Novel 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.

1 . A method for producing a derivative of citric acid which method comprises modifying a eukaryotic host cell wherein said modifying consists of providing said host cell with 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); with a nucleic acid that expresses one or more genes that encode derivatizing enzymes that convert citric acid into said derivative of citric acid in a suitable 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 one or more genes that encode derivatizing enzymes that convert citric acid into said derivative of citric acid. 
     
     
         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. 
     
     
         5 . The method of  claim 1 , wherein said one or more genes encoding derivatizing enxymes is 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. 
     
     
         6 . The method of  claim 1 , wherein said derivative is itaconic acid and the method comprises overexpressing a gene encoding said citric acid synthase and a gene encoding a protein that is involved in the production or transport of itaconate or any precursor thereof selected from the group consisting of cis-aconitic acid decarboxylase (CAD), ATEG_09969.1, ATEG_09970.1 and ATEG_09972.1.