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(12) Patent Application Publication (10) Pub. No.: US 2010/0311132 A1 Van Derwerf Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2010/0311132 A1 Van Derwerf Et Al US 20100311 132A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0311132 A1 Van DerWerf et al. (43) Pub. Date: Dec. 9, 2010 (54) PRODUCTION OF ITACONIC ACID (30) Foreign Application Priority Data (76) Inventors: Maria Johanna VanDerWerf. Jul. 20, 2007 (EP) .................................. O7112895.3 Wageningen (NL); Martinus O O Petrus Maria Caspers, Rijswijk Publication Classification (NL); Nicole Van Luijk, Utrecht (51) Int. Cl. (NL); Peter Jan Punt, Houten (NL) CI2P 7/46 (2006.01) CI2N 9/88 (2006.01) Correspondence Address: CI2N I/00 (2006.01) MORRISON & FOERSTER LLP CI2N 15/63 (2006.01) 12531 HIGH BLUFF DRIVE, SUITE 100 C7H 2L/04 (2006.01) SAN DIEGO, CA 92130-2040 (US) (52) U.S. Cl. ...................... 435/145; 435/232; 435/254.3: 435/320.1; 536/23.2 (21) Appl. No.: 12/669,955 (57) ABSTRACT (22) PCT Filed: Jul. 21, 2008 The invention relates to the production of itaconic acid in micro-organisms by introducting into a Suitable host cell a (86). PCT No.: PCT/NL2008/050499 gene coding for the enzyme cis-aconitic acid decarboxylase, preferably derived from A. terreus. Further part of the inven S371 (c)(1), tion are host cells, preferably from A. niger, provided with the (2), (4) Date: Aug. 23, 2010 above mentioned genes. Patent Application Publication Dec. 9, 2010 Sheet 1 of 9 US 2010/0311132 A1 Mitochondrion Glucose Pyruvate Pyruvate-a-Acetyl-CoA L-Malate C Citrate Citrate 92 2 92 cis-Aconitates -- - - - - - O w was no is us CiS-Aconitate *g 2 3 Citraconate(?) SOcitrate 5 TACONCACD Fig. 1 Patent Application Publication Dec. 9, 2010 Sheet 3 of 9 US 2010/0311132 A1 Fig. 3 10 20 30 40 50 50 Aterreus CAD !--------------------------------------------- XPOOL209273 AAD34.563 BAE66063 XP001393.934 XPOO38945 XP391.395 XPOOL33345 XPOO 27495 NP961.87 YP880.96 YP890so 2P064868. YPOO15249 Aterreus CAD XPOO292.73 AAD34.563 BAE66063 XPOO .393.934 XPOO138945 XP39.1396 XP0 0383 451 XPOO 27495 NP96.87 YP880.968 YP89.060 2PO164868 YPO 06249 Aterreus CAD XPO 0209273 AAD34.563 BAE65063 XP)039.3934 4 - - a MVTITAKSEAASATSPISSNTITTTINGVGDPKNKEKDLQLQEKE XPOO38945 XP39396 e- or - - - - - - - an as or r us war - w app a so a vs - MSHGCGNYRSF- - - - -WNSFRT - - - - - - - XPOO383451 - a . a vs as a - as a w- - - - ar as on 1 - as a ar - an ar a r - a r - r on -MOSP- - - - - - - - - XP0023,7495 LRENGGFKYGLNRRRNVYMPPRGFLGHGPHRYFYQIVALSERIEQSQLSAPATKEEVWRC NPS 61.87 MTA YP880968 MTA YP89.060 M APO 64,858 YPO 06249 Patent Application Publication Dec. 9, 2010 Sheet 7 of 9 US 2010/0311132 A1 Patent Application Publication Dec. 9, 2010 Sheet 8 of 9 US 2010/0311132 A1 70 kDa CAD protein id Fig. 5 Patent Application Publication Dec. 9, 2010 Sheet 9 of 9 US 2010/0311132 A1 | 2?SeaQT3IAS,SANT! | }+,++),º US 2010/0311 132 A1 Dec. 9, 2010 PRODUCTION OF TACONCACD tous fungi, yeasts and bacteria, more preferably from Escherichia coli, Aspergillus niger and citrate- or lovastatin producing hosts. 0001. The invention relates to the field of microbial pro 0007 Further, the invention pertains to the use of the duction, more specifically production of itaconic acid (ita enzyme cis-aconitic acid decarboxylase for the production of conate), more specifically production of itaconate in micro itaconic acid in a Suitable host cell. organisms. 0008 Also comprised in the invention is a method as 0002 Production and metabolism of itaconic acid in described above, wherein the production and/or transport of microbial cells has been studied extensively for several itaconic acid is further increased by increasing the intracel decades (Calam, C. T. et al., 1939, Thom. J. Biochem. lularitaconic acid, using at least one but preferably a combi 33:1488-1495; Bentley, R. and Thiessen, C. P., 1956, J. Biol. nation of the following methods: 1. overexpression of a gene Chem. 226:673-720; Cooper, R. A. and Kornberg, H. L., coding for a protein capable of transporting di?tricarboxylate, 1964, Biochem. J., 91:82–91; Bonnarme, P. et al., 1995, J. preferably cis-aconitate, citrate or isocitrate, from the mito Bacteriol. 1 17:3573-3578; Dwiarti, L. et al., 2002, J. Biosci. chondrion to the cytosol, more preferably the diacrboxylate Bioeng. 1:29-33), but the metabolic pathway for itaconic acid transporter encoded by the nucleic acid sequence of ATEG has not been unequivocally established (Wilke, Th. and Vor 09970.1 (see co-pending application EP 08151584); 2. a lop, K.-D., 2001, Appl. Microbiol. Biotechnol. 56:289-295; method as described above, wherein the activity of a regulator Bonnarme, P. et al., 1995, J. Bacteriol. 177:3573-3578). Two protein that comprises a Zinc finger and a fungal specific complicating factors in this respect are that the biosynthesis transcription factor domain is modulated. Preferably said route for itaconic acid is thought to occur both in the cytosol regulator protein is the protein encoded by the nucleic acid and the mitochondria (Jaklitsch, W. M. et al., 1991, J. Gen. sequence of ATEG 09969.1, located in the same gene cluster Microbiol. Appl. 6:51-61) and that aconitase, the enzyme that as the transporter of the invention; 3 overexpression of a interconverts citric acid into cis-aconitate, and vice versa, and nucleic acid sequence encoding an itaconate transporting other enzymes in the metabolic pathway have been found to Major Facilitator Superfamily Transporter (MFST) gene be present in many isoforms in microbial cells. sequence (hereinafter “the itaconate transporter'). Preferably 0003 Production of itaconic acid is now commercially said nucleic acid comprises the ATEG 09972.1 sequence of achieved in Aspergillus terreus, which has physiological Aspergillus terreus or a nucleic acid that shares more than similarity to A. niger and A. oryzae. However, these latter two about 70%, preferably more than about 80%, preferably more accumulate citric acid, due to the absence of cis-aconic acid than about 90% sequence identity with the sequence of decarboxylase (CAD)activity. Substrates used by these fungi ATEG 09972.1 (as described in co-pending application EP include mono- and disaccharides, such as glucose, Sucrose 08152332). and fructose and starches, as they exist in forms which are degradable by the micro-organism, and molasses. Recently, it LEGENDS TO THE FIGURES has been discovered that also glycerol is a useful Substrate in itaconic acid production by A. terreus (U.S. Pat. No. 5,637, 0009 FIG. 1: Postulated biosynthesis route(s) for itaconic 485). acid in A. terreus. 1, Citrate synthase; 2. Aconitase; 3, cis 0004. The general scheme currently envisioned for ita aconitic acid decarboxylase (itaconate-forming), 4, cis-aco conic acid biosynthesis is given in FIG. 1, wherein clearly the nitic acid decarboxylase (citraconate-forming); 5, citraconate existence of the biosynthetic route both in the cytosol and the isomerase; 6, mitochondrial dicarboxylate-tricarboxylate mitochondria is depicted and the putative connection between antiporter 7, mitochondrial tricarboxylate transporter; 8. these two compartments. At several point of this scheme dicarboxylate transporter; 9, 2-methylcitrate dehydratase. possibilities exist to try to improve the existing commercial 0010 FIG. 2: Genomic nucleic acid sequence (A) and production of itaconic acid in micro-organisms. encoded amino acid sequence (B) of the A. terreus CAD gene. The depicted genomic sequence also shows 500 nucleotides 5' to the ATG start of the coding sequence. Further, the SUMMARY OF THE INVENTION genomic sequence has a Small intron. 0005. The present inventors now have elucidated the gene 0011 FIG. 3.: Sequence comparison of the CAD protein coding for the cis-aconitate decarboxylase enzyme and have from A. terreus and several homologous proteins (see detailed found that overexpression of the cis-aconitate decarboxylase description for the species of which the NCBI accession gene from Aspergillus is able to boost the production of number is given in the figure). itaconic acid. Accordingly, the invention comprises a method 0012 FIG. 4.: Separation of proteins from a G25 chroma for the production of itaconic acid comprising overexpression tography desalted 35-60% ammonium sulfate pellet of cell of a gene coding for the enzyme cis-aconitic acid decarboxy extracts of A. terreus on an anion exchange Source Q15 lase (CAD) in a suitable host cell. Preferably said gene is column. Blue=OD280, black=% solution B (1 MNaCl) salt B derived from Aspergillus terreus. The suitable host cell is in gradient, red-conductivity, green CAD activity. preferably an Aspergillus niger or A. terreus host cell. 0013 FIG. 5: SDS-PAGE of cell extract of recombinant E. According to a further preferred embodiment, the CAD gene coli containing the CAD gene on the pET52 expression vec is under control of its own or other promoters. tor. (+) and (-), with and without IPTG induction: M, protein 0006 Another embodiment of the present invention is markers. formed by a host cell wherein a gene coding for the enzyme 0014 FIG. 6: Overview of Aspergillus terreus genome cis-aconitic acid decarboxylase is introduced, preferably segment with the cluster of genes involved in production of wherein said enzyme is derived from Aspergillus terreus. itaconic acid and lovastatin ranging from ATEG 09961.1 - Said host cell preferably is a host cell selected from filamen ATEG 09975.1. The cluster contains the cis-aconitate decar US 2010/0311 132 A1 Dec. 9, 2010 boxylase (ATEG 09971.1) and the mitochondrial tricar will recognize that a lengthened primer sequence can be boxylate transporter (ATEG 9970.1). employed to increase specificity of binding (i.e. annealing) to a target sequence. Stringent conditions in this respect means DETAILED DESCRIPTION OF THE INVENTION a reactionata temperature of between 60° C. and 65°C. in 0.3 00.15 "Fungi are herein defined as eukaryotic microor strength citrate buffered saline containing 0.1% SDS fol ganisms and include all species of the Subdivision Eumyco lowed by rinsing at the same temperature with 0.3 strength tina (Alexopoulos, C.
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