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Ep 2434019 A1 (19) & (11) EP 2 434 019 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 28.03.2012 Bulletin 2012/13 C12N 15/82 (2006.01) C07K 14/395 (2006.01) C12N 5/10 (2006.01) G01N 33/50 (2006.01) (2006.01) (2006.01) (21) Application number: 11160902.0 C07K 16/14 A01H 5/00 C07K 14/39 (2006.01) (22) Date of filing: 21.07.2004 (84) Designated Contracting States: • Kamlage, Beate AT BE BG CH CY CZ DE DK EE ES FI FR GB GR 12161, Berlin (DE) HU IE IT LI LU MC NL PL PT RO SE SI SK TR • Taman-Chardonnens, Agnes A. 1611, DS Bovenkarspel (NL) (30) Priority: 01.08.2003 EP 03016672 • Shirley, Amber 15.04.2004 PCT/US2004/011887 Durham, NC 27703 (US) • Wang, Xi-Qing (62) Document number(s) of the earlier application(s) in Chapel Hill, NC 27516 (US) accordance with Art. 76 EPC: • Sarria-Millan, Rodrigo 04741185.5 / 1 654 368 West Lafayette, IN 47906 (US) • McKersie, Bryan D (27) Previously filed application: Cary, NC 27519 (US) 21.07.2004 PCT/EP2004/008136 • Chen, Ruoying Duluth, GA 30096 (US) (71) Applicant: BASF Plant Science GmbH 67056 Ludwigshafen (DE) (74) Representative: Heistracher, Elisabeth BASF SE (72) Inventors: Global Intellectual Property • Plesch, Gunnar GVX - C 6 14482, Potsdam (DE) Carl-Bosch-Strasse 38 • Puzio, Piotr 67056 Ludwigshafen (DE) 9030, Mariakerke (Gent) (BE) • Blau, Astrid Remarks: 14532, Stahnsdorf (DE) This application was filed on 01-04-2011 as a • Looser, Ralf divisional application to the application mentioned 13158, Berlin (DE) under INID code 62. • Wendel, Birgit 13437, Berlin (DE) (54) Process for the production of fine chemicals (57) The present invention relates to a process for tors, antisense molecules, antibodies, host cells, plant the production of the fine chemical in a microorgansm, a tissue, propagtion material, harvested material, plants, plant cell, a plant, a plant tissue or in one or more parts microorganisms as well as agricultural compositions and thereof. The invention furthermore relates to nucleic acid to their use. molecules, polypeptides, nucleic acid constructs, vec- EP 2 434 019 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 434 019 A1 Description [0001] The present invention relates to a process for the production of the fine chemical in a microorgansm, a plant cell, a plant, a plant tissue or in one or more parts thereof. The invention furthermore relates to nucleic acid molecules, 5 polypeptides, nucleic acid constructs, vectors, antisense molecules, antibodies, host cells, plant tissue, propagtion ma- terial, harvested material, plants, microorganisms as well as agricultural compositions and to their use. [0002] Certain products and by-products of naturally- occurring metabolic processes in cells have utility in a wide array of industries, including, but not limited to, the food, feed, cosmetics, and pharmaceutical industries and agriculture. These molecules, collectively termed ’fine chemicals’ include molecules such as vitamins for example vitamin A, D, E, K, B1, 10 B2, B6. B12, C, pantothenic acid, biotin or folic acid; substances with vitamin-like character for example vitamin F, lipoic acid, ubiquinones, choline, myoinsositiol, vitamin U (S-methylmethionine), flavours for example vanillin, coumarin, iso- eugenol, eugenol, (R)-carvone, (S)-carvone, menthol, jasmone or farnesol; nutraceuticals for example phytosterols, flavonoids, anthocyanidins, isoflavons or isoprenoids; detergents; fatty acids such as saturated fatty acids, mono un- saturated fatty acids (singular MUFA, plural MUFAS), poly unsaturated fatty acids (singular PUFA, plural PUFAS), waxes 15 or lipids containing said fatty acids; carbohydrates for example cellulose, starch, dextrin, pectin, xanthangum, carra- geenan or alginate; sugars for example monosaccharides such as glucose, fructose, manose, sorbose, ribose, ribulose, xylose, xylulose or galactose, disaccharides such as lactose, sucrose, saccharose, maltose, isomaltose or cellobiose, trisaccharides such as raffinose or maltotriose; carboxylic acids for example citric acid, α-ketoglutaric acid, ferulic acid, sinapic acid or lactic acid; carotinoids for example α-carotene, β-carotene, zeaxanthine, lutein, astaxanthine, lycopene, 20 phyotoene or phytofluene, amino acids for example lysine, threonine, methionine, tryptophane, phenylalanine or tyrosine, cofactors for example heme or quinines, enzymes for example lipases, esterases, proteases, amylases, glucosidases etc. and other compounds [as described e.g. in Kuninaka, A. (1996) Nucleotides and related compounds, p. 561-612, in Biotechnology vol. 6, Rehm et al., eds. VCH: Weinheim, in Industial Microbiology and Biotechnology, Demain et al., second edition, ASM Press Washington, D.C. 1999, in Ullmann’s Encyclopedia of Industrial Chemistry, vol. A27, Vitamins, 25 p. 443-613 (1996) VCH: Weinheim and Ong, A.S., Niki, E. & Packer, L. (1995) Nutrition, Lipids, Health, and Disease Proceedings of the UNESCO/Confederation of Scientific and Technological Associations in Malaysia, and the Society for Free Radical Research, Asia, held Sept. 1-3, 1994 at Penang, Malaysia, AOCS Press, (1995)), enzymes, and all other chemicals described in Gutcho (1983) Chemicals by Fermentation, Noyes Data Corporation, ISBN: 0818805086 and references contained therein]. Carotinoids are added for example to soft drinks, margarines or to animal feed for 30 example to colour egg yolk or the flesh of fish. In the food industry polycarbohydrates are widely used as thickener. Polyunsaturated fatty acids are added for example to infant formulas to create a higher nutrition value of such formulas. PUFAs have for example a positive influence on the cholesterol level of the blood in humans and therefore are useful in the protection of heart diseases. Fine chemicals for example PUFAS can be isolated from animal sources such as for example fish or produced with microorganisms through the large-scale culture of microorganisms developed to 35 produce and accumulate or secrete large quantities of one or more desired molecules. [0003] In large scale fine chemicals are produced with microorganism in the fermentation industry, which is responsible for the manufacturing of at least five major ingredient categories: antibiotics, organic acids, amino acids, enzymes, vitamins and other related products. There are production facilities in all important areas of the world especially in Europe, the US and Asia. Companies continuously try to optimize the production processes, the organisms and thereby increasing 40 the efficiency but, as in the case of amino acids and organic acids, with already high conversion rates based on feeded carbon source, the limitations of such work become evident. All fermentation processes depend on the efficient utilization of carbohydrates, supplied mainly in the form of oils, glucose or molasses. It is therefore the availability and pricing of these raw materials that influence the competitiveness of fermentation products versus production for example in plants. Amino acids, organic acids and vitamins are offered at very low prices. For such products the question is whether it is 45 still economical to continue fermentation production in future. And that is frequently a question of comparing the availability and pricing of carbohydrates with the future markets. [0004] Particularly useful organisms for the production of fine chemicals are microorganisms such as the algae, fungi, bacteria or plants. Through strain selection, a number of mutant strains of microorganisms have been developed which produce an array of desirable compounds including vitamins, amino acids, PUFAs etc. However, selection of strains 50 improved for the production of a particular molecule is a time-consuming and difficult process. [0005] Alternatively the production of fine chemicals can be most conveniently performed via the large scale production of plants developed to produce for example carotinoids, carbohydrates or PUFAS. For example for the production of carotinoids plants such as marigold are used. Particularly well- suited plants for this purpose are sugar producing plants such as sugar beet or sugar cane or oilseed plants containing high amounts of lipid compounds such as rapeseed, 55 canola, linseed, soybean, sunflower, borage and evening primrose. But also other crop plants containing sugars, oils or lipids and fatty acids are well suited as mentioned in the detailed description of this invention. Through conventional breeding, a number of mutant plants have been developed which produce an array of desirable lipids and fatty acids, cofactors and enzymes. However, selection of new plant cultivars improved for the production of a particular molecule 2 EP 2 434 019 A1 is a time-consuming and difficult process or even impossible if the compound does not naturally occur in the respective plant as for example in the case of C20 and higher C-carbon chain polyunsaturated fatty acids. [0006] Carbohydrates are an important dietary nutrient, which is mostly used to supply energy to the body, as well as, a carbon source for synthesis of other compounds such as fats or proteins. Furthermore mono- and disaccharides 5 are widely used in the food and feed industry as sweetener. Saccharides have varying degrees of sweetness on a relative scale. Fructose is the sweetest. For example in the United States 22 million tons of sugar and other sweeteners were consumed in 1999. Said natural sweetener consumption includes the consumption of sugar, corn sweeteners such as high fructose corn syrup as main product and others such as honey or maple syrup. All natural sugar based sweeteners have a market shar of around 36 to 40 percent. 10 [0007] Whether unsaturated or saturated fatty acids are preferred in the food and feed industry depends on the intended purpose; thus, for example, lipids with unsaturated fatty acids, specifically polyunsaturated fatty acids, are preferred in human nutrition since they have a positive effect on the cholesterol level in the blood and thus on the possibility of heart disease. They are used in a variety of dietetic foodstuffs or medicaments. In addition PUFAs are commonly used in food, feed and in the cosmetic industry. Poly unsaturated ω-3- and/or ω-6-fatty acids are an important part of animal feed and 15 human food.
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