is formed by the sense and antisense sequence. The spacer then serves to form the hairpin loop connecting both sense and antisense sequence. The order of the sense and antisense sequence is not important. It is also possible to combine more than one sense-antisense combination in one and the same construct. If the simple form is depicted as: prom - S - spac - AS - term, also the following constructs can be applied: prom - Si - spac - ASl - spac - S2 - spac - AS2 - term, or prom - S2 - spac - Si - spac - ASl - spac - AS2 - term.

Variations in the built up of the construct are possible, as long as the end product of the transcription of said constructs yields one or more dsRNAs. Alternatively, the double stranded structure may be formed by two separate constructs coding for complementary RNA strands, where RNA duplex formation occurs in the cell. In short notation these constructs then look like: proml-Sl-terml and prom2-ASl-term2. Proml and prom2 can be the same or different but should both be constitutive or fruit- specific promoters, terml and term2 can be the same or different. Both constructs can be introduced into the cell on the same vector, but can also be introduced using two different vectors. RNA containing nucleotide sequences identical to a portion of the target female suppressor gene are preferred for inhibition. RNA sequences with insertions, deletions and single point mutations relative to the target sequence have also been found effective for inhibition. Thus, sequences with a sequence identity of less than 100% may be used. Sequence identity may be calculated by sequence comparison and alignment algorithms known in the art (see Gribskov and Devereux, Sequence Analysis Primer, Stockton Press, 1991, and references cited therein), for instance by using the Smith- Waterman algorithm as implemented in the BESTFIT software program using default parameters (e.g. University of Wisconsin Computing Group). Thus, the duplex region of the RNA may be defined functionally as a (double stranded) nucleotide sequence that is capable of hybridizing with a portion of the target gene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C to 65°C hybridization for 12-16 hours; followed by

washing). The length of the identical nucleotide sequences should be at least 23 nucleotides, but preferably larger: 40, 50, 100, 200, 300 or 400 bases.

As disclosed herein, 100% sequence identity between the inhibiting construct and the target endogenous gene is not required to practice the present invention. Thus the invention has the advantage of being able to tolerate sequence variations that might be expected due to genetic mutation, strain polymorphism or evolutionary divergence.

Thus also included in the invention are constructs having a nucleotide sequence under control of a suitable promoter wherein said nucleotide sequence comprises a part of 40 or more nucleotides in a sense direction, or in an antisense direction or in an inverted repeat form, of the sequence of the gene coding for the enzyme itaconyl-CoA transferase and/or citramalyl- CoA hydro-lyase (itaconyl-CoA hydratase) and/or citramalyl-CoA lyase, or homologues or variants thereof. Alternatively, transcription is prevented by means of the expression of a negatively acting transcription factor acting on the target gene promoter. Such negatively acting transcription factor can be natural or artificial. Artificial negatively acting transcription factors can be employed by the overexpression of an engineered polydactyl zinc-finger transcription factor coupled to a general transcription repressor. According to a further embodiment, the interfering with the target gene consists of destabilizing the target gene mRNA, in particular' by means of nucleic acid molecules that are complementary to the target gene mRNA selected from the group consisting of antisense RNA, RNAi molecules. Virus Induced Gene Silencing (VIGS) molecules, co- suppressor molecules, RNA oligonucleotides or DNA oligonucleotides.

In another embodiment the interfering with the target gene consists of inhibiting the target gene expression product. This can be achieved by means of the expression product(s) of one or more dominant negative nucleic acid constructs, overexpression of one or more suppressors which interact with the target gene product, or by means of one or more chemical compounds. Novel ways to introduce site-specific alterations in transcription of an (eukaryotic) gene is by a variation in the recently described CRISPR- Cas genetic engineering, homologous recombination system. (Cong L et al. Science 2013; 339: 819-823; Mali P et al. Science 2013; 339: 823-826; Cho SW et al. Nat Biotechnol 2013; 31: 230-232; Jinek M et al. Elife 2013; 2: e00471). This variation entails the use of a Cas enzyme that is defective in endonuclease activity, but which retains its ability, when co-expressed with a gRNA, to specifically interfere with transcriptional elongation, RNA polymerase binding or transcription factor binding. This system is also indicated as CRISPRi.(Qi LS et al. Cell 2013; 152: 1173-1183; Larson, MH et al 2013, Nature Protocols 8:2180-2196; Amelio, I. and Melino G., 2015, Cell Death & Differentiation, 22: 3-5). The above-described systems are all systems that act on expression and do not change the underlying genetic sequence of the gene. In that respect these systems are also relatively easy to switch on or switch off at moments when suppression of expression is needed or when suppression of expression is no longer needed. Such a switch can e.g. advantageously be effected by putting the expression of one or all of the components of the silencing system under control of a specific time- or location-restrained promoter.

Next to changes in the expression of the gene, the gene itself may be changed in such a way that no longer a functional protein is expressed. This may be achieved by mutating the gene. The one or more mutations can be introduced randomly by means of one or more chemical compounds and/or physical means and/or by insertion of genetic elements. Suitable chemical compounds are ethyl methanesulfonate, nitrosomethylurea, hydroxylamine, proflavine, N-methyl-N-nitrosoguanidine, N-ethyl-N-nitrosourea, N-methyl- N-nitro" nitrosoguanidine, diethyl sulfate, ethylene imine, sodium azide, formaline, urethane, phenol and ethylene oxide, Physical means that can be used comprise UV-irradiation, fast-neutron exposures X-rays and gamma irradiation. The genetic element is a transposon, T-DNA, or retroviral element.

More efficient and targeted techniques are provided for by so-called site-directed mutagenesis techniques. Many systems for site-directed mutagenesis (SDM) are known to the skilled person, the most notorious being nuclease based SDM systems such as zinc finger nucleases,

transcription activator-like effector nucleases (TALENs), and LAGLIDADG homing endonucleases (Curtin, S.J. et al., 2012, The Plant Genome 5:42-50). Another technology for SDM is based on homologous recombination with the target gene. Very recently, the above discussed CRISPR-Cas system has been proven very effective for SDM based on homologous recombination (see e.g. WO2014/144155). In an embodiment, TALEN (Transcription Activator-like Effector Nuclease) protein or enzyme is used to disrupt or inactivate one or more genes of the itaconic acid degradation or biochemical conversion pathways of a cell. In such an embodiment, TALEN (Transcription Activator-like

Effector Nuclease) protein or enzyme is used to disrupt or inactivate or mutate gene selected from the group comprising itaconyl-CoA transferase, itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase. A TALEN protein is made of a DNA binding domain and a nuclease domain. The DNA binding domain also has 2 parts - the TAL domain that identifies sequences left to the double strand break (DSB) target is termed TAL-L and the TAL domain that identifies sequence right to the DSB target is termed TAL-R. Both TAL-L and TAL-R domains are expressed as fusion protein with the nuclease domain. The natural TAL effector proteins have two domains: an effector domain and a DNA-binding domain. The structure of the DNA-binding domain can be manipulated such that the domain binds specifically to any DNA sequence in the genome. These DNA-binding protein domains can be linked to a customized effector domain such as a nuclease, thus producing a chimeric TALEN (Transcription Activator-like Effector Nuclease) protein. The DNA-binding domain which provides DNA sequence specificity of TALE/TALEN, consists of a variable number of amino acid repeats. Each repeat contains 33-35 amino acids and recognizes a single DNA base pair. The DNA recognition occurs via 2 hypervariable amino acid residues at positions 12 and 13 within each repeat, called

Repeat- Variable Di-Residues (RVDs), which are critical for recognizing specific DNA sequences. The RVDs of the repeats in TAL effectors can be varied to create a TAL protein that recognizes a specific target DNA sequence. RVD is specific to a simple cipher like, NI = A, HD = C, NG = T, NN = G or A (Boch, 2009; Moscou, 2009). N, I, H, D, and G represent one letter amino acid codes. The repeats of DNA binding domain are assembled in a TALE expression vector and co- expressed with a nuclease Fokl endonuclease catalytic domain to create TALE nuclease (TALEN). Such TALENs, once expressed in the cell, bind sequence specifically and create a double stranded break which is repaired by Non Homologous End Joining (NHEJ). During such cellular processes, mutations, i.e. either deletions and/or insertions within the gene sequence render nonfunctional protein products.

Next to induced mutations also spontaneous mutations can occur that result in improved itaconic acid resistance and improved itaconic acid levels due to reduced degradation and or biochemical conversion. For the resistant mutants that are exemplified in the present application the exact nature of the mutation is not (yet) known, but the mutation is resided in one of the degradation or biochemical conversion pathways of itaconic acid.

"Clustered Regularly Interspaced Short Palindromic Repeats" and "CRISPRs", as used interchangeably herein refers to loci containing multiple short direct repeats that are found in the genomes of

approximately 40% of sequenced bacteria and 90% of sequenced archaea. The CRISPR system is a microbial nuclease system involved in defense against invading phages and plasmids that provides a form of acquired immunity. The CRISPR loci in microbial hosts contain a combination of

CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. Short segments of foreign DNA, called spacers, are incorporated into the genome between CRISPR repeats, and serve as a 'memory' of past exposures. Cas9 forms a complex with the 3' end of the sgRNA, and the protein-RNA pair recognizes its genomic target by complementary base pairing between the 5 ' end of the sgRNA sequence and a predefined 20 bp DNA sequence, known as the protospacer. In nature this complex is directed to homologous loci of pathogen DNA via regions encoded within the crRNA, i.e., the protospacers, and protospacer-adjacent motifs (PAMs) within the pathogen genome. The non-coding CRISPR array is transcribed and cleaved within direct repeats into short crRNAs containing individual spacer sequences, which direct Cas nucleases to the target site (protospacer). By simply exchanging the 20 bp recognition sequence of the expressed sgRNA, the Cas9 nuclease can be directed to new genomic targets. CRISPR spacers are used to recognize and silence exogenous genetic elements in a manner analogous to RNAi in eukaryotic organisms.

Three classes of CRISPR systems (Types I, II and III effector systems) are known. The Type II effector system carries out a targeted DNA double- strand break in four sequential steps, using a single effector enzyme, Cas9, to cleave dsDNA. Compared to the Type I and Type III effector systems, which require multiple distinct effectors acting as a complex, the Type II effector system may function in alternative contexts such as eukaryotic cells. The Type II effector system consists of a long pre-crRNA, which is transcribed from the spacer-containing CRISPR locus, the Cas9 protein, and a tracrRNA, which is involved in pre-crRNA processing. The tracrRNAs hybridize to the repeat regions separating the spacers of the pre-crRNA, thus initiating dsRNA cleavage by endogenous RNase III. This cleavage is followed by a second cleavage event within each spacer by Cas9, producing mature crRNAs that remain associated with the tracrRNA and Cas9, forming a Cas9:crRNA-tracrRNA complex.

The Cas9:crRNA-tracrRNA complex unwinds the DNA duplex and searches for sequences matching the crRNA to cleave. Target recognition occurs upon detection of complementarity between a "protospacer" sequence in the target DNA and the remaining spacer sequence in the crRNA. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3 ' end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage. Different Type II systems have differing PAM requirements. The S. pyogenes CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5'-NRG-3', where R is either A or G, and

characterized the specificity of this system in human cells. A unique capability of the CRISPR/Cas9 system is the straightforward ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, the

Streptococcus pyogenes Type II system naturally prefers to use an "NGG" sequence, where "N" can be any nucleotide, but also accepts other PAM sequences, such as "NAG" in engineered systems (Hsu et al, Nature

Biotechnology (2013) doi: 10.1038/nbt.2647). Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9) normally has a native PAM of NNNNGATT, but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN PAM (Esvelt et al. Nature Methods (2013) doi: 10.1038/nmeth.2681).

An engineered form of the Type II effector system of Streptococcus pyogenes was shown to function in eukaryotic cells for genome engineering. In this system, the Cas9 protein was directed to genomic target sites by a

synthetically reconstituted "guide RNA" ("gRNA", also used interchangeably herein as a chimeric single guide RNA ("sgRNA")), which is a crRNA- tracrRNA fusion that obviates the need for RNase III and crRNA processing in general.

In the present invention CRISPR/Cas9-based engineered systems may be used in genome editing of the target organism. The CRISPR/Cas9-based engineered systems may be designed to target any gene, but for the use in the present invention especially a gene selected from the group consisting of itaconyl-CoA transferase, itaconyl-CoA hydratase (citramalyl-CoA hydrolase) and/or citramalyl-CoA lyase. The CRISPR/Cas9- based systems may include a Cas9 protein or Cas9 fusion protein and at least one gRNA. The Cas9 fusion protein may, for example, include a domain that has a different activity that what is endogenous to Cas9, such as a transactivation domain. The CRISPR/Cas9-based system may include a Cas9 protein or a Cas9 fusion protein. Cas9 protein is an endonuclease that cleaves nucleic acid and is encoded by the CRISPR loci and is involved in the Type II CRISPR system. The Cas9 protein may be from any bacterial or archaea species, such as Streptococcus pyogenes. The Cas9 protein may be mutated so that the nuclease activity is inactivated. An inactivated Cas9 protein from Streptococcus pyogenes (iCas9, also referred to as "dCas9") with no endonuclease activity has been recently targeted to genes in bacteria, yeast, and human cells by gRNAs to silence gene expression through steric hindrance. As used herein, "iCas9" and "dCas9" both refer to a Cas9 protein that has the amino acid substitutions DIOA and H840A and has its nuclease activity inactivated. The CRISPR/Cas9-based system may alternatively include a Cas fusion protein. The fusion protein may comprise two heterologous polypeptide domains, wherein the first polypeptide domain comprises a Cas protein and the second polypeptide domain has nuclease activity that is different from the nuclease activity of the Cas9 protein. The fusion protein may include a Cas9 protein or a mutated Cas9 protein, as described above, fused to a second polypeptide domain that has nuclease activity. A nuclease, or a protein having nuclease activity, is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits of nucleic acids. Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall in both categories. Well known nucleases are deoxyribonuclease and

ribonuclease.

The gRNA provides the targeting of the CRISPR/Cas9-based system. The gRNA is a fusion of two noncoding RNAs: a crRNA and a tracrRNA. The sgRNA may target any desired DNA sequence by exchanging the sequence encoding a 20 bp protospacer which confers targeting specificity through complementary base pairing with the desired DNA target. gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II effector system. This duplex, which may include, for example, a 42-nucleotide crRNA and a 75 -nucleotide tracrRNA, acts as a guide for the Cas9 to cleave the target nucleic acid. The "target region", "target sequence" or "protospacer" as used interchangeably herein refers to the region of the target gene to which the CRISPR/Cas9-based system targets. In the present invention this would be a target region in a gene selected from the group consisting of itaconyl-CoA transferase, itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase. The CRISPR/Cas9-based system may include at least one gRNA, wherein the gRNAs target different DNA sequences. The target DNA sequences may be overlapping. The target sequence or protospacer is followed by a PAM sequence at the 3 ' end of the protospacer. Different Type II systems have differing PAM requirements. For example, the Streptococcus pyogenes Type II system uses an "NGG" sequence, where "N" can be any nucleotide.

The gRNA may target any nucleic acid sequence such as the genes mentioned above. The CRISPR/Cas9-based system may use gRNA of varying sequences and lengths. The gRNA may comprise a complementary polynucleotide sequence of the target DNA sequence followed by a PAM sequence. The gRNA may comprise a "G" at the 5' end of the complementary polynucleotide sequence. The gRNA may comprise at least a 10 base pair, at least an 1 1 base pair, at least a 12 base pair, at least a 13 base pair, at least a 14 base pair, at least a 15 base pair, at least a 16 base pair, at least a 17 base pair, at least an 18 base pair, at least a 19 base pair, at least a 20 base pair, at least a 21 base pair, at least a 22 base pair, at least a 23 base pair, at least a 24 base pair, at least a 25 base pair, at least a 30 base pair, or at least a 35 base pair complementary polynucleotide sequence of the target DNA sequence, wherein said target sequence is derived from the coding sequence of a gene selected from the group consisting of itaconyl-CoA transferase, itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase, followed by a PAM sequence. The PAM sequence may be "NGG", where "N" can be any nucleotide. The gRNA may target at least one of the promoter region, the enhancer region or the transcribed region of the target gene.

Two or more sequences (polynucleotide or amino acid) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between two aligned sequences divided by the length of the shorter sequences and multiplied by 100. An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research

Foundation, Washington, D.C., USA, and normalized by Gribskov, Nucl. Acids Res. 14(6):6745-6763 (1986). An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility

application. The default parameters for this method are described in the Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, Wis.). A preferred method of establishing percent identity in the context of the present disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S.

Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif.). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects sequence identity. Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; expect=10;

Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+Swiss protein+Spupdate+PIR. Details of these programs can be found at the following internet address: http://www.ncbi.nlm.gov/cgi- bin/BLAST. With respect to sequences described herein, the range of desired degrees of sequence identity is approximately 80% to 100% and any integer value there between. Typically the percent identities between sequences are at least 70-75%, preferably 80-82%, more preferably 85-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity. Alternatively, the degree of sequence similarity between polynucleotides can be determined by hybridization of polynucleotides under conditions that allow formation of stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments. Two nucleic acid, or two polypeptide sequences are substantially homologous to each other when the sequences exhibit at least about 70%- 75%, preferably 80%- 82%, more preferably 85%-90%, even more preferably 92%, still more preferably 95%, and most preferably 98% sequence identity over a defined length of the molecules, as determined using the methods above. As used herein, substantially homologous also refers to sequences showing complete identity to a specified DNA or polypeptide sequence. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra; Nucleic Acid Hybridization: A Practical Approach, editors B. D. Hames and S. J. Higgins, (1985) Oxford; Washington, D.C.; IRL Press).

Selective hybridization of two nucleic acid fragments can be determined as follows. The degree of sequence identity between two nucleic acid molecules affects the efficiency and strength of hybridization events between such molecules. A partially identical nucleic acid sequence will at least partially inhibit the hybridization of a completely identical sequence to a target molecule. Inhibition of hybridization of the completely identical sequence can be assessed using hybridization assays that are well known in the art (e.g., Southern (DNA) blot, Northern (RNA) blot, solution

hybridization, or the like, see Sambrook, et al., Molecular Cloning: A

Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). Such assays can be conducted using varying degrees of selectivity, for example, using conditions varying from low to high stringency. If conditions of low stringency are employed, the absence of non-specific binding can be assessed using a secondary probe that lacks even a partial degree of sequence identity (for example, a probe having less than about 30% sequence identity with the target molecule), such that, in the absence of non-specific binding events, the secondary probe will not hybridize to the target.

The (recombinant DNA) constructs for use in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The recombinant gene constructs may be inserted into vectors, which may be commercially available, suitable for introducing into micro-organisms and suitable for expression of the gene product in the transformed cells.

Selectable markers, which may be included as a part of the introduced recombinant DNA, are used to select transformed or transfected cells (those containing recombinant DNA) over untransformed cells. Examples of suitable markers include genes that provide antibiotic resistance. Cells containing the recombinant DNA are capable of surviving in the presence of antibiotic concentrations that kill

untransformed/untransfected cells.

The target organism is a micro-organism which is capable of producing itaconic acid. This property may be endogenous, or it may be introduced into the cell by recombinant genetic technologies. Examples of endogenously itaconic acid producing cells are cells of Aspergillus terreus, more particularly A. terreus strain TN484-M1 (high yield) and strain CM85J (low yield) (Dwiarti, L. et al., 2002, J. Biosci. Bioeng. 1:29-33), strain NRRL 1960 (Riscaldati, E. et al., 2000, J. Biotechnol. 83(3):219-230; Bonarme, P. et al., 1995, J. Bacteriol. 177(12):3573-3578) and strain L.S.H.T.M. Cat. No. Am. 1 (Calam, C.T. et al., 1939, Thorn. J. Biochem. 33:1488-1495) and of A. itaconicus (Kinoshita, 1931, Bot. Mag. 45:30), Ustilago maydis (WO

2015/140314; Geiser, E. et al., 2016, Microb Biotechnol. (9)1:116-126) and Aspergillus oryzae (Jimenez-Quero, A. et al., 2016, J Microbiol Biotechnol., doi: 10.4014/jmb.l603.03073).

Alternatively, a non-natural itaconic acid producing strain can be constructed by introducing the gene for the enzyme cis-aconitic acid decarboxylase derived from any of the above mentioned strains in another micro-organism, preferably A. niger. This enzyme converts cis-aconitic acid into itaconic acid. If the host organism lacks sufficient substrate, then it can be contemplated to also introduce the gene(s) coding for the enzyme aconitase and/or the enzyme 2-methylcitrate dehydratase into the same host organism, which enzymes convert the ubiquitous substrate citrate into cis- aconitic acid. The genes coding for the above-mentioned enzymes can be derived from A. terreus according to techniques, which are generally known to a person skilled in the art. Also the construction of an expression vector with these genes and the introduction of such a vector into the host organism lies within the skill of the artisans.

In both cases, production of itaconic acid may be further enhanced by the presence of a transporter protein capable of transporting itaconic acid or precursors thereof over the mitochondrial membrane or to export itaconic acid out of the cell. Examples of such transporter proteins and genes are Aspergillus terreus ATEG_09970.1 or ATEG_09972.1 as described in WO

2009/110796 and WO 2009/104958.

With respect to Ustilago maydis it should be remarked that an alternative itaconic acid biosynthetic pathway was discovered in this microorganism (see Geiser, E. et al., 2016, Microb Biotechnol. (9)1:116-126).

Based on genome mining paralogous genes to this pathway are also present in A. niger:

Trans-aconitase decarboxylase An01g02970 Aconitate isomerase Anl3g01480 However the low level of homology and different genomic organisation of the pathway genes make it unlikely that this pathway is functional in A. niger. Key in the biosynthetic pathway for itaconic acid is the localisation of the various substrates. It is thought that production of itaconic acid mainly occurs in the cytosol . In many biochemical pathways, the end-product is inhibiting its own production to prevent excess end- product in the biological system. Excess end-product will not only lead to loss of energy in an economical sense, it can also give rise to unwanted side effects such as toxicity. As is shown in the experimental part, it has been demonstrated that at the end of the batch culture of itaconic acid the production levels decrease, which is deemed due to degradation or biochemical conversion of itaconic acid. Moreover, in high level itaconic acid producing strains the biomass formation also decreases which is deemed to be due to the toxic effects of itaconic acid and/or one of its metabolites. It is contemplated that by depleting the cell of itaconic acid the formation of new itaconic acid will continue without end-product inhibition, thus giving—in total- an increase yield of itaconic acid. This can be established by

additionally providing the culturing organism with transporters that transport the itaconic acid precursors to the cytosol and which transport the itaconic acid outside the cell. Such transporters have been shown and exemplified in WO 2009/104958 and WO 2009/110796. The genes and methods that are claimed herein are incorporated in the present application by reference.

Additionally the present invention enables an alternative approach by providing mutants with reduced itaconic acid sensitivity alleviating unwanted toxic side effects. Next to the effect of a decrease of end-product inhibition, the present invention also prevents toxic effects caused by an accumulation of products that are produced by the enzymes that break down or convert the itaconic acid or its precursoers, the enzymes itaconyl-CoA transferase and/or itaconyl-CoA hydratase (citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase . It is believed that the effect of the so-called organic acid toxicity (which then would be caused by the presence of the itaconic acid) is not caused by the itaconic acid itself, but by the CoA-ester generated in the first step of the metabolic pathway departing from the organic acid. Hence, inhibition of the enzymes as indicated will give rise to a higher concentration of itaconate without the toxic effects that are attributed to this higher amount of the organic acid, both inside the cell as well as excreted to the culture medium.

Although the enzymes as they occur in Aspergillus have a localization signal which targets the expression of these enzymes to the mitochondrion, inhibition of these genes has been found advantageous even if the itaconic acid is predominantly present in the cytosol. The theory to explain this is that probably there is still a form of active transport between cytosol and mitochondrion and if the itaconic acid is degraded in the mitochondrion and thus depleted, the balance should be restored by transporting itaconic acid to the mitochondrion. Accordingly, the amount of itaconic acid in the cytosol is thereby decreasing.

Degradation of itaconic acid has been shown to occur in the pathogenic bacteria Yersinia pestis and Pseudomonas aeruginosa. This pathway consists of three steps which degrade itaconic acid in the cellular building block chemicals pyruvate and acetyl- CoA. Itaconic acid is converted to itaconyl-CoA by the action of itaconyl-CoA transferase (ICT). Itaconyl- CoA is subsequently converted to citramalyl-CoA by the action of itaconyl- CoA hydratase (ICH) (citramalyl-CoA hydro-lyase) and citramalyl-CoA is cleaved by citramalyl-CoA lyase (CCL) into pyruvate and acetyl-CoA (Fig. 22) (adapted from Sasikaran et al., 2014, Nat. Chem. Biol. 10:371-377). Accordingly, inhibition of expression of any of these three genes blocks degradation and thereby increases the amount of itaconic acid and diminishes the toxic effects caused by the degradation products of itaconic acid.

As is extensively described herein, inhibition of expression of a gene can be achieved in many different ways. Of course, the inhibition is preferably directed against one or more of these enzymes as they occur in the itaconic acid producing micro-organism.

Micro-organisms used in the invention are preferably microorganisms that naturally produce itaconic acid. Preferably overexpression of the genes encoding the above described protein(s) and enzyme(s) is accomplished in filamentous fungi, yeasts and/or bacteria, such as, but not limited to Aspergillus sp., such as the fungi A. terreus, A. itaconicus and A. niger, Aspergillus nidulans, Aspergillus oryzae or Aspergillus fumigatus, Ustilago zeae, Ustilago maydis, Ustilago sp., Candida sp., Yarrowia lipolytica, Rhodotorula sp. and Pseudozyma antarctica, the bacterium E. coli and the yeast Saccharomyces cerevisiae. Especially preferred are

heterologous citric acid producing organisms in which the substrates are available in the host organism.

It has also been established (see US 2004/0033570) that the so- called D4B segment of Aspergillus terreus, which comprises the CAD gene is responsible for the synthesis of lovastatin (see Fig. 2 in US 2004/0033570). Thus, it is submitted that also these micro-organisms which are known to produce lovastatin would be suitable candidates for the production of itaconic acid. Such micro-organisms include Monascus spp. (such as M.

ruber, M. purpureus, M. pilosus, M. vitreus and M. pubigerus), Penicillium spp. (such as P. citrinum, P. chrysogenum), Hypomyces spp., Doratomyces spp. (such as D. stemonitis), Phoma spp., Eupenicillium spp., Gymnoascus spp., Pichia labacensis, Candida cariosilognicola, Paecilomyces virioti, Scopulariopsis brevicaulis and Trichoderma spp. (such as T. viride).

Consequently also the CAD encoding part of the D4B segment and the enzyme with CAD activity for which it codes from these above- mentioned lovastatin producing micro-organisms are deemed to be suitable for use in the present invention. It further is contemplated that a

heterologous organism, which in nature does not or hardly produce itaconic acid like Aspergillus niger or Aspergillus oryzae can be used when providing such an organism with a functional pathway for expression of itaconic acid, by overexpression of the above mentioned genes.

A functional pathway for the expression of itaconic acid can be produced by transforming the organisms with the gene coding for cis- aconitate decarboxylase CAD (EC 4.1.1.6) such as such as the enzyme encoded by the nucleic acid sequence of ATEG_09971.1, this further comprises enzymes with similar activities (see EP07112895). The organism can then be further equipped with genes that influence the transport of the metabolic products, such as di/tricarboxylate transporters, capable of transporting, among others, cis-aconitate, citrate or isocitrate from the mitochondrion to the cytosol, preferably the gene encoded by the nucleic acid sequence of ATEG_09970.1. These subsequent processes will lead to an increase in cis-aconitate in the cytosol, which can be further converted to itaconic acid, using overexpression of the gene encoding the enzyme CAD.

Also optionally such organisms may also comprise an enzyme that is able to transport itaconic acid or itaconate over the cell membrane, such as the enzyme coded by the nucleic acid sequence of ATEG_09972.1.

Even further optimisation of the present invention can be achieved by modulating the activity of the regulator protein that comprises a zinc finger and a fungal specific transcription factor domain as can be found on the gene cluster that also comprises ATEG_09970, wherein this regulator protein is indicated as ATEG_09969.1.

Micro-organisms overexpressing these enzymes, methods to provide such micro-organisms and the sequence information of the genes from the ATEG cluster have been extensively described in the applications WO 2009/014437, WO 2009/104958 and WO 2009/110796, which are hereby incorporated by reference.

The above described processes alone or in combination lead to a subsequent increase of itaconic acid. The combination of improved

production and reduction of toxicity leads to an increase in itaconic acid yield by a suitable host. The above described genes are preferably derived from Aspergillus sp. like, Aspergillus terreus, Aspergillus niger, Aspergillus nidulans, Aspergillus oryzae or Aspergillus fumigatus. However, it is also possible to derive the genes from other itaconate producing micro-organisms such as XJstilago zeae, Ustilago maydis, Ustilago sp., Pseudozyma antarctica, Candida sp., Yarrowia lipolytica, and Rhodotorula sp. Recombinant host cells as described above can be obtained using methods known in the art for providing cells with recombinant nucleic acids. These include transformation, transconjugation, transfection or

electroporation of a host cell with a suitable plasmid (also referred to as vector) comprising the nucleic acid construct of interest operationally coupled to a promoter sequence to drive expression. Host cells of the invention are preferably transformed with a nucleic acid construct as further defined below and may comprise a single but preferably comprises multiple copies of the nucleic acid construct. The nucleic acid construct may be maintained episomally and thus 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, Biotechnology 9: 968-975) plasmids. Preferably, however, the nucleic acid construct is integrated in one or more copies into the genome of the host cell. Integration into the host cell's genome may occur at random by illegitimate recombination but preferably the nucleic acid construct is integrated into the host cell's genome by homologous recombination as is well known in the art of fungal molecular genetics (see e.g. WO 90/14423, EP-A-0 481 008, EP- A-0 635 574 and US 6,265,186) Most preferably for homologous

recombination the techniques is used as described for instance in WO 02/052026 and Krappmann, 2007, Fungal Biol. Rev. 21:25-29).

Transformation of host cells with the nucleic acid constructs of the invention and additional genetic modification of the fungal host cells of the invention as described above may be carried out by methods well known in the art. Such methods are e.g. known from standard handbooks, such as

Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of fungal host cells are known from e.g. EP-A-0 635 574, WO 98/46772, WO 99/60102 and WO 00/37671.

In a further aspect the invention relates to fermentation processes in which the transformed host cells of the invention are used for the conversion of a substrate into itaconic acid. A preferred fermentation process is an aerobic fermentation process. The fermentation process may either be a submerged or a solid state fermentation process.

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., "Special

Transformation Processes Using Fungal Spores and Immobilized Cells", Adv. Biochem. Eng. Biotech., (1997), Vol 55, pp. 179; Roussos et al.,

"Zymotis: A large Scale Solid State Fermenter", Applied Biochemistry and Biotechnology, (1993), Vol. 42, pp. 37-52; Smits et al., "Solid-State

Fermentation-A Mini Review, 1998), Agro-Food-Industry Hi-Tech,

March/April, pp. 29-36). 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 fermenter 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).

Preferred in the invention is a submerged fermentation process, which is performed in a fed-batch or repeated (fed-)batch mode. In a fed- batch fermentation there is a continuous input of feed containing a carbon source and/or other relevant nutrients in order to improve itaconic acid yields. The input of the feed can, for example, be at a constant rate or when the concentration of a specific substrate or fermentation parameter falls below some set point. In a repeated batch fermentation the culture is harvested at regular time-intervals by stopping the fermentation and retrieving the produced product from the medium. Next to refreshing the medium often also part of the microbial culture is discarded, while the rest is used as a new inoculum for a following batch culture.

It is preferred to use a host cell that naturally would contain the enzymes/transporters of the itaconic acid pathway as depicted in Fig. 21, and the enzymes/transporters of the citric acid pathways in the cytosol and mitochondrion. However, if the host would lack one or more of these genes, they can be co-introduced with the above described enzymes and proteins. Such a co-introduction can be performed by placing the nucleotide sequence of such a gene on the same plasmid vector as the above described genes, or on a separate plasmid vector.

Further, since the itaconic acid pathway is located partly in the cytosol and partly in the mitochondrion, it is contemplated that

overexpression of the genes/enzymes in either or both of those

compartments would be desirable. The person skilled in the art will know how to achieve overexpression in the cytosol or mitochondria by using the appropriate signal sequences. As is already described above, a further part of the invention is formed by a strain that is 'resistant' to the toxic effects of itaconic acid and/or its metabolites. Such a strain may be obtained through spontaneous mutation and selection of strains growing in the presence of concentrations of itaconic acid that are inhibitory to the non-mutant strains. Among these itaconic acid resistant strains are those strains producing high levels of itaconic acid. In such a way the two strains EE#25 and EE#26 that are deposited under the Budapest Treaty with the Westerdijk Fungal

Biodiversity Institute (formerly known as the Centraal Bureau

Schimmelcultures (CBS)) in Utrecht (The Netherlands) on 17 August 2017 with accession numbers CBS141661 and CBS141662 have been obtained. It is of course also possible to induce mutations by chemical or radiation treatment and selection for high itaconic acid producing strains.

EXAMPLES

Improved production of itaconic acid by reducing/removin itaconic acid degradation and/or biochemical conversion Introduction

The invention relates to the field of microbial production of itaconic acid in fungi. The invention comprises results showing increased itaconic acid levels by reducing toxicity degradation and/or biochemical conversion of itaconic acid, by

(1) reducing/removing itaconyl-CoA transferase, itaconyl-CoA hydratase

(citramalyl-CoA hydro-lyase) and/or citramalyl-CoA lyase activity from the production host.

(2) reducing IA sensitivity of the production host by mutant selection. Besides reducing itaconic acid levels modification in the conversion pathways can also affect the influence of itaconic acid sensitivity of an itaconic acid producing strain, since organic acid toxicity is often not mediated by the organic acid itself but by the CoA-ester generated in the first step of the conversion pathway. Disruption of CoA transferase activity could result in reduced toxicity effects. All strains and transformants used are listed in Table 1.

Strains CBS 141653, CBS 141655, CBS 141657, CBS 141659, CBS 141660, CBS 141661 and CBS 141662 have been deposited as 'safe deposits' on 21 July 2016 with the Westerdijk Fungal Biodiversity Institute (formerly known as the Centraal Bureau Schimmelcultures (CBS)). On 17 August

2017 these deposits were converted into deposits under the Budapest treaty. Strains with CBS no's 143047 to 143057 were deposited under the Budapest Treaty on 24 July 2017 in the CBS collection of the Westerdijk Fungal Biodiversity Institute (The Netherlands).

Example I:

Controlled fermentation of itaconic acid producing host strains shows reduced IA levels at the end of the fermentation Controlled fermentations were carried out with an itaconic acid producing Aspergillus terreus strain and itaconic acid producing A. niger transformant strains carrying the A. terreus cadA, mttA, and mfsA genes and

overexpressing the A. niger citB gene. As shown in Figure 1, Figure 2 and Figure 3 significant decrease in the itaconic acid levels was observed after prolonged fermentation suggesting itaconic acid degradation and/or biochemical conversion as a means of this decrease.

Fermentation conditions of A. terreus

The following conditions were used unless stated otherwise:

37°C

pH start 3.5, set point 2.3

DO set points Day 1: 75%

Day 2, 3, 4: 50%

Subsequent days: 25%

Preculture: 100 ml of the same medium as used in the fermentation medium (10 ⁷ spores/ml) in 500 ml Erlenmeyer flask with baffles, overnight, 37°C, 150 rpm

pH control: 4M KOH (Base), 1.5 M H ₃ PO ₄ (Acid)

Antifoam: Struktol (Schill & Seilacher)

Medium composition:

Per litre demineralised water: 2.36 g of NH4SO4, 0.11 g of KH2PO4, 2.08 g of MgSO ₄ *7H ₂ O, 0.13 g of CaCl ₂ *2H ₂ O, 0.074 g of NaCl, 0.2 mg of CuSO ₄ *5H ₂ O, 5.5 mg of Fe(III)SO ₄ *7H ₂ O, 0.7 mg of MnCl ₂ *4H ₂ O and 1.3 mg of ZnSO4*7H ₂ O and 100 g of glucose as a carbon source.

Controlled batch-cultivations with A. niger strains CitB #77 and #101 were performed on 5L scale benchtop New Brunswick Scientific fermenters

(BioFlo 3000) at 33°C. Starting pH was 3.5 after inoculation and medium was allowed to naturally acidify till pH 2.3 and then kept at pH 2.3 by addition of 4M KOH. Dissolved oxygen (DO) tension was 25% at moment of inoculation and DO dropped till 20% and kept at 20%. The system was calibrated with 100% sterile air as 100% DO and 100% N2 as 0% DO. The fermenter was inoculated by 72h old 100 mL baffled shakeflask cultures containing 1.0*10^8 spores. Medium composition for fermentation and pre- culture (M12+Cu) is listed in Table 2.

Controlled batch-cultivation with Q199 (CBS 143051), a transformant derived from an industrial citric acid production strain (CBS 140903), carrying the IA gene cluster (cadA, mttA, and mfsA) and overexpressing citB was performed at 35°C instead. The fermenter was inoculated with a 72h old 100 mL non-baffled shakeflask culture containing 1.0*10^6 spores. Medium composition for fermentation and pre-culture is listed in Table 2 with the following modifications: 160 g/L glucose was used instead of 100 g/L glucose and 1.43 g/L NH4NO3 and 0.00058 g/L FeC13*6 H2O were used instead of (NH4)2SO4 and FeSO4.

Strain Q199 (CBS 143051) was constructed via co-transformation of strain Aceet#l (CBS 143053) with separate expression cassettes containing mttA and mfsA and a pyrE selection marker. Strain Aceet #1 (CBS 143053) was constructed via co-transformation of AN39 pyrE #4 (CBS 143052), a pyrE mutant (created by fluoroorotic acid selection) of an industrial citric acid production strain (CBS 140903), with separate expression cassettes containing cadA-amdS and citB. Sequences of expression cassettes and transformations are described in Hossain et al., 2016, Microb. Cell Fact., 15:130.

Table 2: Composition of Medium 12+Cu, which is used as production medium for IA. Adapted from Li et al. 2012, BMC Biotechnol., 12:57.

Example 2:

Itaconic acid toxicity

Shakeflasks (500 mL) were prepared with 100 niL M12+Cu supplemented with the following IA concentrations: 0, 10, 20, 40, 75 g/1 and pH was adjusted to 2.3 by addition of KOH/H2SO4. The flasks were inoculated with 1 mL of overnight grown pre-culture. After 7 days the mycelium was harvested and the biomass was determined by measuring the dry weight. The effect of IA on the growth of A. niger was tested using the AB1.13 CAD 4.1 (CBS 141653) strain. As shown in Figure 4 the growth of A. niger is already hampered at concentrations of 10 g/1 IA in the extracellular medium. At even higher concentrations (20, 40 and 75 g/1) the detrimental effects of IA on growth become more evident. Based on this result we hypothesize that IA toxicity may indeed limit its production.

Example 3:

Increased IA levels result in reduced biomass formation during controlled fermentation

Again A. niger controlled batch- cultivations were performed on 5L scale benchtop New Brunswick Scientific fermenters (BioFlo 3000) at 33°C .

Starting pH was 3.5 after inoculation and medium was allowed to naturally acidify till pH 2.3 and then kept at pH 2.3 by addition of 4M KOH. Dissolved oxygen (DO) tension was 25% at moment of inoculation and DO dropped till 20% and kept at 20%. The system was calibrated with 100% sterile air as 100% DO and 100% N ₂ as 0% DO. The fermenter was inoculated by 72h old 100 mL baffled shakeflask cultures containing 1.0*10^8 spores. Medium composition for fermentation and pre-culture are listed in Table 2 above (adapted from Li et al. 2012, BMC Biotechnol., 12:57).

As shown in Figure 5 in two high IA producing A. niger transformants carrying the IA gene cluster (cadA, mttA, and mfsA) and overexpressing the citB gene concomitant to IA production the amount of biomass produced during controlled fermentation is lower than that of a low IA producing strain AB1.13 #49B (CBS 141657) showing the toxic effect of IA on fungal growth under these conditions. Example 4:

Isolation of A. niger mutant strains with reduced growth inhibition towards I A

In a laboratory evolution experiment performed with CitB #99 (CBS 141659) strain we have identified and isolated mutant strains with increased resistance towards IA. Selection was performed in several round of growth selection on agar plates with 50-100 g/1 of IA. From this evolution

experiment mutants were characterized on their IA sensitivity, IA

consumption and IA productivity. In a shakeflask experiment these strains were evaluated on their ability to grow in production medium with and without I A (40 g/1). The results of this experiment are shown in Figure 6 and 7. From Figure 6 it becomes apparent that all evolution mutants produce approximately the same amount of biomass when grown in medium devoid of IA. In medium that contains 40 g/1 IA evolution mutants EE #25

(CBS141661) and EE #26 (CBS141662) consistently show more biomass in the presence of 40 g/1 IA when compared to the parental strain CitB #99. Figure 7 shows the average inhibition of mycelial growth, confirming less growth inhibition of EE #25 and EE #26 compared to the parental strain CitB #99. Moreover, the ability of EE #25 and EE #26 to degrade IA, i.e. to take up IA from the medium and metabolize, was assessed together with CitB #99 and AB1.13 CAD 4.1 (CBS 141653), a strain carrying only the heterologous cadA gene. Shakeflasks containing production medium were supplemented with IA (20 g/1) and glucose (1 g/1 and 5 g/1). The supplementation with glucose was necessary for conidial germination. Shakeflasks containing production media and supplemented with 1 g/1 glucose was used as positive control. All four strains used in this experiment showed consumption of IA (Fig. 8). In medium supplemented with 20 g/1 IA and 1 g/1 glucose evolution mutant strains EE#25 and EE #26 consumed less IA from the medium compared to CitB #99 and AB1.13 CAD 4.1 (CBS 141653). The same effect can be seen in production medium (Table 2) supplemented with IA and 0.5% glucose (Fig. 8). Together IA evolution mutants EE #25 and EE #26 show decreased sensitivity towards IA and decreased consumption, making them

appropriate strains for improved IA production.

The two evolution mutants EE #25 and EE #26 were also compared to their parental strain CitB #99 for IA and CA production. Shakeflasks (125 mL) were filled with 25 mL working volume of IA production medium and inoculated with l.OxlO ⁶ spores per ml and incubated at 33°C for 5 days. After incubation supernatant was filtered through 0.22 μΜ filter and analyzed on HPLC. Biomass was determined by filtering on Whatmann filters and drying at 105°C for dry biomass. The two mutant under these conditions produced more IA while CA levels were lower in different cultivation media. In the M12+Cu reference medium 4-fold more IA and 2- fold less CA was produced (Fig. 9), corresponding with a 4-5 fold increase specific productivity (Fig. 10).

Example 5

Genome mining for itaconic acid catabolism In filamentous fungi the catabolic pathway for itaconic acid has recently been described based on homology searches using proteins encoded by itaconic acid catabolic pathway genes from bacteria such as

Pseudomonas / Yersinia, in which the degradation pathway has been described (Chen, M. et al., 2016, Appl. Microbiol. Biotechnol. DOI

10.1007/s00253-016-7554-0).

For both itaconyl-CoA transferase and itaconyl-CoA hydratase at least two possible orthologous genes were identified in Aspergillus niger and A.

terreus. Based on the results published by Chen et al. for A. terreus functional homologues for the pathway were identified by expression in E.coli.

For A. niger the orthologous genes (75-85% identity) were identified (see Table 3). Protein analysis of all three pathway genes indicate mitochondrial localization of the degradation pathway.

Table 3: Itaconic acid catabolic pathway protein sequences.

In bold are those protein sequences described in Chen et al., 2016 for A. terreus (ATEG_XXXXX) and their closest homologues in A. niger

(AnXXgXXXXX). The other sequences represent genes encoding paralogous proteins of the same gene family.

Itaconyl-CoA transferase

Blast alignments for these genes are provided in Fig. 11.

Blast alignments for these genes are provided in Fig. 12.

Blast alignments for these genes are provided in Fig. 13. Example 6:

Genome mining for Itaconic acid esterification

In relation to conversion of itaconic acid towards related methyl-itaconic esters recent publications (Zhao et al., 2015, WO2015181310 /

WO2015181312) showed the role of trans-aconitate methyltransferases (tmt-1) in the production of mono- and di-esters. Genome mining in A. terreus revealed the presence of a tmt-1 like methyl-transferase homologue (30% identity to S. cerevisiae tmt-1), with no mitochondrial targeting sequence prediction. In A. niger no close homologue to this protein was discovered. Many weak methyltransferase homologues are observed. Some of these genes are part of gene clusters. In A. niger ATCC 1015 additional / different homologues are present than in A. niger CBS 513.88. In both A. terreus and A. niger also a cis-aconitate 2-methyltransferase homologue is present, predicted to be mitochondrially localized (Table 4).

Table 4: Itaconic acid methyltransferase pathway.

Trans-aconitate 3-methyltransferase (tmt-1) ATEG_06208

Trans-aconitate 2-methyltransferase (tmt-2) ATEG_04223, Anl6g06510

Blast alignments for these genes are provided in Fig. 14. Example 7:

Genome mining for alternative IA biosynthetic pathway

Based on data by Geiser et al. (Microb Biotechnol. 2016 Jan; 9(1): 116-126.) in the filamentous fungus Ustilago maydis an alternative IA biosynthetic pathway was discovered. Based on genome mining paralogous genes to this pathway are also present in A. niger (see Table 5). Table 5: Alternative itaconic acid pathway.

The low level of homology and different genomic organization of these pathway genes make it somewhat unlikely that this pathway is functional in A. niger.

Blast alignments for these genes are provided in Fig. 15A and 15B.

Example 8:

Gene expression analysis

Based on transcriptome analysis in A. terreus (see accession GSE73033; Chen, M. et al., 2016, Appl. Microbiol. Biotechnol. DOI 10.1007/s00253-016- 7554-0) expression of the three catabolic pathway genes was induced under conditions of IA degradation (40 g/1 IA addition to culture medium) causing high itaconic acid levels, whereas the methyltransferase pathway genes were not induced (see Table 6).

Similarly, genome wide gene expression analysis of Aspergillus niger strains expressing a functional itaconic acid pathway grown in fermenters was carried out to identify genes co-regulated by the expression of the itaconic acid gene cluster. The following strains were investigated: AB1.13 WT, AB1.13 CAD 4.1 (CBS141653), AB1.13 #49B (CBS141657), CitB #99

(CBS141659), and CitB #113.

Controlled batch-cultivations were performed on 5L scale benchtop New Brunswick Scientific fermenters (BioFlo 3000) at 33°C. Starting pH was 3.5 after inoculation and medium was allowed to naturally acidify till pH 2.3 and then kept at pH 2.3 by addition of 4M KOH. Dissolved oxygen (DO) tension was 25% at moment of inoculation and DO dropped till 20% and kept at 20%. The system was calibrated with 100% sterile air as 100% DO and 100% N2 as 0% DO. The fermenter was inoculated by 72h old 100 mL baffled shakeflask cultures containing 1.0*10^8 spores. Medium

composition for fermentation and pre-culture (M12+Cu) are listed in Table 2. Biomass samples for RNA isolation were taken at several time points during fermentation and washed with distilled water and frozen in liquid N2. The mycelium was disrupted by bead-beating with 0.1 mm acid- washed Zirconium- Silica beads and RNA extraction proceeded using the

ChargeSwitch RNA extraction protocol from Invitrogen (Carlsbad, CA,

USA). Quality control was checked on lx MOPS/6% Formaldehyde agarose gels and stained with ethidium bromide.

BaseClear in Leiden, NL performed digital gene expression profiling experiments based on RNA-Seq with an Illumina HiSeq 2000 System.

Approximately 8-32 M unfiltered paired-end (PE) reads (99bp/read on ~320bp cDNA inserts) were obtained. Reads were trimmed of the first 2 bases of the 5' end because these bases showed an aberrantly low GC content. The reads were then further filtered, such that all quality phred scores after filtering are at least 22, with a read-length of at least 40 bases. Around 70-80% of the bases passed these criteria (including a 2% loss because of clipping). After filtering the # PE-reads/samples were between 7.6M and 19.8M for all the samples respectively.

Reads were aligned to the 20 contigs in a FastA file of the Aspergillus niger reference genome (from http://www.ebi.ac.uk/ena). Source EMBL

annotations were converted to GFF format. The EMBL data appeared to be derived from multiple sources with different feature tags. These were converted to one uniform GFF format that could be accepted by our third- party software (consistent gene_ids across all contigs). Missing gene definitions (e.g. for CAD) were inserted. The reads were aligned to the reference genome using software based on a Burrows-Wheeler Transform (BWT) algorithm. A mismatch rate of 4% was allowed for the alignment. The maximum insertion length was 3. The maximum deletion length was 3. All samples had more than 85% of the reads aligned, resulting in SAM alignment files. Gene expression was measured as the number of aligned reads to reference genes and was normalized to RPKM values (Reads Per Kb per Million reads; Mortazavi et al., 2008, Nature Methods, 5:621-628).

Several genes possibly related to itaconic acid breakdown were identified (Table 7). In addition, the gene encoding the trans-aconitate 2- methyltransierase (Anl6g06510) appeared to be induced by itaconic acid. It was also noted that in A. niger for all genes the induction levels were different for strains overexpressing different parts of the IA biosynthetic pathway and/or citB overexpression, indicating a role for organic acid transport and localization of product intermediates.

As indicated in Table 7 two genes of the proposed IA degradation pathway and the gene encoding a step in the itaconic acid methyltransferase pathway were clearly induced in IA producing A. niger transformant strains indicating a role of both pathways in the degradation and/or biochemical conversion of IA during controlled fermentation.

Example 9:

Host strain modification

Based on the results described in Tables 6 and 7 three genes were identified to be targeted for gene disruption, since their observed overexpression could lead to reduction of the level of itaconic acid produced, namely itaconyl- CoA transferase (An07g00760) and itaconyl-CoA hydratase (An07g09220)and citramalyl-CoA lyase (An01g08610) .

Targeted gene disruption of single genes in the catabolic pathway is deemed to result in accumulation of pathway intermediates (in particular CoA esters) which could lead to increased toxicity, therefore also combined disruption mutations are contemplated. Based on the annotated gene sequences four so-called splitmarker gene disruption cassettes were developed for these 3 genes (see Fig. 16) according to the method described in Arentshorst et al., 2015, Genetic Transformation Systems in Fungi, Vol 1, Fungal Biology, DOI 10.1007/978-3-319-10142- 2_25.

Transformations were done in two strains. CitB #99 pyrE #67 (CBS 143054) (selected pyrE mutant of high itaconic acid producing lab-strain CitB #99 (CBS 141659)) was co-transformed with pyrE splitmarkers to create ictA (An07g00760) deletion strain CitB #99 AICT #RD1 (CBS 143055), ichA (An07g09220) deletion strain CitB #99 AICH #RB2 (CBS 143056), and tmtA (Anl6g06510) deletion strain CitB #99 ΔΤΜΤ #6 (CBS 143057). Strain Q199 (CBS 143051) (high itaconic acid producing strain, derived from an industrial citric acid production strain) was co-transformed with hph splitmarkers to create ictA deletion strain Q199 AICT #4 (CBS 143050). In our fermentation experiments we saw that the highest producing strains also grew the poorest (see Fig. 2, 3 and 5) due to toxic effects of itaconic acid or one of its degradation products on Aspergillus niger growth. Another ictA deletion strain was created in the industrial citric acid production strain background using a different route. First, strain CAD MFS #15 (CBS 143047) was created by co-transformation of strain AN39 pyrE #4 (CBS 143052) with separate expression cassettes containing cadA-amdS and mfsA. Next, a selected pyrE mutant of CAD MFS #15, namely CAD MFS #15 pyrE #8 (CBS 143048) was co-transformed with separate expression cassettes containing cadA, mttA, mfsA, citB, and the hph splitmarkers to create ictA (An07g00760) deletion strain transformant CAD MFS #15 MTT CITB CAD MFS AICThygB #21 (CBS 143049). Co-transformations of the splitmarker fragments and expression cassettes were performed with PCR products. Sequences of expression cassettes and transformations are described in Hossain et al., 2016, Microb. Cell Fact., 15:130. A selection of transformants was purified and analysed by diagnostic PCR for the disruption of the respective target gene and presence of transformed genes. Correct gene- disruption transformants were identified and used for further research. Example 10:

Improved itaconic acid production

Itaconic acid producing A. niger strains carrying a deletion of itaconyl-CoA transferase (An07g00760) and/or itaconyl-CoA hydratase (An07g09220) and/or citramalyl CoA lyase (An01g08610)led to improved itaconic acid production by reducing/removing itaconic acid degradation and/or

biochemical conversion. As can be seen in Figure 17 and 18 significant improved production of itaconic acid levels were observed in deletion strains of itaconyl-CoA transferase (An07g00760) or itaconyl-CoA hydratase (An07g09220) in both shaking and static flasks. Similar results were obtained with the ictA (An07g00760) deletion strain CAD MFS #15 MTT CITB CAD MFS AICThygB #21 (CBS 143049) that was created in the industrial citric acid production strain background using a different route.

Deletion strains and their parental strains were cultivated in 500 mL Erlenmeyer flasks filled with 100 mL production medium. Medium composition is listed in Table 2 with the following modifications: 160 g/L glucose (Fig. 17) or 200 g/L glucose (Fig. 18) was used instead of 100 g/L glucose and 1.43 g/L NH4N03 and 0.00058 g/L FeC13*6 H20 were used instead of (NH4)2S04 and FeS04. Flasks were inoculated with 1.0*10^6 (Fig. 17) or 1.0*10^5 (Fig. 18) spores/mL and incubated at 33°C (Fig. 17) or 35°C (Fig. 18) in an incubator at 250 or 0 RPM (Fig. 17B). Organic acid levels (g/1) were obtained in time by HPLC analysis.

Controlled fermentation experiments were carried out with strains carrying a deletion of itaconyl-CoA transferase (An07g00760) or itaconyl-CoA hydratase (An07g09220). Controlled fermentations with deletion strain CitB #99 AICT #RD1 (CBS 143055) and deletion strain CitB #99 AICH #RB2 (CBS 143056) were performed as described in Example 3. However, the production medium (Table 2) contained 1.43 g/L NH4N03 and 0.00058 g/L FeC13*6 H20 instead of (NH4)2S04 and FeS04. Controlled fermentation with deletion strain Q199 AICT #4 (CBS 143050) (see Figure 20) was performed as described for strain Q199 (CBS 143051) in Example 1, but with 200 g/L glucose instead of 160 g/L glucose.

Itaconic acid levels were determined from these fermentations and compared with similar samples from the parental host strain (see Figures 3 and 5). From Figures 17 and 19 it is clear that deletion of itaconyl-CoA transferase (An07g00760) improves itaconic acid production more than deletion of itaconyl-CoA hydratase (An07g09220), showing that omission of the whole degradation pathway is more beneficial.

Deletion of any of the three genes from the degradation pathway not only results in less or no itaconic acid degradation, but also less intracellular accumulation of pathway intermediates resulting in less inhibited growth in Aspergillus niger. Moreover, increased levels of itaconic acid are observed in these strains.

Nucleotide & Protein sequences

Coding sequences are highlighted.