(19) TZZ _T (11) EP 2 894 224 A1 (12) EUROPEAN PATENT APPLICATION published in accordance with Art. 153(4) EPC (43) Date of publication: (51) Int Cl.: 15.07.2015 Bulletin 2015/29 C12P 7/62 (2006.01) C12N 15/09 (2006.01) (21) Application number: 13835104.4 (86) International application number: PCT/JP2013/005359 (22) Date of filing: 10.09.2013 (87) International publication number: WO 2014/038216 (13.03.2014 Gazette 2014/11) (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • SATO, Eiji GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO Yokohama-shi PL PT RO RS SE SI SK SM TR Kanagawa 227-8502 (JP) Designated Extension States: • YAMAZAKI, Michiko BA ME Yokohama-shi Kanagawa 227-8502 (JP) (30) Priority: 10.09.2012 JP 2012198840 • NAKAJIMA, Eiji 10.09.2012 JP 2012198841 Yokohama-shi 31.01.2013 JP 2013016947 Kanagawa 227-8502 (JP) 30.07.2013 JP 2013157306 • YU, Fujio 01.08.2013 JP 2013160301 Yokohama-shi 01.08.2013 JP 2013160300 Kanagawa 227-8502 (JP) 20.08.2013 JP 2013170404 • FUJITA, Toshio Yokohama-shi (83) Declaration under Rule 32(1) EPC (expert Kanagawa 227-8502 (JP) solution) • MIZUNASHI, Wataru Yokohama-shi (71) Applicant: Mitsubishi Rayon Co., Ltd. Kanagawa 227-8502 (JP) Tokyo 100-8253 (JP) (74) Representative: Hoffmann Eitle Patent- und Rechtsanwälte PartmbB Arabellastraße 30 81925 München (DE) (54) METHOD FOR PRODUCING METHACRYLIC ACID AND/OR ESTER THEREOF (57) To provide a method for directly and efficiently producing methacrylic acid in a single step from renew- able raw materials and/or biomass arising from the utili- zation of the renewable raw materials. Further provided is a method for producing methacrylic acid using mi- crobes having the ability to produce methacrylic acid, from renewable raw materials and/or biomass arising from the utilization of the renewable raw materials, as a carbonsource and/or energysource. The methodfor pro- ducing methacrylic acid enables methacrylic acid to be safely and easily produced from biomass, without using petroleum-derived raw materials, by utilizing microbes having the ability to produce methacrylic acid. EP 2 894 224 A1 Printed by Jouve, 75001 PARIS (FR) EP 2 894 224 A1 Description TECHNICAL FIELD 5 [0001] The present invention relates to a method for producing methacrylic acid and/or an ester thereof. More specif- ically, it relates to a method for producing methacrylic acid using microbes having the ability to produce methacrylic acid, the microbes, methacrylic acid obtained by the production method, and the like. BACKGROUND ART 10 [0002] Methacrylic acid is useful as a raw material for paint or resin modifying agent, and its esters are an industrially significantly important compound as a raw material for acrylic resin. [0003] As a method for chemical production of methacrylic acid derivatives, the ACH method intermediated by acetone cyanhydrin using cyanide and acetone as a raw material, the C4 oxidation using isobutylene or tert-butanol as a raw 15 material, and the like are practically used. Those chemical production methods rely on fossil fuels and also require a large amount of energy. [0004] Recently, from the viewpoint of preventing global warming and environmental protection, use of renewable biogenous resources (renewable raw materials) as a carbon source to be a substitute of fossil fuels of the related art receives attention. For example, a method for producing 2-hydroxyisobutyric acid and 3-hydroxyisobutyric acid, which 20 are a precursor of methacrylic acid, from a natural product such as sugar using microbes existing in nature has been suggested (see, Patent Documents 1 and 2 and Non-Patent Document 1). However, those methods still rely on a chemical method in terms of the process for producing methacrylic acid by dehydration of a precursor. [0005] Further, although a method for producing methacrylic acid from glucose using a recombinant microbe introduced with plural enzyme genes, which is not exist in nature, has been suggested, it is a mere suggestion of simple combination 25 between known enzyme reactions and imaginary enzyme reactions deduced therefrom, and has not been practically proved (see, Patent Documents 3 to 5). [0006] Meanwhile, as a naturally occurring microbe, a photosynthetic microbe having the ability to produce methacrylic acid is known (Non-Patent Document 2). However, the photosynthetic microbe only converts 3-mercaptoisobutyric acid, which is a special compound not existing in nature, to methacrylic acid. 30 [0007] Further, in Patent Document 6, a method for producing an acrylic acid ester according to an action of a hydrolase in the presence of Acryloyl-CoA and an alcohol is disclosed. It is suggested in the same literature that a methacrylic acid ester can be also similarly produced. However, considering diversity and substrate specificity of an enzyme, it only demonstrates that an acrylic acid ester can be produced with few limited hydrolases, and it remains uncertain whether or not a methacrylic acid ester having a different structure can be also similarly produced by the hydrolase. Further, it 35 remains completely uncertain whether or not production can be made with a different kind of enzyme having a different reaction mechanism. Further, when an ester is synthesized with the hydrolase described in Patent Document 6, it is expected that an ester produced is basically dissociated due to hydrolysis activity, and thus it is difficult to be considered as an effective production method. [0008] Meanwhile, alcohol acyl transferase (hereinbelow, "ATT") is known as an enzyme for synthesis of fruity flavor. 40 In Patent Document 7, a method for producing various esters as fruit flavor by identifying the gene of the same enzyme, which is contained in specific fruits, is suggested. However, the possibility of synthesizing a methacrylic acid ester with the enzyme is not described and remains completely uncertain. [0009] As described above, although several suggestions or discussions have been made, there is no example showing actual production of methacrylic acid derivatives using microbes, and thus an effective production method needs to be 45 established. CITATION LIST PATENT DOCUMENT 50 [0010] Patent Document 1: WO 2007/110394 A Patent Document 2: WO 2008/145737 A 55 Patent Document 3: WO 2009/135074 A Patent Document 4: WO 2011/031897 A Patent Document 5: WO 2012/135789 A Patent Document 6: WO 2007/039415 A 2 EP 2 894 224 A1 Patent Document 7: WO 00/32789 A Patent Document 8: JP 2011-519561 A Patent Document 9: JP 2011-200133 A Patent Document 10: JP 5-64589 A 5 Patent Document 11: JP 10-337185 A NON-PATENT DOCUMENT [0011] 10 Non-Patent Document 1: Green Chemistry, 2012, 14, 1942-1948 Non-Patent Document 2: Bioorganic & Medicinal Chemistry, 1994, 7, 589-593 Non-Patent Document 3: Methods in Enzymology, 2000, 324, 241-258 Non-Patent Document 4: Fungal Genet. Biol., 2008, 45, 180-189 15 Non-Patent Document 5: Microbiology, 1999,145, 2323-2334 Non-Patent Document 6: Bacteriol. Review, 1976, 40, 42-54 (1976) Non-Patent Document 7: J. Mol. Evol., 2009, 69, 176-193 Non-Patent Document 8: Methods in Enzymology, 2000, 324, 129-138 20 DISCLOSURE OF THE INVENTION PROBLEM TO BE SOLVED BY THE INVENTION [0012] According to a chemical production method of the related art, for example, the ACH method, an oxidation 25 treatment of acetone cyanhydrin with an acid is required so that a waste acid is generated in a large amount. Further, as separation or purification is required for each step, there is a problem of having high energy consumption. For such reasons, a method for directly and efficiently producing methacrylic acid in a single step using microbes capable of producing methacrylic acid from renewable raw materials and/or biomass arising from the utilization of the renewable raw materials, as a carbon source and/or energy source, is needed. 30 MEANS FOR SOLVING PROBLEM [0013] To solve the problems described above, inventors of the invention conducted extensive searches for microbes having the ability to produce methacrylic acid derivatives, and as a result, found that methacrylic acid can be produced 35 with many microbes. [0014] Further, as described below, the inventors found that the microbes having the ability to produce methacrylic acid derivatives are a group of microbes having a certain specific function and a constant common property, and they also succeeded in finding a method for searching them. [0015] First, the inventors of the invention tried to produce methacrylic acid derivatives, which are an intermediate of 40 the valine metabolism pathway, from 2-oxoisovaleric acid based on a microbial reaction by producing recombinant E. coli which has been introduced with a gene of the valine metabolism pathway (branched chain keto acid dehydrogenase (hereinbelow, "BCKAD") and isobutyryl-CoA dehydrogenase (hereinbelow, "ACD")). However, production of the meth- acrylic acid derivatives as a target product was not observed while production of isobutyric acid was observed. Further, when a cell extract is prepared from the recombinant and the in vitro activity of BCKAD and ACD is measured after 45 adding a cofactor or the like, coactivity was observed. Based on such results, it was determined that the BCKAD reaction, but not the ACD reaction, has occurred under in vivo conditions. [0016] In mammals, it is known that the dehydrogenase reaction known as metabolism of branched amino acid or β- oxidation of fatty acid occurs in a mitochondria and liberated electrons are transferred to a respiratory chain via an electron transferring flavoprotein. In other words, it was assumed that the main reaction does not occur only by adding 50 foreign ACD to a microbe which does not have the same function as them.