# Patent

## Title
Organic acid pathway

## Abstract

The invention relates to the use of a cytosolic citric acid synthase for the heterologous production of citrate outside the mitochondrion of a micro-organism or algae, wherein the protein is selected from A. niger An08g10920, An01g09940, An09g03570, or an ortholog of these genes. Such production is achieved by introducing the nucleic acid encoding such a protein into a suitable host cell. Preferably the protein is A. niger An08g10920, An01g09940, An09g03570 or an ortholog thereof, more particularly, wherein such an ortholog is chosen from the group of proteins listed in FIG. 9 and proteins having a percentage identity of 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99% with An08g10920, An01g09940 or An09g03570.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is the national phase of PCT application PCT/NL2014/050284 having an international filing date of 2 May 2014, which claims benefit of European patent application No. 13166305.6 filed 2 May 2013. The contents of the above patent applications are incorporated by reference herein in their entirety.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 313632019900_SeqList.txt, date recorded: Oct. 30, 2015, size: 284,842 KB).
The invention relates to the field of microbial production, more specifically production of organic acids, such as citric acid and its derivatives, such as oxaloacetic acid, itaconic acid (itaconate) and metacrylic acid, more specifically the production thereof in micro-organisms.
One of the most fundamental and ubiquitous metabolic pathways in living organisms is the citric acid cycle, or Krebs cycle, named after the discoverer. In eukaryotes this process takes place in the mitochondrion and is fed mainly by pyruvate and acetyl-CoA that are transported from the cytoplasm into the mitochondrion. Some of the constituents of the Krebs cycle or derivatives thereof may be transported back to the cytoplasm by active transport mechanisms, in general by tricarboxylic acid transporters. This basically means that cytoplasmatic metabolic routes that are dependent on, or starting from organic acids such as citric acid (citrate), malic acid (malate) oxaloacetic acid (oxaloacetate) heavily depend, and in general are limited by the activity within the Krebs cycle and the availability of the tricarboxylic acid transporters.
Hitherto no specific metabolic route for the production of extramitochondrial citric acid was elucidated. Yet, the availability of citric acid in the cytoplasm is extremely useful for the production of this acid itself and, even more importantly, for the production of derivatives and metabolites of citric acid. Citric acid is the starting point for many metabolic routes. One important metabolic route is the production of itaconic acid.
Production and metabolism of itaconic acid in microbial cells has been studied extensively for several decades (Calam, C. T. et al., 1939, Thom. J. Biochem., 33:1488-1495; Bentley, R. and Thiessen, C. P., 1956, J. Biol. Chem. 226:673-720; Cooper, R. A. and Kornberg, 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.
The general scheme currently envisioned for itaconic acid biosynthesis is given in FIG. 1, wherein clearly the existence of the biosynthetic route both in the cytosol and the mitochondria is depicted and the putative connection between these two compartments. At several point of this scheme possibilities exist to try to improve the existing commercial production of itaconic acid in micro-organisms.
The production of itaconic acid from citrate has been achieved in Aspergillus (and also other micro-organisms) with technology as described in WO 2009/014437, WO 2009/104958 and WO 2009/110796.
However, next to itaconate, citrate can also be used as a starting point for the production of malate, succinate, glutamate and metacrylic acid.
Further, citric acid can also lead to a metabolic route for lysine and from there to penicillin and similar compounds. Alternatively, citric acid can lead to the synthesis of fatty acids and thus be a source for biodiesel production. Moreover metabolites of citric acid, in particular acetyl-CoA, form the basis of biosynthetic routes towards fatty acids, polyketides and the mevalonate pathway towards terpenoids and other compounds
Yet, however, there is still need for an enzyme capable of production or overproduction of citric acid.
SUMMARY OF THE INVENTION
The present inventors now have elucidated a gene coding for an enzyme that is able to catalyze the reaction from oxaloacetate to citric acid, a so-called citrate synthase enzyme, which is present and functional outside the mitochondrion (and probably in the cytoplasm) of eukaryotic organisms.
The invention therefore comprises the use of a protein having cytosolic citric acid synthase activity for the heterologous production of citrate in the cytosol of a micro-organism or algae, preferably wherein said micro-organism is a fungus or a yeast or a plant or algal cell, more preferably when said micro-organism is selected from the group of Aspergillus spp., more particularly, A. niger, A. nidulans, A. terreus, A. clavatus, A. oryzae or A. flavus, Neurospora spp., more particularly N. crassa or N. tetrasperma, Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus, Penicillium, Trichoderma, Sordaria, Colleotrichum, Verticillium, Arthrobotrys, Nectria, Leptosphaeria, Fusarium, Glomerella, Geomyces, Myceliophthora, Pichia, Saccharomyces spp., such as S. pastorianus, S. cerevisiae, S. boulardii, S. carlsbergensis, S. kudriavzevii, and S. paradoxus, Zygosaccharomyces, Schizosaccharomyces pombe, Kluyveromyces spp., Yarrowia lipolytica, Monascus spp. (such as M. rubber, M. purpureus, M. pilosus, M. vitreus and M. pubigerus), Penicillium spp. (such as P. citrinum, P. chrysogenum), Hansenula spp., Torulaspora delbrueckii, Hypomyces spp., Dotatomyces spp. (such as D. stemonitis), Issatchenko orientalis, Phoma spp., Eupenicillium spp., Gymnoascus spp., Pichia labacensis, Pichia anomala, Wickerhamomyces anomalus, Candida cariosilognicola, Paecilomyces virioti, Scopulariopsis brevicaulis, Brettanomyces spp., such as B. bruxellensis, B. anomalus, B. custersianus, B. naardenensis and Brettanomyces nanus, Dekkera bruxellensis, Dekkera anoma and Trichoderma spp. (such as T. viride).
Preferably in said use the protein is derived from A. niger. It is also preferred when the protein is A. niger An08g10920 or an ortholog thereof, more particularly, wherein such an ortholog is chosen from the group of CAK45764.1/An08g10920, XP001393195.2/ANI1_1474074, EHA18674.1/Aspni5_176409, NP_001142237.1, GAA88109.1/AKAW_06223, EIT75413.1/Ao3042_08560, XP_001827205.1/AOR_1_298024, AO090010000170, XP_002384448.1/AFLA_117410, AFL2G_11427, XP_002148678.1/PMAA_091390, Aspfo1_0085419, Acar5010_212258, Acar5010_171837, Aspbr1_0068777, Asptu1_0164827, and proteins having a percentage identity of 70%, more preferably 75%, more preferably 80%, more preferably 85%, more preferably 90%, more preferably 95%, more preferably 98%, more preferably 99% with An08g10920.
Further part of the invention is a vector for transforming a micro-organism comprising a nucleic acid sequence coding for a protein as defined above. Also part of the invention is a transgenic organism transformed with such a vector or comprising a heterologous citric acid synthase as defined above.
The invention also comprises a method for the production of citric acid comprising overexpression of a gene coding for a citric acid synthase as defined above.
In a further preferred embodiment, the invention comprises a method for the production of itaconic acid comprising overexpression of a gene coding for a citric acid synthase as defined above and overexpression of a gene coding for the enzyme cis-aconitic acid decarboxylase (CAD) in a suitable host cell.
In an also preferred embodiment of the present invention, the invention also comprises a method for the production of a derivative of citric acid comprising overexpression of a gene coding for a citric acid synthase as defined in any of claim 1, 2 or 3 and overexpression of one or more genes that encode enzymes that are capable of converting citric acid into said derivative of citric acid in a suitable host cell. Preferably in such a method said one or more genes are selected from the group comprising An08g10860 (fatty acid synthase subunit beta), An08g10870 (2-methylcitrate dehydratase, prpD)( ); An08g10880 (GAL4; GAL4-like Zn2Cys6 binuclear), An08g10930 (3-oxoacyl-[acyl-carrier-protein] synthase); An08g10970 (MFS multidrug transporter); An08g10980 (transcription factor acetate regulatory DNA binding protein facB), An01g09950, An09g06220, An15g01780, An02g14730 (cytosolic prpD family), An05g02230 and An08g10530 (cytosolic aconitases) An02g12430, (non-mitochondrial isocitrate dehydrogenase) An04g06210 (homocitrate synthase), An11g00510 and An11g00530 (citrate lyase). Further preferred in such a method said suitable host cell is a micro-organism or algae, more preferably a fungus or a yeast or a plant or algal cell, preferably selected from the group of Aspergillus spp., more particularly, A. niger, A. nidulans, A. terreus, A. clavatus, A. oryzae or A. flavus, Neurospora spp., more particularly N. crassa or N. tetrasperma, Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus, Penicillium, Trichoderma, Sordaria, Colleotrichum, Verticillium, Arthrobotrys, Nectria, Leptosphaeria, Fusarium, Glomerella, Geomyces, Myceliophthora, Pichia, Saccharomyces spp., such as S. pastorianus, S. cerevisiae, S. boulardii, S. carlsbergensis, S. kudriavzevii, and S. paradoxus, Zygosaccharomyces, Schizosaccharomyces pombe, Kluyveromyces spp., Yarrowia lipolytica, Monascus spp. (such as M. rubber, M. purpureus, M. pilosus, M. vitreus and M. pubigerus), Penicillium spp. (such as P. citrinum, P. chrysogenum), Hansenula spp., Torulaspora delbrueckii, Hypomyces spp., Dotatomyces spp. (such as D. stemonitis), Issatchenko orientalis, Phoma spp., Eupenicillium spp., Gymnoascus spp., Pichia labacensis, Pichia anomala, Wickerhamomyces anomalus, Candida cariosilognicola, Paecilomyces virioti, Scopulariopsis brevicaulis, Brettanomyces spp., such as B. bruxellensis, B. anomalus, B. custersianus, B. naardenensis and Brettanomyces nanus, Dekkera bruxellensis, Dekkera anoma and Trichoderma spp. (such as T. viride), most preferably wherein said micro-organism is chosen from the group consisting of Aspergillus niger, A. acidus, A. tubigensis, A. oryzae, A. kawachii, A. flavus, A. acidus, A. carbonarius, A. brasiliensis, S. cerevisiae, Talaromyces marneffei, Zea mays, Pichia anomala and Dekkera bruxellensis. 

The invention also comprises a method for the production of a derivative of citric acid, preferably wherein said derivative is itaconic acid comprising overexpression of a gene coding for a citric acid synthase as defined in any of claims 1, 2 or 3 and a gene encoding for protein that is involved in the production or transport of itaconate or any precursor thereof, preferably wherein the gene is selected from the group of cis-aconitic acid decarboxylase (CAD), ATEG_09969.1, ATEG_09970.1 and ATEG_09972.1.

In a further preferred embodiment the invention comprises a method as defined above, wherein said host cell is cultured under anaerobic conditions. Alternatively or additionally, the invention comprises a method as defined above, wherein said host cell is cultured under anaerobic conditions in the presence of nitrate as N-source.



LEGENDS TO THE FIGURES
FIG. 1: Postulated biosynthesis route(s) for itaconic acid in A. terreus. 1, Citrate synthase; 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.
FIG. 2. Schematic projection of the lysine biosynthetic pathway.
FIG. 3: CitB cluster analysis of five Aspergillus species (citB gene indicated in the fifth column from the left and as identified as AFL2G_11427, AO090010000170, An08g10920, 176409-mRNA, Aspfo1_0085419, Aspbr1_10068777 and Asptu1_0164827). From top to bottom A. flavus NRRL 3357, A. oryzae RIB40/ATCC 42149, A. niger CBS 51388, A. niger ATCC 1015, A. acidus, A. carbonarius ITEM 5010. The “black Aspergilli”, A. niger, A. acidus and A. carbonarius, show similar clustering of the genes surrounding the citB gene, whereas the genomic region in A. oryzae and A. flavus only contains the citB (An08g10920) ortholog and the orthologs of An08g10880 and An08g10930. For A. terreus (not depicted) no corresponding gene cluster was found at all.
FIG. 4 Mevalonate pathway.
FIG. 5A-B. Nucleotide sequence of the Aspergillus expression vector pABgpd-I.
FIG. 6. Glucose consumption and organic acid production of A. niger CitB #99.
FIG. 7. Glucose consumption and organic acid production of A. niger CAD+MTT+MFS.
FIG. 8. Growth and itaconic acid production of A. niger AB1.13 CitB #99 on glycerol.
FIG. 9A-L. Amino acid sequences of orthologous citB citrate synthase like proteins.
FIG. 10. A. Homology tree of Aspergillus citrate synthase like proteins; B. Homology tree of Aspergillus niger, A. kawachii, A. ruber citrate synthase like proteins.



DETAILED DESCRIPTION OF THE INVENTION
“Fungi” are herein defined as eukaryotic microorganisms 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 microorganisms 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 microorganisms and include all species of the subdivision Eumycotina that predominantly grow in unicellular form. Yeasts may either grow by budding of a unicellular thallus or may grow by fission of the organism.
The term “fungal”, when referring to a protein or nucleic acid molecule thus means a protein or nucleic acid whose amino acid or nucleotide sequence, respectively, naturally occurs in a fungus.
The core of the invention resides in the discovery of a new, alternative parallel pathway for the production of citric acid from oxaloacetic acid. Most surprisingly, said production takes place outside of the mitochondrion, and probably in the cytoplasm. Accordingly, this route is active even under conditions where the mitochondrial citric acid cycle is inactive, which means that production of citric acid can advantageously take place in the absence of an active TCA cycle under anaerobic or low oxygen conditions. Further, it has appeared that the enzyme is expressed during the stationary phase, which means that an overexpression of citric acid can be achieved without any biomass growth of the producing organism. Moreover, previous research (Rafledge C., 2000, FEMS Microbiol. Lett. 189(2):317-319; Ruijter G. et al., 2000, FEMS Microbiol. Lett. 184(1): 35-40; Murray, S. and Hynes, M. 2010, Eukaryotic Cell 9(4):656-666) has shown that improvement of product fluxes through rational manipulation of the central metabolism (citrate synthase) has not been successful, which is in contrast to the results obtained in the present invention
For sake of easy reference, the enzyme will be addressed in this specification as the citrate synthase B or citB-enzyme, or just citB.
The citB gene as originally isolated was derived from Aspergillus niger. However, also comprised in the invention are homologous proteins that are derived from other micro-organisms (also called orthologs) and the nucleotide sequences coding for these. It will be clear for a person skilled in the art that on basis of the nucleotide sequences coding for the CitB enzyme of A. niger orthologs from other micro-organism species can be easily found through database searching in the NCBI GenBank based on sequence similarity and alignment analysis using minimal gap size in the alignment. A list of these orthologs is presented in Table 1a.







TABLE 1a







List of citB orthologs found in the NCBI 


GenBank database and orthologous genes in 


Aspergillus species (AspGD database, Broad institute). 


The sequences of these genes are given in FIG. 9.










Accession
Species







CAK45764.1/An08g10920
Aspergillus niger



XP001393195.2/ANI1_1474074
Aspergillus niger



EHA18674.1/Aspni5_176409
Aspergillus niger



NP_001142237.1
Zea mays



GAA88109.1/AKAW_06223
Aspergillus kawachii



EIT75413.1/Ao3042_08560
Aspergillus oryzae



XP_001827205.1/AOR_1_298024
Aspergillus oryzae



AO090010000170
A. oryzae



XP_002384448.1/AFLA_117410
Aspergillus flavus



AFL2G_11427
Aspergillus flavus



XP_002148678.1/PMAA_091390
Talaromyces marneffei



Aspfo1_0085419
A. acidus



Acar5010_212258
A. carbonarius_



Acar5010_171837
A. carbonarius



Aspbr1_0068777
A. brasiliensis



Asptu1_0164827
A. tubingensis



ETS77643.1
Pestalotiopsis fici



EOD45286.1
Neofusicoccum parvum



EMR70107.1
Eutypa lata










Also part of the invention are nucleotide sequences which are conservatively modified variants of the above mentioned sequences or polymorphic variants thereof. Those of skill in the art will recognize that the degeneracy of the genetic code allows for a plurality of polynucleotides to encode the identical amino acid. Such “silent variations” can be used, for example, to selectively hybridize and detect allelic variants of the nucleotide sequences of the present invention. Additionally, the present invention provides isolated nucleotide sequences comprising one or more polymorphic (allelic) variants of the above nucleotide sequences. Further part of the invention are polynucleotides still coding for a protein which has a biological function identical to the function of the CitB enzyme, which are the product of amplification from a nucleotide library using primer pairs which selectively hybridize under stringent conditions to loci within the above mentioned nucleotide sequences. The primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50. Those of skill in the art will recognize that a lengthened primer sequence can be employed to increase specificity of binding (i.e. annealing) to a target sequence. Stringent conditions in this respect means a reaction at a temperature of between 60° C. and 65° C. in 0.3 strength citrate buffered saline containing 0.1% SDS followed by rinsing at the same temperature with 0.3 strength citrate buffered saline containing 0.1% SDS.
Thus, also part of the invention are polynucleotides which selectively hybridize, under selective hybridization conditions, to one or more of the above discussed nucleotide sequences, and which code for an amino acid sequence which has a biological function similar to the function of the CitB enzyme disclosed in the present invention. With “a biological function similar to the function of CitB” it is meant the ability to convert oxaloacetate into citrate and to perform this conversion outside the mitochondrion, in the cytoplasm.
Another way to indicate hybridization potential is on sequence identity. In this sense, the present invention provides also for nucleotide sequences which have a percentage of identity related to the above mentioned sequences of 65% to 95%. Thus, for example, the percentage of identity can be at least, 65%, 70%, 75%, 80%, 85%, 90%, or 95%. Sequence identity on nucleotide sequences can be calculated by using the BLASTN computer program (which is publicly available, for instance through the National Center for Biotechnological Information, accessible via the internet on http://www.ncbi.nlm.nih.gov/) using the default settings of 11 for wordlength (W), 10 for expectation (E), 5 as reward score for a pair of matching residues (M), −4 as penalty score for mismatches (N) and a cutoff of 100.
Similarly, the homology can be calculated on basis of the amino acid sequence of the enzyme encoded by said nucleotide sequences. For amino acids, the sequence identity can be calculated through the BLASTP computer program (also available through http://www.ncbi.nlm.nih.gov/). On the amino acid level homologues or orthologs are defined as amino acid sequences having a biological function similar to the CitB enzyme and having a sequence identity of at least 50%, preferably at least 55%, preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 95% to the amino acid sequence of the A. niger CitB enzyme as depicted in FIG. 3.
Further included in the invention are enzymes, and nucleotide sequences coding for such enzymes, with a functional citrate synthase activity, but which lack the signal sequence that normally would cause them to be expressed or to be functional in the mitochondrion. Further included in the present invention and within the definition of citB according to the invention are mitochondrial citrate synthase enzymes in which the signal sequence has been replaced with the signal sequences of the A. niger citB enzyme (An08g10920).
As is shown in the Examples, also the enzymes with annotation An01 g09940 and An09g03570 lack the mitochondrial signal protein part. It is thus envisaged that these proteins and orthologs of these proteins would also qualify as citB enzymes according to the invention. These enzymes and their orthologs, listed in the Table 1 b below, are also depicted in FIG. 9.







TABLE 1b







Protein sequences of predicted non-mitochondrial citrate


synthases homologous to An09g03570 and An01g09940 (these also 


consists of several citB homologues)










Homology to




GAA91575




(besthit of




An09g03570





citrate synthase [Aspergillus kawachii IFO 4308] An09g03570 BEST hit
100% 
GAA91575.1


citrate synthase [Neosartorva fischeri NRRL 181] >gb|EAW19096.1| citrate
71%
XP_001260993.1


synthase [Neosartorva fischeri NRRL 181]




citrate synthase [Aspergillus fumigatus Af293] >gb|EBA27504.1| citrate
69%
XP_001481680.1


synthase, putative [Aspergillus fumigatus Af293] >gb|EDP55036.1| citrate




synthase [Aspergillus fumigatus A1163]




citrate synthase, putative [Aspergillus flavus NRRL3357] >gb|EED54329.1| 
68%
XP_002375601.1


citrate synthase, putative [Aspergillus flavus NRRL3357]




citrate synthase [Aspergillus oryzae RIB40]
68%
XP_001727354.2


TPA: citrate synthase, putative (AFU orthologue; AFUA 2G15312) [Aspergillus
69%
CBF79704.1


nidulaus FGSC A4]




hypothetical protein AN7593.2 [Aspergillus nidulaus FGSC 
69%
XP_680862.1


A4] >gb|EAA62173.1| hypothetical protein AN7593.2 [Aspergillus nidulaus




FGSC A4]




Citrate synthase-like [Penicillium roqueforti]
61%
CMD33221.1


Pc12g00660 [Penicillium chrysogenum Wisconsin 54-1255] >emb|CAP79693.1| 
61%
XP_002556968.1


Pc12g00660 [Penicillium chrysogenum Wisconsin 54-1255]




hypothetical protein COCCADRAFT 104929 [Bipolaris zeicola 26-R-13]
59%
EUC30035.1


hypothetical protein COCMIDRAFT 107988 [Bipolaris oryzae ATCC 44560]
59%
EUC40740.1


citrate synthase, putative [Aspergillus clavatus NRRL 1] >gb|EAW14389.1|
67%
XP_001275815.1


citrate synthase, putative [Aspergillus clavatus NRRL 1]




hypothetical protein COCVIDRAFT 107070 [Bipolaris victoriae FI3]
59%
EUN24125.1


citrate synthase [Aspergillus ruber CBS 135680]
59%
EYE90286.1


hypothetical protein COCHEDRAFT 1118493 [Bipolaris maydis
58%
EMD85580.1


C5] >gb|ENH9968.1| hypothetical protein COCC4DRAFT 151813 [Bipolaris maydis




ATCC 48331]




citrate synthase, putative [Talaromyces stipitatus ATCC 10500] >gb|
52%
XP_002482831.1


EED18839.1| citrate synthase, putative [Talaromyces stipitatus ATCC 




10500]




citrate synthase, putative [Talaromyces stipitatus ATCC 10500] >gb|
42%
XP_002485362.1


EED15409.1| citrate synthase, putative [Talaromyces stipitatus ATCC 




10500] 




conserved hypothetical protein [Aspergillus terreus NIH2624] >gb|EAU32144.1|
68%
XP_001216503.1


conserved hypothetical protein [Aspergillus terreus NIH2624] 




Citrate synthase [Penicillium digitatum PHI26] >gb|EKV21626.1| Citrate 
61%
EKV06554.1


synthase [Penicillium digitatum Pd1]




citrate synthase [Colletotrichum graminicola M1.001]
39%
EFQ27732.1


citrate synthase [Auricularia delicata TFB-10046 SS5] >gb|EJD44900.1| citrate
39%
XP_007347043.1


synthase [Auricularia delicata TFB-10046 SS5]




citrate synthase [Aspergillus oryzae RIB40] >dbj|BAE66072.1| unnamed protein
38%
XP_001827205.1


product [Aspergillus oryzae RIB40]




citrate synthase [Aspergillus oryzae 3.042]
38%
EIT75413.1


citrate synthase [Aspergillus kawachii IFO 4308]
38%
GAA88109.1


uncharacterized protein LOC100274406 [Zea mays] >gb|ACF87962.1| unnamed
38%
NP_001142237.1


[Zea mays]




citrate synthase [Aspergillus niger CBS 513.88] An08g10920 = citB
37%
XP_001393195.2


unnamed protein product [Aspergillus niger] An08g10920 = citB
37%
CAK45764.1


citrate synthase [Aspergillus niger ATCC 1015] 
37%
EHA18674.1


citrate synthase, putative [Talaromyces marneffei ATCC 18224] >gb|EEA22411.1|
38%
XP_002148678.1


citrate synthase, putative [Talaromyces marneffei ATCC 18224]




citrate synthase, putative [Aspergillus flavus NRRL3357] >gb|EED45512.1|
37%
XP_002384448.1


citrate synthase, putative [Aspergillus flavus NRRL3357]




putative citrate synthase protein [Eutypa lata UCREL1]
38%
EMR66249.1


putative citrate synthase protein [Neofusicoccum paryum UCRNP2]
36%
EOD45286.1


putative citrate synthase protein [Eutypa lata UCREL1]
38%
EMR70107.1


unnamed protein product [Aspergillus niger] >gb|EHA26823.1| citrate synthase
36%
CAK37177.1


[Aspergillus niger ATCC 1015]




citrate synthase [Aspergillus kawachii IFO 4308]
35%
GAA82055.1


citrate synthase [Aspergillus niger CBS 513.88] An01g09940
36%
XP_001389414.2









All of these proteins, and orthologs and homologs as defined, are deemed to be encompassed in the term “citB protein” or “citB enzyme” as used herein.

It is further contemplated that overexpression of the gene in a heterologous organism, which in nature does not or hardly produce extramitochondrial citric acid, is able to provide such an organism with a functional pathway for expression of citric acid outside the mitochondrion, and preferably in the cytoplasm. Preferably such overexpression 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, A. oryzae and A niger, Ustilago zeae, Ustilago maydis, Ustilago sp., Candida sp., Mortierella sp., Yarrowia sp., Rgizopus sp. Yarrowia lipolytica, Rhodotorula sp. and Pseudozyma Antarctica, the bacterium E. coli and the yeast Saccharomyces sp, e.g. S. cerevisiae, Pichia sp, e.g. P. pastoris or P. anomala. Also plant cells and algal cells and cell cultures may be used as host. Especially preferred are heterologous organisms in which the substrate oxaloacetate is abundantly available in the host organism. Also applicable in the present invention are hosts that may grow anaerobically while using NO3 as source of nitrogen, such as the yeasts P. anomala and Dekkera bruxellensis. In such a case the acceptor nitrate can yield reductive compounds in the same was as oxygen produces reductive compounds in aerobic fermentation. Similarly also Aspergillus species, such as A. terreus can grow at very low oxygen levels using dissimilatory nitrate reduction (Stief, P., Fuchs-Ocklenhurg, S., Kamp, A., Manohar, C.-S., Houbraken, J., Boekhout, T., De Beer, D., Stoeck, T. Dissimilatory nitrate reduction by Aspergillus terreus isolated from the seasonal oxygen minimum zone in the Arabian Sea (2014) BMC Microbiology, 14 (1), art. no. 35) allowing the production of organic acids as described in the present invention under these conditions.
Further preferred are host organisms which next to the heterologous citB enzyme further contain enzymes that specifically metabolize citric acid further. One of the pathways which is very suitable for this is the pathway to form itaconic acid, wherein from citric acid cis-aconitate is formed by the enzyme aconitase or the enzyme (2-methyl)citrate dehydratase, which enzymes are considered to be functional in the cytosol (see FIG. 1). The production of itaconic acid the can be achieved by the enzyme CAD, which provides cis-aconitic acid decarboxylase activity, thereby converting cis-aconitate into itaconic acid. An advantageous method of producing itaconic acid, the enzymes used therein and the sequences thereof and/or hosts for performing this metabolic pathway have been described in WO 2009/014437. Further optimisation of the present invention in the aspect of the invention dealing with the production of itaconic acid 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 Further, overexpression of a nucleic acid sequence encoding an itaconate transporting Major Facilitator Superfamily Transporter (MFST) gene sequence (hereinafter “the itaconate transporter”) enhances the production/transport of itaconate as described herein as described in WO 2009/104958 and WO 2009/110796. Also the sequences of these enzymes may be found in these references. Preferably said nucleic acid comprises the ATEG_09972.1 sequence of Aspergillus terreus or a nucleic acid that shares more than about 70%, preferably more than about 80%, preferably more than about 90% sequence identity with the sequence of ATEG_09972.1. This process can be even further optimised by combining the overexpression of a CAD gene as described in WO 2009/014437, with overexpression of 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. Overexpression of this transporter will lead to an increase in cis-aconitate in the cytosol, which can be further converted to itaconic acid (see also WO 2009/104958). Accordingly, the combination of an heterologous citB gene and a gene selected from the group of di/tricarboxylate transporters is not only advantageous for the production of itaconate, but it may also cause an increase in the expression of other citrate derivatives as discussed above.
Of course, ideally, combinations of citB with one or more and preferably all of the genes that have been specified above as enhancing the production of itaconic acid maybe applied to act in concert to boost the production and transport of itaconic acid and/or its derivatives.
Recombinant host cells 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 pKD1 (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 U.S. Pat. No. 6,265,186).
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 another aspect the invention relates to a nucleic acid construct comprising a nucleotide sequence encoding a CitB enzyme or homolog or ortholog thereof as defined above and used for transformation of a host cell as defined above. In the nucleic acid construct, the nucleotide sequence encoding the CitB protein preferably is operably linked to a promoter for control and initiation of transcription of the nucleotide sequence in a host cell as defined below. The promoter preferably is capable of causing sufficient expression of the CitB enzyme in the host cell. Promoters useful in the nucleic acid constructs of the invention include the promoter that in nature provides for expression of the CitB gene. Further, both constitutive and inducible natural promoters as well as engineered promoters can be used. Promoters suitable to drive expression of the CitB gene in the hosts of the invention include e.g. GAL7, GAL10, or GAL 1, CYC1, HIS3, PGL, PH05, ADC1, TRP1, URA3, LEU2, ENO, TPI, and A0X1. Other suitable promoters include PDC, GPD1, PGK1, TEF, TDH, promoters from glycolytic genes (e.g. from a glyceraldehyde-3-phosphate dehydrogenase gene), ribosomal protein encoding gene promoters, alcohol dehydrogenase promoters (ADH1, ADH4, and the like), promoters from genes encoding amylo- or cellulolytic enzymes (glucoamylase, TAKA-amylase and cellobiohydrolase). Other promoters, both constitutive and inducible and enhancers or upstream activating sequences will be known to those of skill in the art. The promoters used in the nucleic acid constructs of the present invention may be modified, if desired, to affect their control characteristics. Preferably, the promoter used in the nucleic acid construct for expression of the CitB gene is homologous to the host cell in which the CitB protein is expressed.
In the nucleic acid construct, the 3′-end of the nucleotide acid sequence encoding the CitB enzyme preferably is operably linked to a transcription terminator sequence. Preferably the terminator sequence is operable in a host cell of choice. In any case the choice of the terminator is not critical; it may e.g. be from any fungal gene, although terminators may sometimes work if from a non-fungal, eukaryotic, gene. The transcription termination sequence further preferably comprises a polyadenylation signal.
Optionally, a selectable marker may be present in the nucleic acid construct. As used herein, the term “marker” refers to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a host cell containing the marker. A variety of selectable marker genes are available for use in the transformation of fungi. Suitable markers include auxotrophic marker genes involved in amino acid or nucleotide metabolism, such as e.g. genes encoding ornithine-transcarbamylases (argB), orotidine-5′-decaboxylases (pyrG, URA3) or glutamine-amido-transferase indoleglycerol-phosphate-synthase phosphoribosyl-anthranilate isomerases (trpC), or involved in carbon or nitrogen metabolism, such e.g. niaD or facA, and antibiotic resistance markers such as genes providing resistance against phleomycin, bleomycin or neomycin (G418). Preferably, bidirectional selection markers are used for which both a positive and a negative genetic selection is possible. Examples of such bidirectional markers are the pyrG (URA3), facA and amdS genes. Due to their bidirectionality these markers can be deleted from transformed filamentous fungus while leaving the introduced recombinant DNA molecule in place, in order to obtain fungi that do not contain selectable markers. This essence of this MARKER GENE FREETM transformation technology is disclosed in EP-A-0 635 574, which is herein incorporated by reference. Of these selectable markers the use of dominant and bidirectional selectable markers such as acetamidase genes like the amdS genes of A. nidulans, A. niger and P. chrysogenum is most preferred. In addition to their bidirectionality these markers provide the advantage that they are dominant selectable markers that, the use of which does not require mutant (auxotrophic) strains, but which can be used directly in wild type strains.
Optional further elements that may be present in the nucleic acid constructs of the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences. The nucleic acid constructs of the invention may further comprise a sequence for autonomous replication, such as an ARS sequence. Suitable episomal nucleic acid constructs may e.g. be based on the yeast 2μ or pKD1 (Fleer et al., 1991, Biotechnology 9: 968-975) plasmids. Alternatively the nucleic acid construct may comprise sequences for integration, preferably by homologous recombination (see e.g. WO98/46772). Such sequences may thus be sequences homologous to the target site for integration in the host cell's genome. The nucleic acid constructs of the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the 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).
In a further aspect the invention relates to fermentation processes in which the transformed host cells of the invention are used for the production of citric acid and products that can be derived from further metabolic routes from citric acid. Such a fermentation process may be an aerobic fermentation process, but since the location of the enzyme is outside the mitochondrion advantageously an oxygen-limited or anaerobic fermentation process may be applied. This enables a lot of possible circumstances or conditions under which production of the citric acid can still occur. In particular circumstances with an inactive TCA cycle which are normally believed to be incompatible with citric acid production. In particular various yeast species are able to grow anaerobically by fermentation. For pertaining a suitable cofactor balance in particular yeast strains able to use NO3 such as Dekkera bruxellensis and Pichia anomola are preferred. 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. U.S. Pat. No. 6,746,862).
Preferred in the invention is a submerged fermentation process, which is performed in batch or fed-batch. This means that there is a continuous input of feed containing a carbon source and/or other relevant nutrients in order to improve citric 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.
There are also fermentation processes where a fungus grows on a solid support in an aqueous phase in so-called biofilm processes. In those conditions there will be an oxygen limitation meaning that the metabolism in such a case at least partially will be anaerobically. Biofilms have been used for a long time in water treatment facilities where they were called slime, mats or sludge, but no other practical use was seen until recently. This has brought that most of the available information is on bacterial and, in recent years, on yeast biofilms. Filamentous fungi are naturally adapted to growth on surfaces and in these conditions they show a particular physiological behaviour which it is different to that in a submerged culture; thus, they can be considered as biofilm forming organisms according to the above concept. Differential physiological behaviour of most attached fungi corresponds principally to a higher production and secretion of enzymes and also to a morphological differentiation which is absent in submerged cultures (Akao, T. et al., Curr. Genet. 41:275-281, 2002; Biesebeke, R. et al., FEMS Yeast Res. 2:245-248, 2002). The advantages of this form of growth have been industrially exploited by two culture systems: SSF and cell immobilization (Gutierrez-Correa, M. and Villena, G., Rev. peru. Boil. 10(2):113-124, 2003). Once citric acid is produced in the host cell this citric acid can be used as a substrate for further metabolic processes. One of these metabolic processes is the production of itaconic acid. For this a conversion from citric acid via cis-aconitate to itaconic acid has to be performed via the enzymes aconitase and CAD (cis-aconitate decarboxylase). Such a conversion and further methods of additionally increasing the production of itaconic acid from cis-aconitate has been described in WO 2009/014437, WO 2009/104958 and WO 2009/110796.
Next to a pathway to itaconic acid, citric acid can also be used as a starting point for other metabolic routes. One of the most commercially interesting routes is the production of methacrylic acid. Methacrylic acid can be produced directly by decarboxylation of itaconic acid, but it can also be produced through other metabolic routes (see Carlsson, M. et al., Ind. Eng. Chem. Res. 33:1989-1996, 1994). Citric acid is also one of the basic building blocks in the biosynthesis of fatty acids and triglycerides. Fatty acids form important storage molecules for energy, which energy later can be used, e.g. when fatty acids have been used as a source of biodiesel. The exact nature of the fatty acid is—for use as biofuel—less relevant, since all types of plant fatty acids may be used as such. Fatty acids can, of course, also be used as such, e.g. as food additives. This is especially important in the case of the essential fatty acids, like linoleic acid and α-linolenic acid. for the production of other compounds, such as biodegradable plastics.
Further, citric acid can be used to be a starting point for the biosynthesis of lysine (via homocitrate and homo-cis-aconitate). FIG. 2 gives an overview of the chemical reactions and pathways that can be used to convert citrate into lysine.
This pathway is also required for the biosynthetic production of aminoadipate which is an intermediate for penicillin and other antibiotic compounds. Lysine, in turn, may be used as a starting point for the production of caprolactam (see US 2009/005532, and from there be used for the production of plastics (such as nylon-6, a polyamide).
Further, citrate can also be used as a precursor for the mevalonate pathway (see FIG. 4). This can lead to the production of terpenoids. Lastly, citrate (and acetyl CoA) may function as precursor for a polyketide pathway, resembling fatty acid biosynthesis. Polyketide antibiotics, antifungals, cytostatics, anticholesteremic, antiparasitics, coccidiostats, animal growth promoters and natural insecticides that may be produced following such a pathway are commercially important compounds.
It has further been found that the cluster in which the citB gene is residing contains other genes that have a relation with the pathways in which citrate is involved. In the citB (An08g10920) cluster that is present in a particular A. niger strain the following genes can be found: An08g10860 (fatty acid synthase) An08g10870 (2-methylcitrate dehydratase (prpD)),); An08g10880 (GAL4; GAL4-like Zn2Cys6 binuclear), An08g10930 (3-oxoacyl-[acyl-carrier-protein] synthase, fatty acid synthase); An08g10970 (MFS multidrug transporter); An08g10980 (transcription factor acetate regulatory DNA binding protein facB). (Over)expression of any of these genes, next to the expression of citB is thought to be especially favorable to increase the production and usurpation of intracellular citrate.
EXAMPLES
Example 1 Enzyme Analysis in Itaconic Acid Producing Strain
The table 2 below gives an overview of proteins/genes in Aspergillus niger and S. cerevisiae belonging to the enzyme classes citrate synthase, aconitase and cis-aconitase decarboxylase. For all members in A. niger the results of expression analysis is given by RNA sequencing results in the WT strain and an itaconic acid producing strain (transgenic for CAD by carrying extra gene copies of the cis-aconitate decarboxylase from A. terreus as described in WO 2009/014437). This shows that An08g10920, tentatively called citB, encoding a citrate synthase without a predicted mitochondrial localization, is highly induced in the itaconic acid production strain. This gene has no homologue in S. cerevisiae. Only in closely related black Aspergilli homologues are present (see below under genome mining citB gene cluster). Also one of the predicted cytosolic cis-aconitase decarboxylase (prpD) genes from A. niger, An08g10870 which is clustered with citB is highly induced. Also the canonical cytosolic aconitase An08g10530 more distantly linked is induced in the CAD strain.
In S. cerevisiae all three citrate synthase proteins and the single prpD gene are mitochondrial, while both aconitases are cytosolic as predicted for the common metabolic pathways.
Table 2 Citrate synthase, aconitase and cis-aconitate decarboxylase (prpD) genes in A. niger. In hold the genes induced in the CAD-expressing strain. BG=expression values are at background levels, thus calculation of ratio value (R; expressed as 2 Log R) is not relevant







TABLE 2







Citrate synthase, aconitase and cis-aconitate decarboxylase (prpD) genes


in A. niger.












Subcell. Loc.
RNAseq results












Gene_ID
Protein
WolfPsort
CAD
WT
2LogR















An09g06680/ANI_1_876084
citrate synthase citA 
mito: 19.0
9501
8977
−0.02


An15g01920/ANI_1_1226134
citrate synthase
mito: 22.0
345
616
−0.87


An09g03570
citrate synthase
unknown0
4
BG



An08g10920/ANI_1_1474074
citrate synthase citB
cyto: 10.0
9515
917
3.34


An01g09940/ANI_1_2950014
citrate synthase
cysk: 11, cyto: 8
11
62
BG


An02g11040/ANI_1_3018024
aconitase
mito: 20.5
0
0
BG


An08g10530/ANI_1_1410074
aconitase
cyto: 19.0
11203
5897
0.89


An09g03870/ANI_1_470084
aconitase
mito: 26.5
571
1254
−1.17


An16g05760/ANI_1_1808144
aconitase
mito: 24.0
22
16
BG


An05g02230/ANI_1_578044
aconitase
cyto: 12.5
234
89
1.36


An01g09950/ANI_1_2952014
prpD
cyto: 13.0
50
59
BG


An09g06220/ANI_1_1536084
prpD
cyto: 12.5
67
318
−2.28


An15g01780/ANI_1_306134
prpD
cyto: 13.5
1565
1632
−0.1


An08g10870/ANI_1_2490074
prpD
cyto: 16.5
6099
570
3.38


An02g14730/ANI_1_3352024
prpD
cyto: 13.5
48
47
BG


An01g09930/ANI_1_29480
prpD
mito: 10.0
12
45
BG





In bold the genes induced in the CAD-expressing strain.


BG = expression values are at background levels, thus calculation of ratio value (R; expressed as 2LogR) is not relevant






From this Table it appears that two of the A. niger proteins are clearly located in the mitochondrion (An09g09980 and An15g01920), while two A. niger genes are located outside the mitochondrion (An01g09940 and An08g10920), while the location of one protein is undecided (An09g03570).

In a homology tree (see FIG. 10) it appears that a distinction can also be made between mitochondrial and non-mitochondrial citrate synthase enzymes on basis of homology.

Example 2 RNA Sequence Analysis of A. niger CAD Transformant in Comparison to the Wildtype Strain
RNAseq is a new transcriptomics platform which allows direct sequencing of mRNAs. This means that no arrays are required and all expressed RNA is measured (non-coding, non annotated). Shake flask cultures in fermentation medium described below were grown for 46 hours at 33° C. from which biomass samples were harvested. Total RNA was isolated from biomass samples using Trizol (Invitrogen). The total RNA was send to BaseClear (The Netherlands). Before random mRNA sequencing could be performed, mRNA purification was performed via oligo-dT beads (Illumina TruSeq RNA samp.prep), followed by first-strand cDNA synthesis with random primers. Adaptor ligation, adding 120 bp, was carried out, followed by  ̃270 bp gel-isolation (cDNA inserts  ̃150 bp). Subsequently, paired-end sequencing was performed, resulting in Illumina HiSeq data (28-29 M reads/sample). Data analysis was performed to obtain output files of RNA-Seq alignments (*.clc, *,sam) and RNA-Seq expression tables (*.csv, *.cic, *.xlsx). Sample-normalised expression values were expressed as RKPM/sample (=Reads per Kilobase of exon model per Million mapped reads (Mortazavi et. al 2008)). The data analysis performed at TNO comprised of defining the “Floor” of RPKM values (Excel) (S<1 was defined as S=1). After introducing the “Floor” the differentials of the expression values of the CAD transformant and wildtype were calculated in Excel (R=Sx/Sref; 2 log R ratios). Below table 2 provides the RNAseq data (counts and 2 log R ratios) of the genes directly surrounding the citrate synthase genes which might belong to the putative citrate synthase/prpD gene clusters. AB1.13 data refer to the WT A. niger host strain, while AB1.13CAD refer to the itaconic acid producing strain (CAD) carrying extra gene copies of the cis-aconitate decarboxylase from A. terreus as described in WO 2009/014437. Note that RNAseq values lower than 100-200 represent very low expression. Of the related genes/gene clusters only the citB cluster (An08g10860-An08g1011030, in bold and italics) shows significantly induced expression in the AB1.13CAD strain (Table 3) The other gene regions containing citrate synthase, aconitase and cis-aconitate decarboxylase genes (underlined) show no induction (Table 3)







TABLE 3







RNAseq analysis











value
value















AB1.13CAD
AB1.13

gene name




(Sx)
(Sref)
value
CBS 513.88




count
count
2log(Sx/Sref)
code
ATCC 1015 code
Protein















60113
12
12.26
—
—
ATEG_09971_A. terreus cad gene


0
0
0.00
An08g10760
ANI_1_2472074
hypothetical protein


574
1409
−1.33
An08g10780
ANI_1_2476074
glycosyl hydrolase family 43 protein


0
3
0.00
An08g10800
ANI_1_2480074
L-amino acid oxidase LaoA


26
34
−0.42
An08g10810
ANI_1_2482074
appr-1-p processing enzyme family protein


126
146
−0.25
An08g10820
ANI_1_2484074
aldehyde dehydrogenase


19
135
−2.86
An08g10830
ANI_1_2486074
geranylgeranyl pyrophosphate synthase


17445
1403
3.60
An08g10860
ANI_1_2488074
sterigmatocystin biosynthesis fatty acid synthase







subunit beta


6099
570
3.38
An08g10870
ANI_1_2490074
2-methylcitrate dehydratase (prpD)


2893
141
4.32
An08g10880
ANI_1_2492074
GAL4; GAL4-like Zn2Cys6 binuclear cluster DNA-







binding domain; found in transcription regulators







like GAL4


9515
917
3.34
An08g10920
ANI_1_1474074
citrate synthase citB


21473
1212
4.11
An08g10930
ANI_1_2494074
3-oxoacyl-[acyl-carrier-protein] synthase


11804
2195
2.39
An08g10970
ANI_1_2500074
MFS multidrug transporter


1850
1490
0.28
An08g10980
ANI_1_2502074
acetate regulatory DNA binding protein facB


57671
15873
1.83
An08g10990
ANI_1_1484074
dienelactone hydrolase family protein


956
377
1.31
An08g11000
—
GAL4-like Zn2Cys6 binuclear cluster DNA-binding







domain; similarity to the A. niger xlnR gene


776
473
0.68
An08g11010
ANI_1_2504074
hypothetical protein


140252
64632
1.08
An08g11030
ANI_1_1486074
3-phytase B


287
408
−0.54
An08g11040
ANI_1_1488074
zinc finger protein ZPR1


157
330
−1.11
An08g11060
ANI_1_2506074
hypothetical protein


6
11
0.00
An08g11070
ANI_1_2508074
extracellular invertase


1386
1163
0.22
An01g09730
ANI_1_2924014
choline transport protein


2
1
0.00
An01g09740
ANI_1_2926014
3-hydroxyacyl-CoA dehyrogenase


1
7
0.00
An01g09750
ANI_1_1318014
cytochrome B5


31
30
0.01
An01g09760
ANI_1_1320014
cytochrome P450 monooxygenase


37
55
−0.61
An01g09770
ANI_1_2928014
Zn(II)2Cys6 transcription factor






ANI_1_2930014
calcium/calmodulin dependent protein kinase


434
1331
−1.65
An01g09780
ANI_1_2932014
D-lactate dehydrogenase


356
597
−0.78
An01g09800
ANI_1_1328014
acylamide-delta3(E)-desaturase


342
226
0.56
An01g09810
ANI_1_2934014
glycosyl transferase


595
313
0.89
An01g09820
ANI_1_2936014
udp-glucose 6-dehydrogenase


267
304
−0.22
An01g09830
ANI_1_1332014
glutathione S-transferase


634
677
−0.13
An01g09840
ANI_1_1334014
NADH:ubiquinone oxidoreductase 6.6kD subunit


90
84
0.06
An01g09850
ANI_1_2938014
sin3-associated polypeptide Sap18


155
137
0.14
An01g09860
ANI_1_1338014
mRNA splicing factor (Prp18)


0
1
0.00
An01g09870
ANI_1_2940014
hypothetical protein


303
168
0.82
An01g09880
ANI_1_2942014
hypothetical protein


248
182
0.41
An01g09890
ANI_1_1344014
ADP-ribosylation factor family protein


63
73
−0.25
An01g09900
ANI_1_2944014
hypothetical protein


236
215
0.10
An01g09910
ANI_1_2946014
phosphatidylinositol N-acetylglucosaminyltransferase







gpi3 subunit


169
138
0.26
An01g09920
ANI_1_1350014
ATP-dependent RNA helicase dbp6


12
45
−1.94
An01g09930
ANI_1_948014
2-methylcitrate dehydratase (prpD)


11
62
−2.53
An01g09940
ANI_1_2950014
citrate synthase


50
59
−0.27
An01g09950
ANI_1_2952014
immune-responsive protein (prpD)


11
18
−0.01
An01g09960
ANI_1_1358014
exo-1,4-beta-xylosidase xlnD


12
62
−2.40
An01g09970
ANI_1_2954014
hypothetical protein


7
57
−3.06
An01g09980
ANI_1_1362014 
Asp-hemolysin


1106
845
0.35
An01g10000
ANI_1_2956014
ABC multidrug transporter


123
359
−1.58
An01g10010
ANI_1_2958014
cystathionine gamma-synthase


1237
3533
−1.55
An01g10030
ANI_1_1368014
sphinganine hydroxylase BasA


4824
4664
0.01
An01g10050
ANI_1_1370014
hypothetical protein


229
170
0.39
An01g10060
ANI_1_2962014
Zn(II)2Cys6 transcription factor


94
109
−0.25
An01g1C070
ANI_1_1374014
signal recognition particle protein


3
6
0.00
An15g01770
ANI_1_1208134
C-5 cytosine methyltransferase DmtA


1565
1632
−0.10
An15g01780
ANI_1_306134
2-methylcitrate dehydratase (prpD)


0
7
0.00
An15g01790
ANI_1_1210134
pantothenate transporter


5
12
−0.41
An15g01800
ANI_1_1212134
amidohydrolase


144
86
0.71
An15g01810
ANI_1_1214134
C6 zinc finger domain protein


19
18
0.00
An15g01830
ANI_1_1216134
sodium transport ATPase 5


222
186
0.22
An15g01840
ANI_1_1218134 
short-chain dehydrogenase/reductase family


398
460
−0.24
An15g01850
ANI_1_318134
glutamine synthetase


455
1272
−1.52
An15g01860
ANI_1_320134
malate synthase, glyoxysornal


19
14
0.41
An15g01870
ANI_1_1220134
hypothetical protein


1
0
0.00
An15g01880
ANI_1_1222134
hypothetical protein


1283
808
0.63
An15g01890
ANI_1_1224134
beta-glucosidase E


51
117
−1.23
An15g01900
ANI_1_326134
choline transport protein


1392
1000
0.44
An15g01910
ANI_1_328134
[NU]+ prion formation protein 1


345
616
−0.87
An15g01920
ANI_1_1226134
citrate synthase


2
65
−2.57
An15g01930
ANI_1_1228134
hypothetical protein


68
75
−0.18
An15g01940
ANI_1_1230134
FAD binding domain protein


50
31
0.65
An15g01950
ANI_1_1232134
very-long-chain acyl-CoA synthetase family protein







(CefD1)


174
250
−0.56
An15g01960
ANI_1_1234134
sarcosine oxidase


236
357
−0.63
An15g01970
ANI_1_340134
hypothetical protein


116
153
−0.43
An15g01980
ANI_1_342134
8-amino-7-oxononanoate synthase


461
509
−0.18
An15g01990
ANI_1_344134
onanonoxo-7-onima-8-eninoihtemlysoneda


1537
1798
−0.26
An15g02000
ANI_1_1238134
biotin synthase


0
4
0.00
An09g03570
—
similarity to citrate synthase citA


67
318
−2.28
An09g06220
ANI_1_1536084
immune-responsive protein (prpD)


295
853
−1.57
An09g06460
ANI_1_842084
hypothetical protein


2156
2686
−0.35
An09g06480
ANI_1_844084
phosphatidylinositol transfer protein sfh5


756
652
0.18
An09g06490
ANI_1_846084
lanosterol synthase


328
574
−0.84
An09g06500
ANI_1_1562084
SGT1 and CS domain protein


151
210
−0.51
An09g06510
ANI_1_1564084
PQ loop repeat protein


287
286
−0.03
An09g06520
ANI_1_852084
sir2 family transcriptional regulator


657
500
0.36
An09g06530
ANI_1_1566084
negative regulator of the PHO system


48
70
−0.58
An09g06540
ANI_1_856084
spindle pole protein Nnf1


344
278
0.27
An09g06550
ANI_1_858084
hypothetical protein


1482
877
0.72
An09g06570
ANI_1_862084
hypothetical protein


403
513
−0.38
An09g06580
ANI_1_864084
NTF2 and RRM domain protein


10266
15839
−0.66
An09g06590
ANI_1_860084
heat shock protein 90


594
1492
−1.36
An09g06610
ANI_1_866084
hypothetical protein


1424
1100
0.34
An09g06630
ANI_1_868084
HLH transcription factor (PalcA)


289
325
−0.20
An09g06640
ANI_1_870084
DNA-directed RNA polymerase III subunit RPC-3


3021
3534
−0.26
An09g06650
ANI_1_872084
ubiquinol-cytochrome C reductase complex core







protein 2


2866
3756
−0.43
An09g06670
ANI_1_874084
DNA replication protein YHM2


9051
8977
−0.02
An09206680
ANI_1_876084
citrate synthase citA


833
715
0.19
An09g06700
ANI_1_878084
RNA binding protein Nrd1


345
1089
−1.69
An09g06710
ANI_1_1568084
O-acetylhomoserine (thiol)-lyase


187
463
−1.34
An09g06720
ANI_1_1568084
hypothetical protein


283
3532
−3.68
An09g06730
ANI_1_882084
arginine permease


609
607
−0.03
An09g06740
ANI_1_886084
AMP-binding enzyme


460
470
−0.07
An09g06750
ANI_1_1570084
hypothetical protein


1035
698
0.53
An09g06760
ANI_1_1572084
WW domain protein


288
315
−0.16
An09g06770
ANI_1_1574084
WD repeat protein


391
624
−0.71
An09g06780
ANI_1_894084
peruxisomal membrane protein Pmp47


3632
3045
0.22
An09g06790
ANI_1_896084
GIP-binding protein ypt1


751
1026
−0.49
An09g06800
ANI_1_898084
aminopeptidase


375
296
0.31
An09g06810
ANI_1_1576084
TFIIH basal transcription factor complex p47 subunit


290
214
0.40
An09g06820
ANI_1_902084
hypothetical protein


6
7
0.00
An09g06830
ANI_1_1578084
pumilio-family RNA binding repeat protein


1396
963
0.50
An09g06840
ANI_1_906084
hypothetical protein


3021
2861
0.04
An09g06850
ANI_1_908084
NADH-ubiquinone oxidoreductase subunit


255
244
0.03
An09g06860
ANI_1_1580084
hypothetical protein


964
796
0.24
An09g06870
ANI_1_1582084
cytokinesis regulator (Byr4)


2
11
−1.76
An09g06890
ANI_1_1584084
hypothetical protein









Example 3 Isolation of RNA from Fermentation Samples and Shake Flask Samples
A. niger strains were cultured under different fermentation conditions (see table 4). Five-Liter controlled batch fermentations were performed in New Brunswick Scientific Bioflow 3000 fermenters.
The following conditions were used unless stated otherwise:
Temp. 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 (107 spores/ml) in 500 ml baffled Erlenmeyer flasks, overnight, 37° C., 150 rpm.

pH control: 4M KOH (Base), 1.5M H3PO4 (Acid)

Antifoam: Struktol (Schill & Seilacher)

Fermentation Medium Compositions:

Per liter: 2.36 g of NH4SO4, 0.11 g of KH2PO4, 2.08 g of MgSO4*7H2O, 0.13 g of CaCl2*2H2O, 0.074 of NaCl, 0.2 mg of CuSO4*5H2O, 5.5 mg of Fe(III)SO4*7H2O, 0.7 mg of MnCl2*4H2O and 1.3 mg of ZnSO4*7H2O and 100 g of glucose as a carbon source.

All media were prepared in demineralized water.

    
    









TABLE 4







fermentation conditions of A. niger strains















Biomass


fermentation






sample
Strain
medium
condition
glucose
biomass
ita/DWT

















1
2010
N201
M12
100-25% DO, pH start
60.0
12,460
—



exp2 F12


3.5 control 2.3






T3 (70 h)








2
2011
N201
M12 + Cu
100-25% DO, pH start
204
16,930
0.052



exp2 F8
CAD02

3.5 control 2.3






T6 (88.5 h)








3
2011
N201
M12 + Cu
25% DO, pH start
204
17,970
0.101



exp2 F9
CAD02

3.5 control 2.3






T6 (88.5 h)








4
2011
N201
M12 + Cu
10% DO, pH start
201
20,790
0.102



exp4 F9
CAD02

3.5 control 2.3






T2 (27 h)








5
2011
N201
M12 + Cu
15% DO, pH start
213
18,670
0.100



exp7 F8
CAD02

3.5 control 2.3






T5 (68 h)








6
2011
N201
M12 + Cu
20% DO, pH start
201
18,250
0.137



exp4 F8
CAD02

3.5 control 2.3






T2 (27 h)








7
2010
AB1.13 wt
M12
100-25% DO, pH start
60.0
11,600
—



exp2 F11
pyr+ (Cora)

3.5 control 2.3






T3 (70 h)








8
2010
AB1.13
M12
100-25% DO, pH start
60.0
8,880
—



exp2 F13
ΔoahA#76

3.5 control 2.3






T3 (70 h)








9
2011
AB1.13
M12 + Cu
100-25% DO, pH start
204
15,070
0.110



exp2 F10
ΔoahA#76

3.5 control 2.3






T6 (88.5 h)
CAD 05







10
2011
AB1.13 CAD
M12 − Cu
100-25% DO, pH start
210
14,937
0.152



exp1 F8
pyr+

3.5 control 2.3






T5 (49 h)








11
2011
AB1.13 CAD
M12
100-25% DO, pH start
210
17,339
0.106



exp1 F9
pyr+

3.5 control 2.3






T5 (49 h)








12
2011
AB1.13 CAD
M12 + Cu
100-25% DO, pH start
210
27,350
0.081



exp1 F10
pyr+

3.5 control 2.3






T5 (49 h)








13
2011
AB 1.13
M12 + Cu
10% DO, pH start
201
15,200
0.205



exp4 F10
CAD + FHB

3.5 control 2.3






T2 (27 h)
2,5







14
2011
AB 1.13
M12 + Cu
20% DO, pH start
213
16,780
0.153



exp7 F10
CAD + FHB

3.5 control 2.3






T5 (68 h)
2,5







15
2011
AB 1.13
M12 + Cu
5% DO, pH start
210
12,026
0.191



exp6 F10
CAD + FHB

3.5 control 2.3






T3 (27 h)
2,5









For the shake flask cultures the fermentation medium described above was used. The cultures were grown for 46 hours at 33° C. from which biomass samples were harvested.
RNA was isolated from biomass samples using Trizol from Invitrogen. Equal amounts of RNA (8 micrograms) were loaded on a RNA gel and blotted on Hybond N+ membrane from GE Healthcare.
Example 4 Northern Analysis
From the RNAseq data it was shown that several genes in a gene cluster, including the citB gene, were upregulated in the TNO-CAD strain compared to the TNO-WT strain.
To confirm the RNAseq results and to analyze different strains and different fermentation conditions, Northern analysis was carried out.
Primers were designed to amplify gene fragments by PCR of four upregulated genes from the gene cluster (citB, MFS, citR and prpD, seeTable 5) and other interesting genes (gpdA, citA, CAD). The labeling of the gene fragments was carried out using the PCR DIG Probe Synthesis Kit (Roche). Hybridization was performed using the DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche).







TABLE 5





Primers for Northern analysis


















o5886-CitA forward
TGTTGTCGCCGTAGCCGAGC



o5887-CitA reverse
AGCTCCTCCCCAAGGCTCCC



o5888-CitB forward
GCGACGCGGTCCACCTCAAA



o5889-CitB reverse
GCACAGGACTGCCCAACCCC



o5890-An08g10880 forward
GCTTCGCGGCCCATACTGCT



o5891-An08g10880 reverse
TGAAGCTGCCAACACCCCGC



o5892-An08g10870 forward
GATGGACGGCCACCCACTGC



o5893-An08g10870 reverse
TGCGCTCCCTTCAGCAGCAC



o5894-An08g10970 forward
GCAAAGGGTGCCAGGCCGAT



o5895-An08g10970 reverse
CTGGGTGCTGGTCATCGCGG



cadA forward
GGTCTTAGCCGAGCAAGGC



cadA reverse
GCGACACTCATCTGCCCTG



gpdA forward
ATCGAGACCTACGAGGAGGG



gpdA reverse
CCGGGAGTTCCTGCGAAGG










The results of the Northern analysis of the fermentation samples are shown in the table 6 below.








TABLE 6







Results Northern analysis


















citA
citB
citB (RT-qPCR





Strain
gpdA
cadA
(An09g06680)
(An08g10920)
citB/g pdA ratio)
An08g10880
An08g10870
An08g10970


















Shake flask culture










AB1.13pyr+
NA
NA
NA
NA
0.2
NA
NA
NA


AB1.13 CAD pyr+
NA
NA
NA
NA
5.0
NA
NA
NA


Controlled fermentation










AB1.13 CAD pyr+
+++
+++
+
−
0.2-0.5
−
−
−


AB1.13.CAD/MTT/MFS#3
NA
NA
NA
NA
0.8
NA
NA
NA


AB1.13 CAD+ FHB 2,5
+++
+++
+
−/+
0.8
−
−
+


N201
+++
−
+
−
0.2
−
−
−/+


N201 CAD02
++++
+++
+
−
1.0
−
−
−









From the Northern analysis it was concluded that the expression of the geners from the gene clister showed low expression levels, or no expression was detected, or were below detection limits.

Example 5 Quantitative RT-PCR
Northern analysis revealed very low expression levels, most likely below the detection limit of the method. Therefore, quantitative RT-PCR was carried out using the Superscript III platinum One Step Quantitative RT-PCR kit (Life Technologies) to analyze the expression of the citB gene (see Table 6). A primer-probe combination for the citB gene was designed using the software Primer Express 2.0 (Applied Biosystems). To compare the expression level of citB with a highly expressed gene, also gpdA primers were designed. For the normalization of the citB and gpdA data, also a quantitative RT-PCR was carried out using 18S primers. The quantitative PCR was performed on the 7500 Fast Real time PCR system (Applied Biosystems). The total RNA isolated from biomass of fermentation experiments, which was used in the Northern analysis, was analyzed in this method. Also the total RNA used for the RNAseq experiment was analyzed and total RNA isolated from A. niger strains grown in a new shakeflask culture experiment was analyzed. In the normalized RT-qPCR results can been seen that the citB gene is induced in certain conditions. For the samples used for RNAseq RT-qPCR confirmed the RNAseq results.
Example 6 Genome Mining citB Gene Cluster
In table 7 the results of a genome mining effort of the citB gene cluster is given.
Sequences were obtained from NCBI. Alignments were performed using
BLASTX with a BLOSUM62 matrix and the default settings for BLASTX (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi).







TABLE 7







Blast results of genes from the CitB genome cluster








Query
BLASTX best hits

















E


Gene
Accession nr.
Description
Accession nr.
Strain
value





An08g10860
ang: ANI_1_24880 74
sterigmatocystin
GAA88112.1
Aspergillus kawachii
0




biosynthesis fatty
XP_002149767.1
Penicillium marneffei
0




acid synthase
XP_002340041.1
Talaromyces stipitatus
0




subunit beta
XP_002384436.1
Aspergillus flavus
0





XP_001827193.1
Aspergillus oryzae
0


An08g10870
ang: ANI_1_2490074
2-methylcitrate
GAA88111.1
Aspergillus kawachii
0




dehydratase
XP_002384435.1
Aspergillus flavus
0





XP_001827192.1
Aspergillus oryzae
0


An08g10880
ang: ANI_1_2492074
GAL4; GAL4-like
GAA88110.1
Aspergillus kawachii
0




Zn2Cys6
EDP52162.1
Aspergillus fumigatus
E−127




binuclear cluster
XP_001259243.1
Neosartorya fischeri
E−124




DNA-binding
XP_001274697.1
Aspergillus clavatus
E−122




domain; found in
AAD34561.1
Aspergillus terreus
E−50 




transcription







regulators like







GAL4





An08g10920
ang: ANI_1_1474074
citrate synthase
NP_001142237.1
Zea mays
0





GAA88109.1
Aspergillus kawachii
0





XP_002384448.1
Aspergillus flavus
E−164





XP_001827205.1
Aspergillus oryzae
E−164





XP_002148678.1
Penicillium marneffei
E−137


An08g10930
ang: ANI_1_2494074
3-oxoacyl-[acyl-
GAA88108.1
Aspergillus kawachii
0




carrier-protein]
XP_001827206.1
Aspergillus oryzae
0




synthase
XP_002384449.1
Aspergillus flavus
0





EFQ31023.1
Glomerella
0





XP_002836001.1
graminicola
0






Tuber melanosporum



An08g10970
ang: ANI_1_2500074
MFS multidrug
GAA88107.1
Aspergillus kawachii
0




transporter
EHK20962.1
Trichoderma virens
E−171





EGR44089.1
Trichoderma reesei
E−168





EHK50986.1
Trichoderma
E−167





XP_002376688.1
atroviride
E−166





XP_001820954.1
Aspergillus flavus
E−166






Aspergillus oryzae



An08g10980
ang: ANI_1_2502074
similarity to
GAA88106.1
Aspergillus kawachii
0




acetate regulatory
XP_001820955.2
Aspergillus oryzae
E−132




DNA binding
XP_002376689.1
Aspergillus flavus
E−123




protein facB





An08g10990
ang: ANI_1_1484074
dienelactone
GAA88105.1
Aspergillus kawachii
E−109




hydrolase family
EHY59914.1
Exophiala dermatitidis
E−77 




protein
XP_002481994.1
Talaromyces stipitatus
E−64 





XP_002150690.1
Penicillium marneffei
E−61 









It appears that the genes as depicted in NP— 001142237.1, GAA88109.1, XP_002384448.1, XP_001827205.1 and XP_002148678.1 can be considered to be orthologs of the Aspergillus niger citrate synthase.

Using the search tool Sybil on the AspGD website (Broad Institute) (http://aspgd.broadinstitute.org/cgi-bin/asp2_v3/shared/show_protein_cluster.cgi?site=asp2_v8)

orthologous clusters from multiple genomes can be depicted as shown in FIG. 3.

The citrate synthase gene (An08g10920) was further used to search for the ortholog clusters in other Aspergillus genomes. As can be seen in FIG. 3, the “black Aspergilli”, A. niger (2 genomes) A. acidus, A. tubigensis and A. brasiliensis, show similar clustering of the genes surrounding the citB gene, whereas the genomic region in A. oryzae and A. flavus only contain the citB (An08g10920) ortholog and the orthologs of An08g10880 and An08g10930. For A. terreus no corresponding gene cluster was found at all.
Example 7A Overexpression of the A. niger citB Gene in Aspergillus niger 
To establish the overexpression of the citB gene in A. niger, a PCR generated copy of the gene was generated. For this purpose two sets of primers were generated as shown below. PCR amplification based on A. niger genomic DNA resulted in the isolation of PCR fragments from which the complete coding region of the citB gene could be isolated as a BsmBI-NcoI fragment.








Translation of citB genomic seq (1-1874)



Universal code


       BsmBI            citB-F3-ATG+BsmBI


5′-CGTCTCCCATGCCCGACATCGCATCCAAC-3′


`     citB-F1-6


     5′-ATCACTATGCCCGACATCGC-3 









   1
         ATGCCCGACATCGCATCCAACGGTGCCCGCAACGGCGCCTCCCAGAATGCAGAGACCAAG



   1
          M  P  D  I  A  S  N  G  A  R  N  G  A  S  Q  N  A  E  T  K


 


  61
        CCAGAACCCCCCGTTCTCCATGTGGTAGACAGCCGCACGGGGAAGTACTTCCCCATCCCT


  21
         P  E  P  P  V  L  H  V  V  D  S  R  T  G  K  Y  F  P  I  P


 


 121
        ATCGTGCGCAACGCCATCAACGCAAGCGAATTCAAGAAACTCAAGTCCCCCGAGGATCCC


  41
         I  V  R  N  A  I  N  A  S  E  F  K  K  L  K  S  P  E  D  P


 


 181
        GCACATCCTGAAGATCAGAACGAGCAGGGCATCCGGGTGITTGACCCCGGATACTCCAAC


  61
         A  H  P  E  D  Q  N  E  Q  G  I  R  V  F  D  P  G  Y  S  N


 


 241
        ACGGCTGTTAGTGAGAGCCAGGTTACCTACATgtgcgttttctctgctgcataggattga


  81
         T  A  V  S  E  S  Q  V  T  Y  I


 


 301
        tcatggcgaagagtaactgataacggggcgcagCGATGGCCTGAAGGGAACCATCCAGTA



                                           D  G  L  K  G  T  I  Q  Y


 


 361
        CCGTGGTTACAACATCGAGGATATTGTGGGCAAGAAGAAGTTTATTGACACGGCACACCT


 121
          R  G  Y  N  I  E  D  I  V  G  K  K  K  F  I  D  T  A  H  L


 


 421
        GCTCATTTGGGGAGAATGGCCGACGCCGGAACAGGCCAAATCTCTGCAGGAGAAGCTCTC


 141
          L  I  W  G  E  N  P  T  P  E  Q  A  K  S  L  Q  E  K  L  S


 


 481
        CAGCGTACCTGTCCTGGATGAATCCGTCTTCAAAGTCATTCAGGCATTCCCgtaagtttc


 161
          S  V  P  V  L  D  E  S  V  F  K  V  I  Q  A  F  P


 


 541
        accctagttttagcctctagtcctttcccccacggtctaacggctccagTCCCAACTCGT



                                                           P  N  S


 


 601
        CCATTATCGGCATGATGATCGCCGCTCTGTCAGCTGTCCAGAGTACCCAGATGGATCGCA


 200
        S  I  I  G  M  M  I  A  A  L  S  A  V  Q  S  T  Q  M  D  R


 


 661
        TCCCCGCCCATGCGGCCAAGAACCTCTACTTGGGCAATCCTAAGGCCGTCGATGATGAGA


 220
        I  P  A  H  A  A  K  N  L  Y  L  G  N  P  K  A  V  D  D  E


 


 721
        TCGTCCGTCTGATGGGCTCGCTGTCCATGATCACCGCTGCTGTCTACTGCCACCATACCG


 240
        I  V  R  L  M  G  S  L  S  M  I  T  A  A  V  Y  C  H  H  T


 


 781
        GACGGGAATTTACCCCGCCACGTCCGGAACTTTCCTACATCGAGAACTTCCTGTTGATGA


 260
        G  R  E  F  T  P  P  R  P  E  L  S  Y  I  E  N  F  L  L  M


 


 841
        TGGGCCACGTCGAGTCTAGCACAGGACTGCCCAACCCCCAGTACGTCGACCGCATTGAGC


 280
        M  G  H  V  E  S  S  T  G  L  P  N  P  Q  Y  V  D  R  I  E


 


 901
        GTCTCTGGGTCCTCATTGCCGATCACGAGATGACCTGCTCGACTGCCGCGTTCTTGCAGA


 300
        R  L  W  V  L  I  A  D  H  E  M  T  C  S  T  A  A  F  L  Q


 


 961
        CAGCCTCCTCCCTGCCGGATGTATTCTCCTGTATGATCTCCGCACTGTCGGCGCTCTATG


 320
        T  A  S  S  L  P  D  V  F  S  G  M  T  S  A  L  S  A  L  Y


 


1021
        GTCCGCTGCATGGTGGGGCCATTGAGGTAGCTTACAAAAATTTCGAGGAGATTGGCTCGG


 340
        G  P  L  H  G  G  A  I  E  V  A  Y  K  N  F  E  E  I  G  S


 


1081
        TTGAGAACGTCGCGGCCAAGATAGAACGTGTCAAGGCCGGTAAGGAGCGTCTGTACGGCT


 360
        V  E  N  V  A  A  K  I  E  R  V  K  A  G  K  E  R  L  Y  G


 


1141
        ACGGTCACCGCATCTACCGCGTCACAGACCCGCGCTTCATCTTCATCCGCCAGATCTTAG


 380
        Y  G  H  R  I  Y  R  V  T  D  P  R  F  I  F  I  R  Q  I  L


 


1201
        ACGAGTTGAAGGAAGAGATCGCCCGGAACCCGCTGCTGAAGGTGGCGTTTGAGGTGGACC


 400
        D  E  L  K  E  E  I  A  R  N  P  L  L  K  V  A  F  E  V  D


 


1261
        GCGTCGCCTCGGAGGATGAATACTTTGTCACCCGGAAGCTACGGCCCAACGCCGATCTCT


 420
        R  V  A  S  E  D  E  Y  F  V  T  R  K  L  R  P  N  A  D  L


 


1321
        TTGCGGCGCTTGTGTATAGTGCCATgtaggccttccgtgaagtagtggtttcagacatca


 440
        F  A  A  L  V  Y  S  A  M


 


1381
        gacccgctaacgcattgggaatagGGGCTTCCCGACTGAGTTTATTCTACCGTTGTCGCT



                                  G  F  P  T  E  F  I  L  P  L  S  L


 


1441
        GTTGTCCCGCACGCAGGGATTCATGGCCCACTGGAAAGAAGCCATGTgtaagtggcccat


 481
          L  S  R  T  Q  G  F  M  A  H  W  K  E  A  M


 


1501
        tttgccactgcgtgtcccactotgagactaacgatgtgacagCGAGCACGGCACGTATCT



                                                  S  S  T  A  R  I


 



                                                         3′-ATCCGTC



                                                         3′-ATCCGTC


1561
        GGCGGCCCGGCCAGATCTACACCGGACACTTGAACCGCGAGATGGCGTAGgtctaggag


 520
        W  R  P  G  Q  I  Y  T  G  H  L  N  R  E  M  A  *


 



                   NcoI          citB-R1 + NcoI



        AAAGCGAGAGTGGTACC-5′



          citB-R1 + 1631



        AAAGCGAGAT-5′


1621
        tttcgctctcatcggtg


 








Overview primers










citB-F1-6
ATCACTATGCCCGACATCGC 50.6° C.



 


citB-F3-ATG + BsmBI
CGTCTCCCATGCCCGACATCGCATCCAAC 63.3° C.


 


citB-R1 + 1631
TGAGAGCGAAACTGCCTA 54.1° C.


 


citB-R1 + NcoI
CCATGGTGAGAGCGAAACTGCCTA 54.1° C.


 








citB-F3-ATG + BsmBI; 29-mer; 63.3° C.



5′-CGTCTCCCATGCCCGACATCGCATCCAAC-3′ Primer


           |||||| ||||||||||||||


3′-        TACGGGCTGTAGCGTAGGTTG-5′ (21) Strand -


 


citB-F1-6; 20-mer; 50.6° C.


5′-ATCACTATGCCCGACATCGC-3′ Primer


           |||||||| |||||


3′-        TACGGGCTGTAGCG-5′ (14) Strand -


 


citB-R1 + NcoI; 24-mer; 54.1° C.


5′-CCATGGTGAGAGCGAAACTGCCTA-3′ Primer


   |   | || ||||| |||||||||


3′-GTGGCTACTCTCGCTTTGACGGAT-5′ (1614) Strand +


 


citB-R1 + 1631; 18-mer; 54.1° C.


5′-TGAGAGCGAAACTGCCTA-3′ Primer


   |||||||| ||||| |||


3′-ACTCTCGCTTTGACGGAT-5′ (1614) Strand +






The resulting BsmBI-NcoI fragment was cloned in the NcoI site of the Aspergillus expression vector pABgpd-I (FIG. 5). In a derivative of this vector also the Aspergillus auxotrophic selection marker pyrG was cloned.
Subsequently, an itaconic acid producing Aspergillus niger strain (Li, A. et al., Appl. Microbial. Biotechnol. 1-11, 2013; Li, A. et al. Fungal Genet. Biol. 48:602-611, 2011) was transformed with the citB overexpression vector. PyrG+ transformants were purified by single colony purification and retested for their PyrG+ phenotype. Several PyrG+ transformants were subsequently cultured in shake flask cultures from which the expression of the introduced citB expression cassette was analyzed using quantitative RT-PCR. In addition Southern analysis was carried out to confirm the presence of intact copies of the expression cassette in the transformants.
The transformants with the highest copy number and/or highest citB expression level were cultured in batch fermentations. Following up, the media samples were analyzed by HPLC for the amount of itaconic acid produced by the A. niger transformants. Besides this, other organic acids like citric acid and oxalic acid were also analyzed due to their relevance in the assumed itaconate production pathway in Aspergillus niger. 
Cultivation Conditions
For the screening and selection of A. niger transformants, our previously developed screening assay was used (Li et al. 2012). After seeding, all plates were directly sealed with an oxygen permeable film (Sealing film sterile, breathable M20193, Dispolab the Netherlands), placed in a plastic air bag and cultivated in a 33° C., 850 rpm incubator (Microtron, Infos-ht) for 60 h. In the end of the cultivation, culture medium was harvested and used for HPLC analysis.
For shakeflask and controlled batch fermentations, the production medium
(M12) described in our previous study (Li et al. 2012) with the following composition was used (per liter): 100 g glucose, 2.36 g (NH4)2SO4, 0.11 g KH2PO4, 0.5 g MgSO4.7H2O, 0.6 mg FeSO4.7H2O, 2.5 mg CuSO4.5H2O, 0.6 mg ZnSO4.7H2O, 0.074 g NaCl, and 0.13 g CaCl2.2H2O. This medium was prepared in demineralised water. The production medium M12+Cu has an extra addition of 2.5 mg CuSO4.5H2O (0.01 mM). For controlled fermentation pre-cultures were prepared by inoculation of 106 spores per milliliter in 2×100-mL production medium in two 500 mL baffled Erlenmeyer flasks. After 64 h at 33° C. and shaking at 125 rpm, the pre-cultures were used for inoculation of the fermenters. Fermentations were performed in 5-L Benchtop Fermentors (BioFlo 3000, New Brunswick Scientific Co., Inc.) at 33° C. The basic pH regime was initiated at 3.5 and subsequently regulated at 2.3, by addition of 4 M KOH (base). Struktol was applied as antifoam agent (Schill & Seilacher) in all cultures throughout the fermentation. Air was used for sparging the bioreactor at a constant flow of 0.25 vvm [(vol.liquid)−1 min−1]. The solubility of oxygen in the medium is around 225 μMol at 33° C. Pure air sparging was calibrated as 100% D.O., whereas pure nitrogen sparging was calibrated as 0% D.O. In the basic D.O. regime, D.O. was set at 100% from the start of the fermentation. As soon as due to mycelial growth D.O. levels dropped below 25%, stirrer agitation was increased automatically to maintain D.O. at 25%. For studying the influence of oxygen availability on itaconic acid production, D.O was fixed throughout the whole fermentation at 10, 15, 20, and 25% for strain N201 CAD and at 5, 10, and 20% for strain HBD 2.5. The different percentage of D.O. was obtained by varying the mixture of air/nitrogen in the inlet gas.
The cultured transformants were analysed for the presence of citric acid and derivatives in microplate cultures. Based on these cultures several transformants producing increased itaconic acid levels were selected for further research.
Based on the results obtained in microplate screening a selection of transformants was grown in shakeflask cultures as described by Li et al., 2012, 2013 and analysed for itaconic acid productivity and yield














Itaconic acid



Itaconic acid
Yield (mg/g Glucose)


Strain
Productivity (mg/L/hr)
Shake flask







culture




CAD
7.8



CAD + citB#49
8.8



CAD + citB#53
11.4



CAD + citB#71
10.4



CAD + citB#84A
8.7



CAD MFS/MTT#48
17.9



CAD MFS/MTT#49
31.2



CAD MFS/MTT#63 26.7




Controlled fermentation




CAD
8.9
30


CAD
11.2



CAD + citB#53
15.4
42


CAD MFS/MTT#8
20.9



CAD MFS/MTT#63 
50.2









As shown the introduction of the citB gene into an A. niger strain already expressing cadA resulted in increased productivity and yields of secreted itaconic acid.
Introduction of two previously identified organic acid transporters (MTT/MFS) as described in WO 2009/104958 and WO 2009/110796 in a single host strain also resulted in increased productivity of itaconic acid.
Example 7B. Overexpression of A. niger citB in a Host Strain Expressing all Three Genes of the Itaconic Acid Gene Cluster
Strain Construction
In a strain already simultaneously expressing the A. terreus cadA, mfsA and mttA genes, which genes and strains have been described in WO 2009/014437, WO 2009/104958 and WO 2009/110796 as shown in the table in Example 7A, above, (strains CADMFSMTT#63 or #49), the A. niger citB expression vector was introduced by cotransformation using the phleomycin resistence marker for transformant selection. From the resulting tranosformants strain CitB#99 was selected for further analysis

Fermentation Conditions

Controlled batch cultivations were performed in 5 liter batch fermentors (BioFlo 3000, New Brunswick Scientific Co., Inc.). The production medium, as published earlier by An Li et al., 2012, consists of the following (per liter): 100 g glucose, 2.36 g (NH4)2SO4, 0.11 g KH2PO4, 0.5 g MgSO4.7H2O, 0.6 g FeSO4.7H2O, 2.5 mg CuSO4.5H2O, 0.6 mg ZnSO4.7H2O, 0.074 g NaCl and 0.13 g CaCl2.2H2O. This medium was prepared in demineralized water. Inoculum was prepared with 1.0·106 spores/mL in 100 mL production medium in 500 mL baffled Erlenmeyer flasks. Inoculum was then incubated at 33° C. for 72 hours and shaking at 125 rpm. Temperature was kept stable at 33° C. throughout the fermentation. The fermentation starts with a pH of 3.5 and afterwards is kept stable at 2.3 by addition of 4M KOH. The bioreactor was sparged with a constant flow of 1.25 vvm [(vol.liquid)−1 min−1] air. The system was calibrated as 100% D. O. by sparging the bioreactor with pure air whereas pure nitrogen sparging was calibrated as 0% D. O. Throughout the course of the fermentation the D. O. was kept at 20%, which is achieved by applying various mixtures of air and nitrogen in the inlet gas. Struktol (Schill & Seilacher) was used as antifoaming agent. Autosamples were taken every six hours using a 0.22 μM filter (Applikon Biotechnology, USA).

HPLC Analysis

Filter-sterilized fermentation samples were analyzed by high-performance liquid chromatography (HPLC) to quantify metabolites and assess organic acid production. Samples were loaded on a WATERS e2695 Separations Module outfitted with an Aminex HPX-87H column (Bio-Rad) and 5 mM H2SO4 as eluent. Metabolites were detected by a refractive index detector (WATERS 2414) and a dual-wavelength detector (WATERS UV/Vis 2489) simultaneously. Empower Pro was used as software for the processing of data (Empower 2 Software, copyright 2005-2008, Waters Corporation, Milford, Mass., USA).

Results

In order to compare the organic acid production capacity of the CitB #99 strain with the AB1.13 CAD+MTT+MFS strain, controlled batch fermentations were performed. The glucose consumption and organic acid production capacity of the two strains is depicted in FIG. 6 (CitB #99) and FIG. 7 (AB1.13 CAD+MTT+MFS). As can be seen in FIG. 6 and FIG. 7, CitB #99 produces more itaconic acid (higher yield) and has a higher production rate. CitB #99 produces itaconic acid up to 25 grams per liter and, unexpectedly, no citric acid. This shows that the conversion of citric acid to itaconic acid is very efficient in our transformed strain. Furthermore, the improved production rate of itaconic acid is a major step forwards; maximum yield is achieved after 5 days of fermentation, whereas the AB1.13 CAD+MTT+MFS strain needs 7 days to achieve maximum yield of around 12 grams per liter.

Example 7C Itaconic Acid Production on Crude Second Generation Feedstocks
Shakeflask Cultures
Cultivations in shakeflasks were performed in order to assess if the CitB #99 strain can grow on second generation feedstocks e.g. glycerol. For this experiment crude waste glycerol was acquired from a biodiesel production plant. In the biodiesel process a waste product containing glycerol as mayor carbon source is produced as waste product. The experiment was performed in 500 mL baffled Erlenmeyer shakeflasks with a volume of 100 mL. Medium was prepared by adding 10 mL of crude glycerol from the company to 90 mL demineralized water. Preculture was prepared by inoculating 1.0·106 spores/mL in 100 mL production medium in 500 mL baffled Erlenmeyer flasks and grown overnight at 33° C. and shaking at 125 rpm. From this preculture 2 mL was used as inoculum.
Results
In order to assess if the CitB #99 strain can grow on second-generation feedstock, shakeflask cultivations were performed with glycerol as C-source (FIG. 8). The flasks were incubated at 33° C. for 216 hours. In FIG. 8 it can be seen that glycerol consumption starts after 96 hours together with the production of itaconic acid. The first 96 hours of incubation appear to be necessary for the organism to adapt to the environment. This phenomenon may partly be caused due to the fact that the preculture was grown on production medium, which has glucose as C-source, rather than glycerol. Production level of itaconic acid leads up to approximately 1.5 grams per liter indicating that this strain can produce itaconic acid when grown on a second generation feedstocks, such as crude glycerol. For future purposes the CitB #99 strain can be cultivated in controlled batch fermentations to achieve optimal itaconic acid production.
Example 8 Overexpression of the A. niger citB Gene and A. terreus cadA Gene in Saccharomyces cerevisiae 
For the overexpression of A. niger citB and A. terreus cadA in Saccharomyces cerevisiae an expression vector was synthesized at Geneart (Life technologies Europe, Bleiswijk, The Netherlands) containing two expression cassettes. The Saccharomyces codon optimized gene encoding the A. niger CitB protein was inserted between the gpd promotor and CYC1 terminator of Saccharomyces. The Saccharomyces codon optimized gene encoding the A. terreus CAD protein was inserted between the tef promotor and ADH1 terminator of Saccharomyces. In between both expression cassettes the URA3 marker was placed in antisense. The complete fragment was surrounded with URA3 flanking regions for integration at the URA3 locus in Saccharomyces cerevisiae.








5′ URA3 flank-GPD promoter-citB gene (codon optimized for Saccharomyces



cerevisiae)-CYC1 terminator-URA3 marker-TEF promoter-CAD gene (codon


optimized for Saccharomyces cerevisiae)-ADH1 terminator-3′ URA3 flank










 NotI



1
GCGGCCGCGATAAGTTTTGACCATCAAAGAAGGTTAATGTGGCTGTGGTTTCAGGGTCCA



 


61
TAAAGCTTTCAGTTTATCATTATCAATACTCGCCATTTCAAAGAATACGTAAATAATTAA


 


121
TAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAAC


 


181
CCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGG


 


241
GTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCAT


 


301
CCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCAT


 


361
AGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCA


 


421
CAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCC


 


481
ACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGG


 


541
AAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAAT


 


601
AAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTT


 


661
AAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAGAACTTAGTTTCGA


 



       XbaI


721
CGGATTCTAGAATGCCAGATATTGCTTCTAATGGTGCTAGAAATGGTGCTTCTCAAAACG



            M  P  D  I  A  S  N  G  A  R  N  G  A  S  Q  N


 


781
CTGAAACAAAACCAGAACCACCAGTTTTACACGTTGTTGATTCAAGAACTGGTAAGTACT


260
A  E  T  K  P  E  P  P  V  L  H  V  V  C  S  R  T  G  K  Y


 


841
TCCCAATCCCAATCGTTAGAAATGCTATTAACGCCTCCGAGTTCAAGAAGTTGAAATCTC


280
F  P  I  P  I  V  R  N  A  I  N  A  S  E  F  K  K  L  K  S


 


901
CAGAAGATCCAGCTCATCCAGAAGATCAAAACGAACAAGGTATCAGAGTTTTCGATCCAG


300
P  E  D  P  A  H  P  E  D  Q  N  E  Q  G  I  R  V  F  D  P


 


961
GTTACTCTAATACCGCTGTTTCTGAATCTCAAGTTACCTACATTGATGGTTTGAAGGGTA


320
G  Y  S  N  T  A  V  S  E  S  Q  V  T  Y  I  D  G  L  K  G


 


1021
CTATCCAATACAGAGGTTACAACATCGAAGATATCGTCGGTAAGAAGAAGTTCATTGATA


340
T  I  Q  Y  R  G  Y  N  I  E  D  I  V  G  K  K  K  F  I  D


 


1081
CCGCCCATTTGTTGATTTGGGGTGAATGGCCAACTCCAGAACAAGCTAAATCATTGCAAG


360
T  A  H  L  L  I  W  G  E  W  P  T  P  E  Q  A  K  S  L  Q


 


1141
AAAAGTTGTCCTCCGTTCCAGTTTTGGATGAATCTGTTTTCAAGGTTATTCAAGCCTTCC


380
E  K  L  S  S  V  P  V  L  D  E  S  V  F  K  V  I  Q  A  F


 


1201
CACCAAACTCCTCTATTATTGGTATGATGATTGCTGCTTTGTCCGCTGTTCAATCTACTC


400
P  P  N  S  S  I  I  G  M  M  I  A  A  L  S  A  V  Q  S  T


 


1261
AAATGGATAGAATACCAGCTCATGCTGCTAAGAACTTGTATTTGGGTAATCCAAAAGCCG


420
Q  M  D  R  I  P  A  H  A  A  K  N  L  Y  L  G  N  P  K  A


 


1321
TTGATGACGAAATCGTTAGATTGATGGGTTCCTTGTCTATGATTACTGCTGCTGTTTACT


440
V  D  D  E  I  V  R  L  M  G  S  L  S  M  I  T  A  A  V  Y


 


1381
GTCATCATACCGGTAGAGAATTTACTCCACCAAGACCAGAATTGTCCTACATCGAAAATT


460
C  H  H  T  G  R  E  F  T  P  P  R  P  E  L  S  Y  I  E  N


 


1441
TCTTGTTGATGATGGGTCACGTCGAATCTTCTACTGGTTTGCCAAATCCACAATACGTTG


480
F  L  L  M  M  G  H  V  E  S  S  T  G  L  P  N  P  Q  Y  V


 


1501
ACAGAATTGAAAGATTGTGGGTTTTGATTGCCGATCACGAAATGACTTGTTCTACTGCTG


500
D  R  I  E  R  L  W  V  L  I  A  D  H  E  M  T  C  S  T  A


 


1561
CTTTCTTGCAAACTGCTTCTTCATTGCCAGATGITTTCTCTTGTATGATCTCTGCTTTGT


520
A  F  L  Q  T  A  S  S  L  P  D  V  F  S  C  M  I  S  A  L


 


1621
CTGCATTATACGGTCCATTGCATGGTGGTGCTATTGAAGTTGCTTACAAGAACTTCGAAG


540
S  A  L  Y  G  P  L  H  G  G  A  I  E  V  A  Y  K  N  F  E


 


1681
AAATCGGTTCCGTTGAAAATGTTGCTGCCAAAATCGAAAGAGTTAAGGCCGGTAAAGAAA


560
E  I  G  S  V  E  N  V  A  A  K  T  E  R  V  K  A  G  K  E


 


1741
GATTATACGGTTACGGTCATAGAATCTACAGAGTTACTGATCCAAGATTCATCTTCATCA


580
R  L  Y  G  Y  G  H  R  I  Y  R  V  T  D  P  R  F  I  F  I


 


1801
GACAAATCTTGGATGAATTGAAAGAAGAAATCGCCAGAAACCCTTTGTTGAAGGTTGCTT


600
R  Q  I  L  D  E  L  K  E  E  I  A  R  N  P  L  L  K  V  A


 


1861
TTGAAGTTGATAGAGTCGCCTCTGAAGATGAATACTTCGTTACCAGAAAGTTAAGACCAA


620
F  E  V  D  R  V  A  S  E  D  E  Y  F  V  T  R  K  L  R  P


 


1921
ACGCTGATTTGTTTGCTGCCTTGGTTTATTCTGCTATGGGTTTTCCAACCGAGTTCATCT


610
N  A  D  L  F  A  A  L  V  Y  S  A  M  G  F  P  T  E  F  I


 


1981
TGCCATTGTCTTTGTTGTCAAGAACCCAAGGTTTTATGGCCCATTGGAAAGAAGCTATGT


660
L  P  L  S  L  L  S  R  T  Q  G  F  M  A  H  W  K  E  A  M


 


2041
CATCTACTGCTAGAATTTGGAGACCTGGTCAAATCTATACTGGTCACTTGAATAGAGAAA


680
S  S  T  A  R  I  W  R  P  G  Q  I  Y  T  G  H  L  N  R  E


 



        XhoI


2101
TGGCTTAACTCGAGTCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCAC


700
M  A  *


 


2161
ATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTT


 


2221
TTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCT


 


2281
GTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGG


 



                             BamHI


2341
ACGCTCGAAGGCTTTAATTTGCGGCCGGTGGATCCTTTTCTTTCCAATTTTTTTTTTTTC


 


2401
GTCATTATAAAAATCATTACGACCGAGATTCCCGGGTAATAACTGATATAATTAAATTGA


 



                                                    *  N  Q



TCGAGATTAAACACTCAAATCATATGTACGTAAATGAATATTATGTCAAAAAATCAAAAC


2461
AGCTCTAATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTG


 



 Q  G  C  R  R  L  Y  A  E  W  G  A  K  R  Y  R  E  G  E  V



GACCGGCGTAGAAGAGTTTATACGAAGGGTCGGACGAAAAGACATTGCAAGTGGGAGATG


2521
CTGGCCGCATCTTCTCAAATATGCTTCCCAGCCTGCTTTTCTGTAACGTICACCCTCTAC


 



 K  A  D  R  G  K  A  F  L  G  R  G  V  I  I  I  D  S  G  T



GAATCGTAGGGAAGGGAAACGTTTATCAGGAGAAGGTTGTTATTATTACAGTCTAGGACA


2581
CTTAGCATCCCTTCCCTTTGCAAATAGTCCTCTICCAACAATAATAATGICAGATCCTGT


 



 S  V  V  D  D  V  T  R  Y  Q  Q  G  L  A  D  G  K  D  D  L



TCTCTGGTGTAGTAGGTGCCAAGATATGACAACTGGGTTACGCAGAGGGAACAGTAGATT


2641
AGAGACCACATCATCCACGGTTCTATACTGTTGACCCAATGCGTCTCCCTTGTCATCTAA


 



 G  V  G  P  T  M  I  L  W  D  Y  G  E  D  R  G  G  M  D  R



TGGGTGTGGCCCACAGTATTAGTTGGTTAGCATTGGAAGTAGAGAAGGTGGGTACAGAGA


2701
ACCCACACCGGGTGTCATAATCAACCAATCGTAACCTTCATCTCTTCCACCCATGTCTCT


 



 Q  A  I  F  G  I  V  F  D  K  D  S  K  A  I  D  V  T  G  K



AACTCGTTATTTCGGCTATTGTTTTAGAAACAGCGAGAAGCGTTACAGTTGTCATGGGAA


2761
TTGAGCAATAAAGCCGATAACAAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTT


 



 T  Y  E  G  T  S  L  S  G  K  C  S  L  E  A  L  M  L  L  G



TCATATAAGAGGTCATCTATCCCTCGGGAACGTACTGTTAAGACGATTGTAGTTTTCCGG


2821
AGTATATTCTCCAGTAGATAGGGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCC


 



 R  P  E  K  T  V  E  E  A  A  Q  K  L  G  S  V  I  G  P  G



AGATCCAAGGAAACAATGAAGAAGACGGCGGACGAAGTTTGGCGATTGTTATGGACCCGG


2881
TCTAGGTTCCTTTGTTACTTCTTCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGGCC


 



 V  V  G  H  A  N  T  I  D  A  W  E  A  I  R  Y  V  G  A  S



GTGGTGTGGCACACGTAAGCATTACAGACGGGTAAGACGATAAGACATATGTGGGCGTCT


2941
CACCACACCGTGTGCATTCGTAATGTCTGCCCATTCTGCTATTCTGTATACACCCGCAGA


 



 Y  Q  L  K  V  T  N  G  I  D  A  F  K  R  D  E  F  L  L  F



CATGACGTTAAACTGACATAATGGTTACAGTCGTTTAAAAGACAGAAGCTTCTCATTTTT


3001
GTACTGCAATTTGACTGTATTACCAATGTCAGCAAATTTTCTGTCTTCGAAGAGTAAAAA


 



 N  Y  K  A  S  L  A  K  L  P  K  V  T  G  E  M  S  F  D  T



TAACATGAACCGCCTATTACGGAAATCGCCGAATTGACACGGGAGGTACCTTTTTACTCA


3061
ATTGTACTTGGCGGATAATGCCTTTAGCGGCTTAACTGTGCCCTCCATGGAAAAATCAGT


 



 L  I  D  V  H  T  K  L  L  C  I  K  P  G  L  A  E  V  L  E



GTTCTATAGGTGTACACAAAAATCATTTGTTTAAAACCCTGGATTACGAAGTTGATTGAG


3121
CAAGATATCCACATGTGTTTTTAGTAAACAAATTTTGGGACCTAATGCTTCAACTAACTC


 



 L  L  E  K  T  T  R  V  D  L  S  A  C  L  N  T  Q  K  E  H



GTCATTAAGGAACCACCATGCTTGTAGGTTACTTCGTGTGTTCAAACAAACGAAAAGCAC


3181
CAGTAATTCCTTGGTGGTACGAACATCCAATGAAGCACACAAGTTTGTTTGCTTTTCGTG


 



 M  I  N  F  L  K  A  A  V  P  S  P  H  T  A  A  R  E  K  Y



GTACTATAATTTATCGAACCGTCGTTGTCCTGATCCTACTCATCGTCGTGCAAGGAATAT


3241
CATGATATTAAATAGCTTGGCAGCAACAGGACTAGGATGAGTAGCAGCACGTTCCTTATA


 



 T  A  K  S  M



ACATCGAAAGCTGTA


3301
TGTAGCTTTCGACATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGT


 


3361
TAAGAATACTGGGCAATTTCATGTTTCTTCAACACTACATATGCGTATATATACCAATCT


 


3421
AAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATT


 



                                                   EcoRI


3481
TCAAAGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAAGAATTCTTA


 


3541
CCCATAAGGTTGTTTGTGACGGCGTCGTACAAGAGAACGTGGGAACTTTTTAGGCTCACC


 


3601
AAAAAAGAAAGAAAAAATACGAGTTGCTGACAGAAGCCTCAAGAAAAAAAAAATTCTTCT


 


3661
TCGACTATGCTGGAGGCAGAGATGATCGAGCCGGTAGTTAACTATATATAGCTAAATTGG


 


3721
TTCCATCACCTTCTTTTCTGGTGTCGCTCCTTCTAGTGCTATTTCTGGCTTTTCCTATTT


 


3781
TTTTTTTTCCATTTTTCTTTCTCTCTTTCTAATATATAAATTCTCTTGCATTTTCTATTT


 


3841
TTCTCTCTATCTATTCTACTTGTTTATTCCCTTCAAGGTTTTTTTTTAAGGAGTACTTGT


 



                                         XbaI


3901
TTTTAGAATATACGGTCAACGAACTATAATTAACTAAACTCTAGAATGACCAAGCAATCC



                                              M  T  K  Q  S


 


3961
GCTGATTCTAATGCTAAATCTGGTGTTACCGCTGAAATTTGTCATTGGGCTTCTAATTTG


1321
 A  D  S  N  A  K  S  G  V  T  A  E  I  C  H  W  A  S  N  L


 


4021
GCCACCGATGATATTCCATCTGATGTTTTGGAAAGAGCCAAGTACTTGATCTTGGATGGT


1341
 A  T  D  D  I  P  S  D  V  L  E  R  A  K  Y  L  I  L  D  G


 


4081
ATTGCTTGTGCTTGGGTTGGTGCTAGAGTTCCATGGTCTGAAAAGTATGTTCAAGCTACC


1361
 I  A  C  A  W  V  G  A  R  V  P  W  S  E  K  Y  V  Q  A  T


 


4141
ATGTCTTTTGAACCACCAGGTGCTTGTAGAGTTATTGGTTATGGTCAAAAATTGGGTCCA


1381
 M  S  F  E  P  P  G  A  C  R  V  I  G  Y  G  Q  K  L  G  P


 


4201
GTTGCTGCTGCTATGACTAATTCTGCTTTTATTCAAGCCACCGAATTGGATGATTACCAT


1401
 V  A  A  A  M  T  N  S  A  F  I  Q  A  T  E  L  D  D  Y  H


 


4261
TCTGAAGCTCCATTGCATTCTGCTTCTATAGTTTTGCCAGCTGTTTTTGCTGCTTCTGAA


1421
 S  E  A  P  L  H  S  A  S  I  V  L  P  A  V  F  A  A  S  E


 


4321
GTTTTGGCTGAACAAGGTAAAACCATCTCCGGTATTGATGTTATTTTGGCTGCTATCGTT


1441
 V  L  A  E  Q  G  K  T  I  S  G  I  D  V  I  L  A  A  I  V


 


4381
GGTTTCGAATCTGGTCCAAGAATTGGTAAAGCTATCTACGGTTCTGACTTGTTGAACAAT


1461
 G  F  E  S  G  P  R  I  G  K  A  I  Y  G  S  D  L  L  N  N


 


4491
GGTTGGCATTGTGGTGCTGTTTATGGTGCTCCAGCTGGTGCTTTGGCTACTGGTAAGTTG


1481
 G  W  H  C  G  A  V  Y  G  A  P  A  G  A  L  A  T  G  K  L


 


4501
TTGGGTTTGACTCCAGATTCTATGGAAGATGCTTTGGGTATTGCATGTACTCAAGCTTGT


1501
 L  G  L  T  P  D  S  M  E  D  A  L  G  I  A  C  T  Q  A  C


 


4561
GGTTTGATGTCTGCTCAATATGGTGGTATGGTTAAGAGAGTTCAACACGGTTTTGCTGCA


1521
 G  L  M  S  A  Q  Y  G  G  M  V  K  R  V  Q  H  G  F  A  A


 


4621
AGAAATGGTTTGTTGGGTGGTTTGTTGGCTTATGGTGGTTATGAAGCTATGAAGGGTGTA


1541
 R  N  G  L  L  G  G  L  L  A  Y  G  G  Y  E  A  M  K  G  V


 


4681
TTGGAAAGATCTTACGGTGGTTTCTTGAAGATGTTCACTAAGGGTAATGGTAGAGAACCA


1561
 L  E  R  S  Y  G  G  F  L  K  M  F  T  K  G  N  G  R  E  P


 


4741
CCATACAAAGAAGAAGAAGTTGTTGCTGGTTTGGGTTCTTTTTGGCATACTTTCACCATC


1581
 P  Y  K  E  E  E  V  V  A  G  L  G  S  F  W  H  T  F  T  I


 


4801
AGAATCAAGTTGTATGCTTGTTGCGGTTTGGTTCATGGTCCAGTTGAAGCTATTGAAAAG


1601
 R  I  K  L  Y  A  C  C  G  L  V  H  G  P  V  E  A  I  E  K


 


4861
TTGCAAAGAAGATACCCAGAATTATTGAACAGAGCCAACTTGTCCAACATCAGACATGTT


1621
 L  Q  R  R  Y  P  E  L  L  N  R  A  N  L  S  N  I  R  H  V


 


4921
TACGTTCAATTGTCTACCGCCTCTAATTCTCATTGTGGTTGGATTCCAGAAGAAAGACCA


1641
 Y  V  Q  L  S  T  A  S  N  S  H  C  G  W  I  P  E  E  R  P


 


4981
ATTTCTTCTATTGCCGGTCAAATGTCCGTTGCTTACATTTTGGCTGTTCAATTGGTTGAC


1661
 I  S  S  I  A  G  Q  M  S  V  A  Y  I  L  A  V  Q  L  V  D


 


5041
CAACAATGTTTGTTGGCCCAATTCTCCGAATTTGATGACAATTTGGAAAGACCAGAAGTT


1681
 Q  Q  C  L  L  A  Q  F  S  E  F  D  D  N  L  E  R  P  E  V


 


5101
TGGGATTTGGCTAGAAAAGTTACTCCATCCCACTCCGAAGAATTTGATCAAGATGGTAAC


1701
 W  D  L  A  R  K  V  T  P  S  H  S  E  E  F  D  Q  D  G  N


 


5161
TGTTTGTCCGCTGGTAGAGTTAGAATTGAGTTCAACGATGGTTCCTCTGTTACCGAAACT


1721
 C  L  S  A  G  R  V  R  I  E  F  N  D  G  S  S  V  T  E  T


 


5221
GTTGAAAAACCATTGGGTGTCAAAGAACCTATGCCAAACGAAAGAATCTTGCACAAGTAT


1741
 V  E  K  P  L  G  V  K  E  P  M  P  N  E  R  I  L  H  K  Y


 


5281
AGAACTTTGGCTGGTTCTGTTACCGATGAATCAAGAGTCAAAGAAATCGAAGATTTGGTC


1761
 R  T  L  A  G  S  V  T  D  E  S  R  V  K  E  T  E  D  L  V


 


5341
TTGTCCTTGGATAGATTGACTGATATTACCCCTTTGTTGGAATTATTGAATTGCCCAGTT


1781
 L  S  L  D  R  L  T  D  I  T  P  L  L  E  L  L  N  C  P  V


 



                   XhoI


5401
AAGTCCCCATTGGTCTAACTCGAGGCGAATTTCTTATGATTTATGATTTTTATTATTAAA


1801
 K  S  P  L  V  *


 


5461
TAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGA


 


5521
AAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCAT


 


5581
GAGGTCGCTCTTATTGACCACACCTCTACCGGCATGCCGAGCAAATGCCTGCAAATCGCT


 


5641
CCCCATTTCACCCAATTGTAGATATGCTAACTCCAGCAATGAGTTGATGAATCTCGGTGT


 


5701
GTATTTTATGTCCTCAGAGGACAACACCTGTTGTAATCGTTCTTCCACACTCATGGCCTT


 



                                                    NotI


5761
 TATAAAAAGGAACTATCCAATACCTCGCCAGAACCAAGTAACAGTATTTTGCGGCCGC






The citB expression fragment was isolated from the synthesized vector using BamHI-SstI and cloned into the pFL61 yeast expression vector (ATCC77215; http://www.lgcstandards-atcc.org/Products/All/77215.aspx; Minet M. et al. Complementaton of Saccharomyces cerevisiae auxotrophic mutants by Arabidopsis thaliana cDNA. Plant J. 2: 417-422, 1992), which was digested with BamHI and SstI. The cadA expression fragment was isolated using Acc651-EcoRI and cloned into the pFL61 yeast expression vector. The resulting citB and cadA expression vectors were transformed to the Saccharomyces cerevisiae strain CEN.PK113-5D using the electroporation protocol as described in (Transformation of commercial baker's yeast strains by electroporation., Gysler et al., Biotechnology Techniques Vol 4 No 4 285-290 (1990)) The yeast transformants were purified by single colony purification and analyzed with PCR for the presence of the expression vector. Subsequently, the transformants carrying citB or cadA genecopies were cultured in microtitreplate cultures under aerobic and anaerobic conditions and analyzed for the organic acid production using HPLC. In cadA expressing strains under both anaerobic and aerobic conditions itaconic acid production was detected in the culture medium.

## Claims

1. A method for producing itaconic acid which method comprises modifying a eukaryotic host cell wherein said modifying consists of providing said host cell with both a nucleic acid that expresses a gene encoding a citric acid synthase that lacks a signal for expression in the mitochondrion, wherein the citric acid synthase is that encoded by an A. niger gene designated An08g10920 (SEQ ID NO: 18), or An01g09940 (SEQ ID NO: 50), or An09g03570 (SEQ ID NO: 49), or is an ortholog thereof,
wherein the ortholog is a protein that has citric acid synthase activity and a percentage identity of at least 95% with that encoded by An08g10920 (SEQ ID NO: 18), or An01g09940 (SEQ ID NO: 50) or An09g03570 (SEQ ID NO: 49); and
with a nucleic acid that expresses a gene encoding cis-aconitic acid decarboxylase (CAD); and
culturing said eukaryotic host cell.

2. The method of claim 1, wherein said modifying is effected by transforming the host cell with a vector comprising said nucleic acid that expresses sequence encoding said citric acid synthase and a vector comprising said nucleic acid that expresses sequence encoding CAD.

3. The method of claim 1, wherein said host cell is a fungus or a yeast or a plant or algal cell.

4. The method of claim 1, wherein said host cell is selected from the group of Aspergillus spp., Neurospora spp., Sclerotina, Gibberella, Coniothyrium, Psiticum, Magnaporthe, Podospora, Chaetomium, Phaeosphaeria, Botryotinia, Neosartorya, Pyrenophora, Panicum, Aureococcus, Penicillium, Trichoderma, Sordaria, Colleotrichum, Verticillium, Arthrobotrys, Nectria, Leptosphaeria, Fusarium, Glomerella, Geomyces, Myceliophthora, Pichia, Saccharomyces spp., Zygosaccharomyces, Schizosaccharomyces pombe, Kluyveromyces spp., Yarrowia lipolytica, Monascus spp., Penicillium spp., Hansenula spp., Torulaspora delbrueckii, Hypomyces spp., Dotatomyces spp., Issatchenko orientalis, Phoma spp., Eupenicillium spp., Gymnoascus spp., Pichia labacensis, Pichia anomala, Wickerhamomyces anomalus, Candida cariosilognicola, Paecilomyces virioti, Scopulariopsis brevicaulis, Brettanomyces spp., Dekkera bruxellensis, Dekkera anoma and Trichoderma spp.