US 2003O157592A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0157592 A1 Lerchl et al. (43) Pub. Date: Aug. 21, 2003 (54) MOSS GENES FROM PHYSCOMITRELLA (30) Foreign Application Priority Data PATENS ENCODNG PROTEINS INVOLVED IN THE SYNTHESIS OF TOCOPHEROLS Dec. 16, 1999 (US)........................................... 6O171121 AND CAROTENOIDS Publication Classification (76) Inventors: Jens Lerchl, Ladenburg (DE); Andreas Renz, Limburgerhof (DE); Thomas (51) Int. Cl." ............................ C12P 23/00; C12N 9/10; Ehrhardt, Speyer (DE); Andreas AO1H 1/00; C12N 15/82; Reindl, Birkenheide (DE); Petra C12P 21/02; C12N 5/04 Cirpus, Mannheim (DE); Friedrich (52) U.S. Cl. .......................... 435/67; 435/69.1; 435/193; Bischoff, Mannheim (DE); Markus 435/320.1; 435/419; 800/278; Frank, Ludwigshafen (DE); Annette 800/282 Freund, Limburgerhof (DE); Elke Duwenig, Ludwigshafen (DE); (57) ABSTRACT Ralf-Michael Schmidt, Kirrweiler (DE); Ralf Reski, Oberried (DE); Ralf Isolated nucleic acid molecules, designated TCMRP nucleic Badur, Goslar (DE) acid molecules, which encode novel TCMRPs from e.g. Correspondence Address: Phycomitrella patens are described. The invention also KEIL & WEINKAUF provides antisense nucleic acid molecules, recombinant 1350 CONNECTICUT AVENUE, N.W. expression vectors containing TCMRP nucleic acid mol WASHINGTON, DC 20036 (US) ecules, and host cells into which the expression vectors have been introduced. The invention still further provides isolated (21) Appl. No.: 10/149,759 TCMRPs, mutated TCMRPs, fusion proteins, antigenic pep tides and methods for the improvement of production of a (22) PCT Filed: Dec. 14, 2000 desired compound from transformed cells, organisms or plants based on genetic engineering of TCMRP genes in (86) PCT No.: PCT/EP00/12698 these organisms. Patent Application Publication Aug. 21, 2003 Sheet 1 of 4 US 2003/0157592 A1 Figure 1: Xho EcoRI Bamhi O92-260CCs pOE30-092-260cds BamH Kpnl Smal Xmal Sall PSt Xbal Mscl Patent Application Publication Aug. 21, 2003 Sheet 2 of 4 US 2003/0157592 A1 Figure 2: pBinLePTkTp9-092-260Cas EcoR LeB4-Promotor Kpnl wif Bam H. TKTP 092-260cds OCS Bamhi Smal Xmal Sall Patent Application Publication Aug. 21, 2003 Sheet 3 of 4 US 2003/0157592 A1 Figure 3: poE30-087-259Cterm Xhol : EcoR : Bamhl 78pProt187e12Cterm BamH Sphl -----Sac Kpni Smal Xmal Sall Sapi ; Nde Xbal Patent Application Publication Aug. 21, 2003 Sheet 4 of 4 US 2003/0157592 A1 Figure 4: pBinLePTkTp-087-259Cterm EcoR LeB4-Promotor BamHl Smal Xmal Sall Nhel US 2003/O157592 A1 Aug. 21, 2003 MOSS GENES FROM PHYSCOMITRELLA PATENS ENCODING PROTEINS INVOLVED IN THE SYNTHESIS OF TOCOPHEROLS AND (1) CAROTENOIDS HO BACKGROUND OF THE INVENTION 0001 Certain products and by-products of naturally-oc curring metabolic processes in cells have utility in a wide array of industries, including the food, feed, cosmetics, and pharmaceutical industries. These molecules, collectively termed fine chemicals, include organic acids, both protei 0008) 1a, C-tocopherol: R'=R=RCH nogenic and non-proteinogenic amino acids, nucleotides and 0009) 1b, B-tocopherol: R=R-CH, R-H nucleosides, lipids and fatty acids, carotenoids, diols, car 0010) 1c, Y-tocopherol: R'=H, R=R=CH bohydrates, aromatic compounds, Vitamins and cofactors and enzymes. 0011) 1d, 8-tocopherol: R=R-H R =CH 0002 Their production is most conveniently performed (2) through the large-scale culture of bacteria developed to R1 produce and Secrete large quantities of one or more desired molecules. One particularly useful organism for this purpose HO is Corynebacterium glutamicum, a gram positive, nonpatho genic bacterium. R2 O 2 3 0003) Through strain selection, a number of mutant R3 Strains of the respective microorganisms have been devel oped which produce an array of desirable compounds. However, Selection of Strains improved for the production of 0012 2b, C-tocotrienol: R'=R=R CH a particular molecule is a time-consuming and difficult 0013) 2b, B-tocotrienol: R'=R=CH, R=H proceSS. 0014) 2b, Y-tocotrienol: R'=H, R=R =CH 0004 Alternatively the production of fine chemicals can 0015 2b, 8-tocotrienol: R'=R=H, RCH be most conveniently performed via the large Scale produc 0016. In the present invention, tocopherols are to be tion of plants developed to produce one of aforementioned understood as meaning all the abovementioned tocopherols fine chemicals. Of particular interest for this purpose are all and tocotrienols and derivates thereof with vitamin E activ crop plants for food and feed uses. Increased or modulated ity. compositions of fine chemicals like amino acids, Vitamins 0017. These compounds with vitamin E activity (vitamin and nucleotides, in these plants would lead to optimized E compounds) are important natural lipid-Soluble Sub nutritional qualities. stances, which among other activities have especially the function of antioxidants. A lack of Vitamin E in humans and 0005 Through conventional breeding, a number of animals leads to pathophysiological situations. Vitamin E mutant plants have been developed which produce increased compounds therefore have an important economical value as amounts of for example, carotenoids, and amino acids. additives in the food and feed Sectors, in pharmaceutical However, selection of new plant cultivars improved for the formulations and in cosmetic applications. production of a particular molecule is a time-consuming and 0018. An economical method for the production of vita difficult process. min E compounds, and foodstuffs and animal feeds with an elevated Vitamin E content are therefore of great importance. SUMMARY OF THE INVENTION 0019 WO 00/10380 describes the gene sequence encod 0006. This invention provides novel nucleic acid mol ing the 2-methyl-6-phytylplastoquinol-methyltransferase ecules which may be used to modify tocopherols and from the prokaryotic organism Synechocystis Spec. carotenoids in plants, algae and microorganisms. PCC6803. WO97/27285 describes the mapping of the gene 0007. The naturally occurring eight compounds with locus of p-hydroxyphenylpyruvate dioxygenase encoding vitamin E activity are derivatives of 6-chromanol (Ull gene of Arabidopsis thaliana. Speculations are done about mann's Encyclopedia of Industrial Chemistry, Vol. A 27 the effects of overexpression or downregulation of the plant (1996), VCH Verlagsgesellschaft, Chapter 4, 478-488, Vita enzyme on the Vitamin E content or herbicide resistance in min E). The group of the tocopherols (1C-6) has a Saturated transgenic plants. WO 99/04622 and D. DellaPenna et al., Side chain, while the group of the tocotrienols (2C-6) has an Science 1998, 282, 2098-2100 describe gene sequences unsaturated Side chain: encoding a Y-tocopherol methyltransferase from Syn US 2003/O157592 A1 Aug. 21, 2003 echocystis PCC6803 and Arabidopsis thaliana and their ogy on the DNA sequence and polypeptide level allowing incorporation into plants. However, the transgenic plants the use of heterologous screening of DNA molecules with show only a shift in the Spectum of tocopherols, i.e. a shift probes evolving from other mosses or organisms, thus from gamma-tocopherol to alpha-tocopherol because of the enabling the derivation of a consensus Sequence Suitable for higher expression of Y-tocopherol methyltransferase. No heterologous Screening or functional annotation and predic data are shown concerning a higher yield of tocopherols, i. tion of gene functions in third species. The ability to identify e. a quantitative improvement in tocopherol content. Such functions can therefor have significant relevance, e.g., prediction of Substrate Specificity of enzymes. Further, these 0020. To date no economical methods are available for an effective production of tocopherols and/or carotinoids in nucleic acid molecules may serve as reference points for the transgenic organisms, i. e. for effectively increasing the mapping of moSS genomes, or of genomes of related organ metabolite flow in the direction of increased tocopherol SS. and/or carotinoid content in transgenic organisms, for 0026. This invention provides novel nucleic acid mol example in transgenic plants, by overexpressing one or ecules which encode proteins, referred to herein as Toco Several biosynthesis genes, alone or in any combination, pherol, and Carotenoid Metabolism Related Proteins related to the tocopherol and/or carotinoid metabolism. (TCMRP). These TCMRPs are capable of, for example, performing an enzymatic Step involved in the metabolism of 0021 Methods which are particularly economical are certain fine chemicals, including tocopherols and/or caro biotechnological methods which exploit proteins and bio tenoids. Synthesis genes from tocopherol or carotinoid biosynthesis from organisms producing these compounds. 0027) Given the availability of cloning vectors for use in plants and plant transformation, Such as those published in 0022 Microorganisms like Corynebacterium and fungi and cited therein: Plant Molecular Biology and Biotechnol and algae like Phaeodactylum are commonly used in indus ogy (CRC Press, Boca Raton, Fla.), chapter 6/7, S.71-119 try for the large-scale production of a variety of fine chemi (1993); F. F. White, Vectors for Gene Transfer in Higher cals. Plants, in: Transgenic Plants, Vol. 1, Engineering and Uti 0023 Given the availability of cloning vectors for use in lization, eds.: Kung und R. Wu, Academic Press, 1993, Corynebacterium glutamicum, Such as those disclosed in 15-38; B. Jenes et al., Techniques for Gene Transfer, in: Sinskey et al., U.S. Pat. No.