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(12) Patent Application Publication (10) Pub. No.: US 2003/0003528A1 Brzostowicz Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2003/0003528A1 Brzostowicz Et Al US 20030003528A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2003/0003528A1 Brzostowicz et al. (43) Pub. Date: Jan. 2, 2003 (54) CAROTENOID PRODUCTION FROM A (21) Appl. No.: 09/941,947 SINGLE CARBON SUBSTRATE (22) Filed: Aug. 29, 2001 (76) Inventors: Patricia C. Brzostowicz, West Chester, PA (US); Qiong Cheng, Wilmington, Related U.S. Application Data DE (US); Deana Dicosimo, Rockland, DE (US); Mattheos Kofas, (60) Provisional application No. 60/229,907, filed on Sep. Wilmington, DE (US); Edward S. 1, 2000. Provisional application No. 60/229,858, filed Miller, Wilmington, DE (US); James on Sep. 1, 2000. M. Odom, Kennett Square, PA (US); Stephen K. Picataggio, Landenberg, PA Publication Classification (US); Pierre E. Rouviere, Wilmington, (51) Int. Cl. .............................. C12P 23/00; C12N 1/21 DE (US) (52) U.S. Cl. ........................................... 435/67; 435/252.3 Correspondence Address: E DUPONT DE NEMOURS AND (57) ABSTRACT COMPANY LEGAL PATENT RECORDS CENTER A method for the production of carotenoid compounds is BARLEY MILL PLAZA 25/1128 disclosed. The method relies on the use of microorganisms 4417 LANCASTER PIKE which metabolize Single carbon Substrates for the production WILMINGTON, DE 19805 (US) of carotenoid compounds in high yields. Patent Application Publication Jan. 2, 2003 Sheet 1 of 7 US 2003/0003528A1 'cool -- o FIGURE 1 Pyruvate v dys Glyceraldehyde-3P H O OP 1-Deoxyxylulose-5P H discr' 9 H3 : Or 2-C-Methylerythritol-4-phosphate H2 OH CH yghP a 9 Hs GP OH, OH oisorsO O of 4-diphosphocytidyl-methylerythriyol ychB, ygbB, lytB,gcpE, ipk OHOH isopentenyl-plus <ld-> --> Dimethylallyl-PP O-PP ispá OPP ---y ispa Geranyl-PP s s 1s '' pyrophosphate p N cri co GGPP GGPP N crifB Phytoere | cert lycopene criY al-Yerra8-Carotene 2 40 N cra o -D e Zeaxanthir CrO to O Astaxanthin Zeaxanthin-3-o-diglucoside Patent Application Publication Jan. 2, 2003 Sheet 2 of 7 US 2003/0003528A1 Zenôl! Patent Application Publication Jan. 2, 2003 Sheet 3 of 7 US 2003/0003528A1 ****dqGGGII go þjó Eko Patent Application Publication Jan. 2, 2003 Sheet 4 of 7 US 2003/0003528A1 : --- T ca Y. 5 .9 c 3. NY al \ - <g e O Wed --- m 9. sy - s M. Ry a Y as use 7s su : E; > S c Patent Application Publication US 2003/0003528A1 G?un61-I Patent Application Publication Jan. 2, 2003 Sheet 6 of 7 US 2003/0003528A1 eunfil9 Patent Application Publication US 2003/0003528A1 C co na Aug 6tu Jo w Aid fruo fin (sXp US 2003/0003528A1 Jan. 2, 2003 CAROTENOID PRODUCTION FROM ASINGLE No. 5,656,472; U.S. Pat. No. 5,5545,816; U.S. Pat. No. CARBON SUBSTRATE 5.530,189, U.S. Pat. No. 5,530, 188; U.S. Pat. No. 5,429, 0001) This application claims the benefit of U.S. Provi 939). Despite the similarity in operon structure, the DNA sional Application No. 60/229,907, filed Sep. 1, 2000 and sequences of E. uredovora and E. herbicola show no homol the benefit of U.S. Provisional Application No. 60/229,858 ogy by DNA-DNA hybridization (U.S. Pat. No. 5,429,939). filed Sep. 1, 2000. 0007 Although more than 600 different carotenoids have been identified in nature, only a few are used industrially for FIELD OF THE INVENTION food colors, animal feeding, pharmaceuticals and cosmetics. Presently, most of the carotenoids used for industrial pur 0002 The invention relates to the field of molecular poses are produced by chemical Synthesis, however, these biology and microbiology. More Specifically, the invention compounds are very difficult to make chemically (Nelis and describes the production of carotenoid compounds from Leenheer, Appl. Bacteriol. 70:181-191 (1991)). Natural microorganisms which metabolize Single carbon Substrates carotenoids can either be obtained by extraction of plant as a Sole carbon Source. material or by microbial Synthesis. At the present time, only a few plants are widely used for commercial carotenoid BACKGROUND OF THE INVENTION production. However, the productivity of carotenoid Synthe sis in these plants is relatively low and the resulting caro 0.003 Carotenoids represent one of the most widely dis tenoids are very expensive. tributed and structurally diverse classes of natural pigments, producing pigment colors of light yellow to orange to deep 0008. A number of carotenoids have been produced from red. Eye-catching examples of carotenogenic tissues include microbial Sources. For example, Lycopene has been pro carrots, tomatoes, red peppers, and the petals of daffodils duced from genetically engineered E. coli and Candia utilis and marigolds. Carotenoids are Synthesized by all photo (Farmer W. R. and J. C. Liao. (2001) Biotechnol. Prog. 17: Synthetic organisms, as well as Some bacteria and fungi. 57-61; Wang C. et al., (2000) Biotechnol Prog. 16:922–926; These pigments have important functions in photosynthesis, Misawa, N. and H. Shimada. (1998). J. Biotechnol. 59:169 nutrition, and protection against photooxidative damage. For 181; Shimada, H. et al. 1998. Appl. Environm. Microbiol. example, animals do not have the ability to Synthesize 64:2676-2680). B-carotene has been produced from E. coli, carotenoids but must instead obtain these nutritionally Candia utilis and Pfafia rhodozyma (Albrecht, M. et al., important compounds through their dietary Sources. Struc (1999). Biotechnol. Lett. 21: 791-795; Miura, Y. et al., 1998. turally, carotenoids are 40-carbon (C) terpenoids derived Appl. Environm. Microbiol. 64:1226-1229; U.S. Pat. No. from the isoprene biosynthetic pathway and its five-carbon 5,691,190). Zeaxanthin has been produced from recombi universal isoprene building block, isopentenyl pyrophoS nant from E. coli and Candia utilis (Albrecht, M. et al., phate (IPP). This biosynthetic pathway can be divided into (1999). Biotechnol. Lett. 21: 791-795; Miura, Y. et al., 1998. two portions: the upper isoprene pathway, which leads to the Appl. Environm. Microbiol. 64:1226-1229). Astaxanthin has formation of IPP, and the lower carotenoid biosynthetic been produced from E. coli and Pfafia rhodozyma (U.S. Pat. pathway, which converts IPP into long Co and Co carote Nos. 5,466.599; 6,015,684; 5,182,208; 5,972,642). nogenic compounds. Both portions of this pathway are 0009 Additionally genes encoding various elements of shown in FIG. 1. the carotenoid biosynthetic pathway have been cloned and 0004 Various other crt genes are known, which enable expressed in various microbes. For example genes encoding the intramolecular conversion of long Co and Co com lycopene cyclase, geranylgeranyl pyrophosphate Synthase, pounds to produce numerous other carotenoid compounds. It and phytoene dehydrogenase isolated from Erwinia herbi is the degree of the carbon backbone's unsaturation, conju cola have been expressed recombinantly in E. coli (U.S. Pat. gation and isomerization which determines the Specific Nos. 5656472; 5545816; 5530189; 5530188). Similarly carotenoids unique absorption characteristics and colors. genes encoding the carotenoid products geranylgeranyl Several reviews discuss the genetics of carotenoid pigment pyrophosphate, phytoene, lycopene, B-caroteine, and ZeaX biosynthesis, such as those of Armstrong (J. Bact. 176: anthin-diglucoside, isolated from Erwinia uredov'Ora have 4795-4802 (1994); Annu. Rev. Microbiol. 51:629-659 been expressed in E. coli, Zymomonas mobilis, and Saccha (1997)). romyces cerevisiae (U.S. Pat. No. 5,429,939). Similarly, the 0005. In reference to the availability of carotenoid genes, Carotenoid biosynthetic genes crtE (1), crtB (3), crt1 (5), public domain databases Such as GenBank contain crtY (7), and crtz isolated from Flavobacterium have been Sequences isolated from numerous organisms. For example, recombinantly expressed (U.S. Pat. No. 6,124,113). there are currently 26 GenBank Accession numbers relating 0010 Although the above methods of propducing caro to various crtE genes isolated from 19 different organisms. tenoids are useful, these methods suffer from low yields and The leSS frequently encountered crtz gene boasts 6 GenBank reliance on expensive feedstocks. A method that produces Accession numbers with each gene isolated from a different higher yields of carotenoids from an inexpensive feedstock organism. A similarly wide Selection of carotenoid genes is is needed. available for each of the genes discussed above. 0011. There are a number of microorganisms that utilize 0006 The genetics of carotenoid pigment biosynthesis Single carbon Substrates as Sole energy Sources. These has been extremely well Studied in the Gram-negative, Substrates include, methane, methanol, formate, methylated pigmented bacteria of the genera Pantoea, formerly known amines and thiols, and various other reduced carbon com as Erwinia. In both E. herbicola EHO-10 (ATCC 39368) and pounds which lack any carbon-carbon bonds and are gen E. uredovora 20D3 (ATCC 19321), the crt genes are clus erally quite inexpensive. These organisms are referred to as tered in two genetic units, crt Z and crt EXYIB (U.S. Pat. methylotrophs and herein as “C1 metabolizers”. These US 2003/0003528A1 Jan. 2, 2003 organisms are characterized by the ability to use carbon abundance of methane that makes the biotransformation of Substrates lacking carbon to carbon bonds as a Sole Source of methane a potentially unique and valuable process. energy and biomass. A Subset of methylotrophs are the methanotrophs which have the unique ability to utilize 0014. Many methanotrophs contain an inherent iso methane as a Sole energy Source. Although a large number prenoid pathway which enables these organisms to Synthe of these organisms are known, few of these microbes have Size other non-endogenous isoprenoid compounds. Since been Successfully harnessed to industrial processes for the methanotrophs can use one carbon Substrate (methane or Synthesis of materials. Although Single carbon Substrates are methanol) as an energy Source, it is possible to produce cost effective energy Sources, difficulty in genetic manipu carotenoids at low cost. lation of these microorganisms as well as a dearth of 0015 Current knowledge in the field concerning methy information about their genetic machinery has limited their lotrophic organisms and carotenoids leads to the following use primarily to the Synthesis of native products.
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