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WO2003101184A2 - Synthese du carotenoide - Google Patents

Synthese du carotenoide Download PDF

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Publication number
WO2003101184A2
WO2003101184A2 PCT/US2003/017775 US0317775W WO03101184A2 WO 2003101184 A2 WO2003101184 A2 WO 2003101184A2 US 0317775 W US0317775 W US 0317775W WO 03101184 A2 WO03101184 A2 WO 03101184A2
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seq
substitution
carotenoid
plant
sequence
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WO2003101184A3 (fr
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Frances H. Arnold
Daisuke Umeno
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California Institute of Technology
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California Institute of Technology
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis

Definitions

  • Carotenoids are pigments, which occur universally in plants and some microorganisms.
  • the two main classes are carotenes, which are hydrocarbons, and xanthophylls, which contain hydroxyl groups or epoxide rings and are therefore slightly polar.
  • Carotenoids often occur in association with specific proteins. Their functions are varied, including light harvesting in photosynthesis, protection against damaging effects of light and oxygen, and pigments of fruits and flowers.
  • Naturally occurring carotenoids comprise diverse chemical structures and biological functions. But they still represent a tiny fraction of the structures that can be chemically or biochemically produced. Nature has explored this space by evolving new, mostly secondary metabolic pathways. New enzymes, and enzyme pathways for the synthesis of novel carotenoid compounds are needed.
  • the invention provides a substantially purified polypeptide selected from the group consisting of (a) a polypeptide characterized as comprising a sequence that is 80% or more identical to SEQ ID NO:2 and which catalyzes the generation of a carotenoid; (b) a polypeptide comprising a sequence as set forth in SEQ ID NO:2 having one to five amino acid substitutions and wherein the polypeptide catalyzes the condensation of geranlygeranlydiphosphate to form phytoene; (c) a polypeptide comprising a sequence as set forth in SEQ ID NO:2 having one to ten amino acid substititions, wherein at least one substitution is selected from the group consisting of an HI 2X substitution, an F26X substitution, W38X substitution; a E180G substitution, wherein X is any amino acid, and wherein the polypeptide catalyzes the condensation of geranlygeranlydiphosphate to form phytoene; and (d) a polypeptide selected from
  • the invention further provides an isolated polynucleotide encoding the polypeptide above.
  • the isolated polynucleotide is selected from the group consisting: (a) a polynucleotide having a sequence that is at least 80% identical to SEQ ID NO: 1 and that encodes a polypeptide that catalyzes the generation of a carotenoid; (b) a polynucleotide comprising a sequence as set forth in SEQ ID NO:l, wherein nucleotides 34-36 are selected from the group consisting of (i) caa, and (ii) cag; (c) a polynucleotide comprising a sequence as set forth in SEQ ID NO:l, wherein nucleotides 76- 78 are selected from the group consisting of (i) ggn; (ii) gen; (iii) tta, ttg, or ctn; and (iv) a
  • the invention also provides a vector comprising a polynucleotide of the invention.
  • the vector can be an expression vector.
  • the invention provides a recombinant host cell transfected or transformed with a vector a vector of the invention and/or a polynucleotide of the invention.
  • the recombinant host cell further comprises a polynucleotide encoding a carotenoid-modifying enzyme selected from the group consisting of a synthase (e.g., GGDP synthase from E. uredoora having a sequence as set forth in SEQ ID NO:6), a desaturase (e.g., desaturase is from E.
  • a synthase e.g., GGDP synthase from E. uredoora having a sequence as set forth in SEQ ID NO:6
  • a desaturase e.g., desaturase is from E.
  • the invention also provides a method of making a carotenoid.
  • the method includes genetically engineering a host cell to contain at least one mutated enzyme from a carotenoid biosynthetic pathway; providing the genetically engineered host cell with an appropriate substrate or substrates; and identifying a carotenoid product.
  • the carotenoid product made by the method of the invention is selected from the group consisting of a carotene, C 30 carotene, a C 30 xantophyll, a C 35 carotene, and an apocarotenoid.
  • the at least one mutated enzyme is a carotene synthase or a carotene desaturase.
  • the mutated enzyme is a polypeptide of the invention.
  • the carotenoid can be a C 0 carotene, a C 30 xantophyll (e.g., C 30 xantophyll having 5-10 carbon double bonds or salt thereof), a C 35 carotene, or an apocarotenoid.
  • the carotenoid is a C 30 carotenoid comprising 5, 7, 11, 8+ ⁇ , or 6+ ⁇ carbon double bonds.
  • the invention further provides a plant entity, or progeny thereof consisting essentially of a plant cell, seed, or plant produced from the in vitro introduction of a polynucleotide of the invention into a plant cell.
  • the invention also includes a method for producing carotenoids in a plant entity.
  • the method includes transfecting or transforming a plant cell with the polynucleotide of the invention, culturing the plant cell to a plant entity wherein the polynucleotide is expressed in the plant entity.
  • the invention includes a method of producing carotenoids includes transforming or transfecting a plant cell with a polynucleotide of the invention; growing plants from the transformed plant cell; screening the plant for a desired carotenoid type or concentration; and processing the plant having the desired carotenoid type or concentration to obtain carotenoids.
  • the invention provides a method for modifying the carotenoid composition of a plant or plant organ, wherein the method comprises growing a stably transformed, transgenic plant containing a recombinant DNA expression construct encoding a polypeptide of the invention, under conditions wherein the polypeptide is expressed and the carotenoid composition of the plant or plant organ is modified.
  • the invention provides a transgenic plant cell comprising at least one recombinant enzyme from a carotenoid biosynthetic pathway.
  • the invention also provides a method for producing a transgenic plant comprising (a) introducing into a plant cell or plant tissue a polynucleotide of the invention to produce a transformed plant cell or plant tissue; and (b) cultivating said transformed plant cell or transformed plant tissue to produce said transgenic plant.
  • Figure 1 shows polynucleotide and polypeptide sequence of carotenoid pathway enzymes (SEQ ID NOs:l-10, respectively).
  • Figure 2 shows three pathways for carotenoid biosynthesis. In addition to natural C 30 and C 0 carotenoid pathways, a C 35 carotenoid pathway has been constructed in E. coli. The number for each carotenoid corresponds to those in other Figures.
  • Figure 3 shows (A) a cell pellet of XL1 cells harboring ] ⁇ JC-crtM-crtN-crtE, pUC-crtM-crtN, and pUC-crtB-crtN-crtE and (B) a typical view of agar plates of XL1 cells transformed with pUC-crt -[crtN]-crtE library.
  • Figure 4 is an HPLC analysis of carotenoid extract from HB 101 cells carrying plasmids (A) p ⁇ C-crtM-crtE, (B) pUC-crt , and (C) pUC-crtE-crtR. Individual compounds are: Peak 1 represents 4,4'-diapophytoene; Peak 2 represents 4-apophytoene; and Peak 3 represents Phytoene.
  • Figure 5 is an HPLC profile of carotenoid extracts from XL1 cells carrying plasmids (A) p ⁇ C-crtM-crtN, (B) pUC-crtM-crtN-crtE, (C) ipUC-crtM-crtl-crtE, (D) pAC- crtM-N6A-crtE, ( ⁇ ) ⁇ AC-crtM-N10F-crtE, (F) pAC-crtM-I13-crtE, (G) pUC-crtM-crtl-crtE and pUC-crt7, (H) pAC-crtM-crtN6A-crtE and pUC-crtF, (I) pAC-crtM-crt tI13-crtE and pUC-crtF, (I) pAC
  • FIG. 6 shows the natural and unnatural pathways for carotenoid biosynthesis.
  • C 35 , C 5 , and C50 carotenoid pathway were constructed in E. coli.
  • the number assigned to each carotenoid corresponds to those in Fig. 9.
  • Figure 7 shows a reaction mechanism of SqS and CrtM; (b) shows the positions corresponding to F26, W38, and ⁇ 180 of CrtM in the crystal structure of human squalene synthase (SqS). Residues in light gray are involved in the first half-reaction, while those in dark gray are involved in second half-reaction.
  • Figure 8 shows the relationship between C 4 o/C 30 synthase function and Nan der Waals volume of amino acid side chain at (a) position 26 and (b) position 38 in S. ⁇ ureus CrtM.
  • Values for C 0 function are the direct read of peak height (475 nm) of acetone extract from XL1 cells harboring pAC-crtE-crtl.
  • C 30 function solid circles was obtained from the peak height of the acetone extract (470 nm) from XL1 cells harboring pAC-crtN. Each point represents the average of six independent experiments, with standard deviation.
  • Figure 9 is an HPLC analysis of carotenoids produced in E coli HB 101 carrying plasmid (a) BSFPSYSIA, (d) TpUC-crtMwt, (e) pUC-M A w, (f) pUC-A w.
  • Figure 10 shows the (size) distribution of carotenoid backbones synthesized by selected CrtM variants expressed with Y81A mutant of BsFPS.
  • Open bar 2 (C 35 ); Striped bar: 3 (C 40 ); Solid (gray) bar: aaa (C 45 ); Solid (black) bar: bbb (C 50 ). Values were obtained from HPLC peak areas from extract from E. coli HB101 cells carrying pUC-fcrtMJ-
  • Figure 11 is a plot of absorbance units comparing the performance (pigment production) of selected single, double, and triple mutants in C 30 and C 0 pathways. Each point represents the average of four independent experiments.
  • Figure 12 (a) is a schematic depicting the method used to protect specific amino acid residues during random mutagenesis.
  • the residue of interest is replaced by a short, detachable DNA sequence containing a BsaXI recognition site (SEQ ID NO: 11; complementary strand is SEQ ID NO: 12). After random mutagenesis, this sequence is removed. Subsequent intramolecular ligation of the sequences flanking the insert regenerates the original codon. The three-base cohesive ends for ligation correspond to the codon for the target residue, and any variants with mutations at these sites cannot be ligated. As a result, only the variants that are free from mutation at this site can restore their functional reading frame.
  • Figure 12(b) shows a screening setup for the function as C 30 and C 0 carotenoid synthase.
  • Figure 13 is a plotting of amino acid size (v.d. w. volume) of CrtM, CrtBEU, and CrtBEholO on aligned sequences near the reaction pocket for second-half reaction (SaM (SEQ ID NO: 13); yeast SqS (SEQ ID NO: 14); human SqS (SEQ ID NO: 15); EuB (SEQ ID NO: 13).
  • EhB-Ehol3 SEQ ID NO:17
  • EhB-EholO SEQ ID NO:18
  • Figure 14 is an alignment of squalene synthase (SqS), CrtM, CrtBEU, and
  • SqS (SEQ ID NO:20); human SqS (SEQ DD NO:21); EuB (SEQ ID NO:22); EhB-Ehol3
  • the invention provides novel cartoenoids, novel cartenoid synthesis pathways, and novel enzymes for generating carotenoids and carotenoid precursors.
  • Carotenoids are natural pigments that play various biological roles (Ben-Dor et al. Arch. Biochem. Biophys. 391:295-302 (2001); Ishimi et al. J. Clin. Biochem. Nutr. 27 (1999); Mayne, S. T. FASEB Journal 10:690-701 (1996); Nishino et al. Pure Appl. Chem. 71 :2273-2278 (1999)).
  • GenBank Accession Nos. U48963 and X82627 Clarkia xantiana GenBank Accession No. U48962, Arabidopsis thaliana GenBank Accession No. U4896 1, Schizosaccharmyoces pombe GenBank Accession No. U21154, human GenBank Accession No. X17025, Kluyveromyces lactis GenBank Accession No. X14230; geranylgeranyl pyrophosphate synthase fromE. Uredovora Misawa et al. J. Bacteriol.
  • WO 91/13078 see also GenBank Accession Nos. L37405, X95596, D58420, X82458, S71770, and M87280; and from plant sources, including maize (Li et al. Plant Mol. Biol. 30:269-279 (1996)), tomato (Pecker et al. Proc. Nat. Acad. Sci. 89:4962-4966 (1992) , and Aracri et al. Plant Physiol. 106:789 (1994)), and Capisum annuum (bell beppers) (Hugueney et al. J. Biochem. 209: 399-407 (1992)), GenBank Accession Nos.
  • Carotenoids have a number of uses including therapeutic, diagnostic, and uses in industry as coloring agents etc. Carotenoids can be used in the generation of novel pigments and colors both in vivo and in industrial processes, hi addition, carotenoids find use in the treatment of diseases and disorders that affect mammals including, for example, the prevention of oxidative damage and the prevention of damage from exposure to too much sun light (e.g., as sunscreens).
  • Carotenoids may reduce the risk of various cell-proliferative disorders including breast, lung, colon, prostate and cervical cancer, as well as treating diseases and disorders such as heart disease and stroke.
  • carotenoids may retard macular degeneration.
  • beta-carotene is converted to vitamin A and vitamin A analogues or retinoids (see Moon, J Nutr. 119:127-134 (1989)). It is this provitamin A activity and the ability to prevent oxidative damage that has made carotenoids of interest in chemopreventive studies.
  • anti-oxidants are used, amongst other things, to quench free radicals that are by-products of normal metabolism in cells.
  • Beta- carotene has been used in the treatment of erythropoietic protoporphyria, a genetic disease causing an inadequacy in the metabolism of porphyrin compounds that presents as a rapid blistering of the skin on exposure to sunlight.
  • C 30 and C 40 pathways can complement each other. Nevertheless, C 40 carotenoids have not been isolated from C 30 carotenoid-synthesizing organisms, and C 30 carotenoids are not seen in C 40 organisms.
  • the invention provides engineered carotenoid synthases that are capable, for example, of accepting longer diphosphate substrates (e.g., C 20 and longer).
  • longer diphosphate substrates e.g., C 20 and longer.
  • C 40 synthase activity single amino- acid substitutions in, for example, C 30 synthase CrtM (F26L or F26S) were identified that confer a C 0 function.
  • additional amino acid substitutions, W38C and E180G were identified that confer the same phenotype.
  • the invention demonstrates that the specificity of carotenoid synthase CrtM is controlled at a second (rearrangement) step of its two-step reaction.
  • the engineered synthase variants make previously unknown C 45 and C 50 carotenoid backbones (mono- and di-isopentenylphytoenes) from the appropriate C 0 and C 25 isoprenyldiphosphate precursors.
  • carotenoid backbones mono- and di-isopentenylphytoenes
  • the engineered synthase variants make previously unknown C 45 and C 50 carotenoid backbones (mono- and di-isopentenylphytoenes) from the appropriate C 0 and C 25 isoprenyldiphosphate precursors.
  • a wider range of colors may be available from larger carotenoid backbones.
  • the first step of carotenoid synthesis is the condensation of two prenyldiphosphate units, catalyzed by carotene synthases. Products of this step are the linear, colorless hydrocarbons with three conjugated double bonds.
  • the backbone size of the condensation product dictates the possible size of chromophore that can be achieved upon desaturation. The number of the conjugated double bonds that can be contained in each backbone structure is illustrated in Table 1.
  • the benefits of the methods and compositions provided by the invention are significant.
  • the methods of the invention provide the ability to generate novel carotenoids. i addition to the expected higher antioxidant activity (Albrecht et al. Nat. Biotechnol. 18, 843-846 (2000)) and possible hormonal effects (Ishimi et al. J. Clin. Biochem. Nutr. 27. (1999); and Blount et al., Science 300, 125-127 (2003)), carotenoids with larger chromophores (19 conjugated double bonds for C 50 carotenoids, 23 for Ceo) will extend the color range of these natural pigments.
  • isoprenyldiphosphate synthases that produce isoprenyldiphosphates of different size (e.g. C 30 DP, C 35 DP, C 40 DP, C 45 DP, C 55 DP, and natural rubber) are available (Wang et al. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 1529, 33-48 (2000)); these compounds could, in principle, serve as substrates for engineered synthases. Accordingly, the invention provides novel polypeptides, polynucleotides, methods of synthesizing carotenoids, and carotenoid products.
  • the invention provides substantially purified polypeptides comprising a sequences that are 80%-99% identical, but not 100% identical, over the full length of SEQ ID NO:2, and which catalyzes the generation of a C 40 carotenoid.
  • a substantially purified polypeptide of the invention includes a polypeptide that has a sequence as set forth in SEQ ID NO:2 having one to five amino acids substitutions and wherein the polypeptide catalyzes the condensation of geranlygeranlydiphosphate to form phytoene.
  • a polypeptide of the invention also includes a polypeptide comprising a sequence as set forth in SEQ ID NO:2 having a substitution selected from the group consisting of an H12Q substitution, an F26X substitution, W38X substitution; a E180G substitution; and any combination thereof, wherein X is any amino acid.
  • a polypeptide of the invention comprises a substitution of an amino acid at F26 and/or W38 of SEQ ID NO:2 with a smaller amino acid.
  • a substantially purified polypeptide of the invention includes, for example, (i)
  • SEQ ID NO:2 having an F26G substitution (ii) SEQ ID NO:2 having an F26A substitution; (iii) SEQ ID NO:2 having an F26L substitution; (iv) SEQ ID NO:2 having an F26S substitution; (v) SEQ ID NO:2 having a W38C substitution; (vi) SEQ ID NO:2 having a H12Q substitution; (vii) SEQ ID NO:2 having an E180G substitution; (viii) SEQ ID NO:2 having an F26X substitution and a W38C substitution, wherein X is G, A, L, or S; (ix) SEQ ID NO:2 having an F26X substitution, a W38C substitution, and an E180G substitution, wherein X is G, A, L, or S; (x) SEQ ID NO:2 having an F26X substitution and an E180G substitution, wherein X is G, A, L, or S; and (xi) SEQ ID NO:2 having a W38C substitution and an E180
  • a polynucleotide of the invention comprises (i) a polynucleotide having a sequence that is at least 80% identical to SEQ ID NO:l, but less than 100% identical, over the full length of SEQ ID NO:l, and wherein the polynucleotide encodes a polypeptide that catalyzes the generation of a carotenoid; (ii) a polynucleotide comprising a sequence as set forth in SEQ ED NO:l, wherein nucleotides 34-36 are selected from caa and cag; (iii) a polynucleotide comprising a sequence as set forth in SEQ ID NO:l, wherein nucleotides 76- 78 are selected from ggn, gen, tta, ttg, ctn, agt, age, and ten, wherein n is a, g, t, or c; (iv) a polynucleo
  • the polynucleotide encodes a polypeptide as set forth above.
  • a polynucleotide of the invention encodes a polypeptide comprising (i) SEQ ID NO:2 having an F26G substitution; (ii) SEQ ID NO:2 having an F26A substitution; (iii) SEQ ID NO:2 having an F26L substitution; (iv) SEQ ID NO:2 having an F26S substitution; (v) SEQ ID NO:2 having a W38C substitution; (vi) SEQ ID NO:2 having a H12Q substitution; (vii) SEQ ID NO:2 having an E180G substitution; (viii) SEQ ID NO:2 having an F26X substitution and a W38C substitution, wherein X is G, A, L, or S; (ix) SEQ ID NO:2 having an F26X substitution, a W38C substitution, and an E180G substitution, wherein X is G, A, L, or S;
  • a method of the invention comprises making a plant (or other host cell) by incorporating a polynucleotide of the invention and/or incorporating additional members of a cartenoid pathway as described herein.
  • the invention further provides a method for generating novel carotenoids.
  • the method comprises genetically engineering a suitable host cell with complementary enzymes from different carotenoid synthesis pathways or mutating an enzyme of a naturally occurring pathway such that the enzymes substrate specificity and/or product are changed.
  • the invention demonstrates that Staphylococcus CrtM, a synthase from a C 30 pathway, synthesizes C 35 carotenoids in the presence of GGPP.
  • CrtM can condense two FPPs; it can also accept one (or two) molecules of GGPP in the same reaction. In the absence of CrtE, however, CrtM produces C 30 carotenoids (4,4'-diapolycopene) exclusively (see Fig. 4B).
  • a new compound is produced, either as a result of enzyme recruitment or mutation in an existing enzyme (such as those listed above and elsewhere herein), it can be further metabolized by downstream modifying enzymes, thereby allowing a series of novel compounds to emerge ( Komori, 1998; Garcia-Asua, 1998;Garcia-Asua, 2002;Croteau, 1991; Umeno, 2003).
  • modifying enzymes can include, for example, ⁇ -carotene hydroxylase or crtZ (Hundle et al. FEBS Lett. 315:329-334 (1993), GenBank Accession No. M87280), keto-introducing enzymes, such ascrtw (Misawa et al. J. Bacteriol.
  • Artificial carotenoid pathways can be constructed in a target host cell using techniques such as those disclosed in U.S. Patent Application Publication No. 20020051998, which is incorporated herein by reference in its entirety. Things to consider in constructing artificial pathways include, for example, minimizing pathway components; (2) choosing the point that the pathway starts from; (3) toxicity; and (4) possible conversion of the pigments by hosts; and (5) the primary structure of the enzymes for pigment synthesis. [ 0052 ] Depending on the target host cell, the number of genes that can be introduced is limited. Productivities decrease rapidly with increasing numbers of gene added.
  • the artificial pathway will be an extension from a precursor existing in a host cell so that only a small number of polynucleotides (e.g., 1-3 polynucleotides) will need to be transformed or transfected into the host cell.
  • Methods known in the art, as described herein, can be used to transfect or transform a host cell.
  • the enzymatic product resulting from expression of the introduced polynucleotide should minimally burden the host and not seriously hamper its physiological activities.
  • the artificial pathway is engineered such that the pathway branches out from a precursor, which is abundantly and stably supplied (e.g., FPP).
  • FPP stably supplied
  • Overproduction of a polar pigments such as lycopene (C 40 ) should be avoided to reduced toxicity due to the change in the fluidity of the membrane system brought about by the a polar cartenoid products.
  • Some host cells may further metabolize carotenoid products.
  • the products resulting from the artificial carotenoid pathway may be further metabolized by endogenous enzymes in the host cell, resulting in the production of unplanned pigments and unwanted color development. This is more likely in plant systems that may have a preexisting cartenoid pathway that can metabolize enzymatic products of the artificial pathway.
  • Those skilled in the art can readily identify host cells that may prove optimal for the generation of novel carotenoid pathways by generating pathways in host cells that lack a caroteno genie enzyme that can act on the product of the pathway.
  • Carotenoid pathways that generate shorter backbones have the advantage of (1) higher pigment/weight ratio, (2) small number of genes needed for assembly, (3) use of ubiquitous and abundant FPP as precursors, (4) higher solubility in both membrane and aqueous systems, and (5) less probability of being metabolized by the host organism, especially plants with C o-cartenoid pathways.
  • light absorption properties of a given carotenoid can be expressed by the effective number of conjugated double bonds (c.d.b.-number), the larger the effective c.d.b.-number the longer the wavelength of the absorption maximum of the carotenoid. Accordingly, by modulating c.d.b. -number, one can synthesize carotenoids with various different colors in a predictable manner. Because each carotenoid pigment possesses a characteristic color, the novel carotenoids can be identified with reasonable ease.
  • a cartenoid pathway includes three main steps: (1) condensation of prenyl units to complete the artenoid backbone catalzyed by a carotene synthase, (2) stepwise extension of chromophore catalyzed by a carotene desaturase; and (3) cyclization or other modification (e.g., hydroxylation) of the structure.
  • a fourth step may be present that performs oxidative cleavage.
  • C 0 backbone which is produced by the condensation of two units of geranylgeranyldiphosphates (GGPP).
  • GGPP geranylgeranyldiphosphates
  • FPP farnesyldiphosphates
  • the C 30 pathway branches from the ubiquitous isoprenoid precursor, FPP, which is available in all living cells.
  • FPP ubiquitous isoprenoid precursor
  • the natural abundance of precursor FPP also ensures a high production level of pigment.
  • the basic C 30 pathway could therefore be established in any host or organs, irrespective of the presence and level of caroteno genesis, by addition of only two genes, a C 30 synthase and a desaturase. If the host already has a C 30 system, some of the existing modification enzymes (desaturase, and the like) maybe able to accept the new C 30 backbone as substrate and effectively carry out further modifications. Thus, the C 30 pathways require insertion of relatively few genes to support pigment production.
  • C 30 carotenoid production should be possible in any cellular compartment, including the cytoplasm.
  • the generation of C 30 carotenoids consumes 25% less carbon than the C 4 o-counterparts, thus conferring high pigmentation level from limited source of isoprenoid precursors.
  • the solubility of C 30 carotenoids in the membrane is significantly higher than that of C 40 carotenoids. Higher storage capacity by host organisms, and thus more intense color, is therefore expected.
  • C 30 carotenoids with polar groups such as keto-, OH-, or glucoside in the C 30 backbone results in more water-soluble pigments, should those be desired.
  • the C 30 carotenoids in bacteria do not interfere with growth, thus their production will have very limited toxicity in other organisms, including plants.
  • Carotene desaturases are multi-step enzymes that extend the polyene chromophore on carotenoid backbone. Starting from phytoene-type substrates with three conjugated double bonds, each step adds two conjugated double bonds. Thus, carotenoids with different colors are obtained from a single substrate by regulating the desaturation step number. In natural C 40 pathways, there are many desaturases characterized by different step numbers. In contrast, there is only one, three-step desaturase (crNfrom Staphylococcus aureus) available in a C 30 pathway.
  • the invention provides methods to generate various acyclic C 30 carotenoids by mutating enzymes of the carotenoid pathway. For example, in one aspect mutants of crtN were generated with altered step number. Such mutants can then be expressed in combination with crtM (for precursor supply) in E. coli to obtain various pigment colors. Each colony with a different color has a crtN-variants with altered step number, and accumulated carotenoids with different chromophores. Thus, acyclic C 30 - carotenoids with all possible c.d.b.-numbers (1,3,5,7,9 and 11, 1-5) can be quickly accessed. All these pathways can then be transferred to other host systems.
  • Cyclization of the carotenoid end allows the creation of an ⁇ -end by removing one double bond from the chromophore.
  • monocyclic ⁇ -carotenoids possess chromophores with even number c.d.b.
  • ⁇ -end construction does not reduce the c.d.b.-number. Instead, because of steric hindrance between the ring methyl substituent and hydrogen atoms along the polyene, the ring is slightly twisted so that endocyclic double bond is not fully effective. This causes a small (5-10 nm) hypochromic shift in comparison to the linear polyenes with the same number of conjugated double bonds.
  • cyclic carotenoids are in general more stable and resistant to oxidative bleaching. Therefore, if allowed (if the desired color is in the range of lemon-yellow-orange), the use of short-chain cyclic carotenoids might be advantageous with respect to the effectiveness and duration of color in the plant.
  • oxo-functionalities can be integrated into the C 30 carotenoids described above.
  • carotene ketolase crtA, from Rhodobacters
  • crtA catalyzes ketolation of spheroidene at the saturated C (2) position resulting in the extension of chromophore.
  • crtM and crtN ' E. coli three polar carotenoids with effective c.d.b. -numbers of 6, 7 and 8- were observed.
  • the structure and pathways for these compounds are epoxy- (13-15) or diol-type carotenoids.
  • C 35 carotenoid pathways was generated by co-expressing C 3 o-carotenoid enzymes with GGPP (C 20 diphosphate) synthase. These unique asymmetrical carotenoid structures are realized via hetero-condensation of two different prenyldiphosphates (C 20 and C ⁇ 5 ). This novel pathway was further extended by a series of modifying enzymes. Both C 30 and C 40 carotene desaturases (Staphylococcus crtN and Erwinia crtl) converted with the C 35 substrates.
  • a number of acyclic C 35 carotenoids can be generated.
  • the carotenoids can be further converted by ⁇ - cyclases from Erwinia, resulting in two previously unknown cyclic C 35 carotenoids, as disclosed herein.
  • the backbones can be further acted upon by downstream enzymes and further molecule diversity generated.
  • the invention demonstrates that ability to engineer enzymes to generate novel pathways and carotenoid pathways.
  • an evolved C 3 o-carotenoid synthase (crtM from Staphylococcus aureus) was mutated to function in a C 4 o pathway, that is, to synthesize phytoene from two GGPP.
  • a single round of directed evolution generated hundreds of crt -mutants with this C 40 -synthase activity. From sequence analysis of these mutants, several amino acid residues were identified that determine the substrate/product specificity of the synthase enzyme, including F26.
  • C 30 and shorter versions of carotenoids can be established in plants without the need for extra genes to be transferred to form the precursors.
  • ⁇ -Carotene dioxygenase ( ⁇ -CD) is used to create additional sets of carotenoid pigments with smaller backbones.
  • This enzyme is know to accept various carotenoids as substrates. However, for any substrates, it acts right at 15-15' (center of backbone) specifically and precisely.
  • the invention also provides methods to modify the specificity of the ⁇ -CD that would allow further novel carotenoid pathways and products to be generated.
  • Naturally occurring ⁇ -CD is known to act on ⁇ -end carotenes, however, by engineering ⁇ -CD it is possible to derive a ⁇ -CD that possesses activity toward ⁇ -end carotenoids (62-65, table 1). It is also possible to increase the activity of ⁇ -CD toward a variety of acyclic C 40 carotenoids which are converted by wild type ⁇ -CD, but at a very low rate. Carotenoids with different chain lengths can be targeted, too. Thus, various vitamin A- like pigments will be accessed (66-74, table 1). In addition, it is possible to obtain ⁇ -CD variants, which conduct eccentric (non-15-15') cleavage (e.g., to generate carotenoid pigments such as 75, 76, table 1).
  • pigments described herein are small and polar, i addition, the polarity can be localized on one side. These pigments are characterized by high solubility both in aqueous and membrane systems. The location and the way these carotenoids accumulate in the host cell would differ significantly from other, less polar types, so that the apparent color in vivo would be unique.
  • carotenoids frequently occur in association with proteins, and this sometimes gives rise to a variety of colors, even purple and blue.
  • the most well known carotenoprotein is called crastacycanin (from lobster).
  • astaxanthin forms a stable but reversible complex with small (18-20kDa) polypeptide units of crustacyanin(s), causing large (-150nm) bathochromic shift in the light absorption spectrum.
  • the invention further provides methods for engineering and expressing crustacyanin-complexes as an attractive alternative strategy for coloring plants and flowers in the blue to purple range.
  • a host cell is genetically engineered with one or more carotenoid pathway enzymes.
  • the host cell is then cultured to obtain colonies (e.g., bacterial colonies).
  • colonies e.g., bacterial colonies.
  • the host cell and/or colonies are then screened for colonies exhibiting a different color than colonies of an untransformed genetically engineered host cell.
  • suitable hosts include E. coli, cyanobacteria such as Synechococcus and Synechocystis, alga and plant cells.
  • the host cell will be a photosynthetic organisms.
  • color complementation test can be enhanced by using mutants which are either (1) deficient in at least one carotenoid biosynthetic gene or (2) overexpress at least one carotenoid biosynthetic gene. In either case, such mutants will accumulate carotenoid precursors.
  • carotenoids include, but are not limited to, the carotenoids shown in Table 1 below. Of particular interest are those carotenoids in Table 1 labeled with an asterisks.
  • the carotenoids of the invention are pigments useful as feed additives, food additives, cosmetics, and the like.
  • the carotenoids of the invention are valuable from an industrial point of view as a feed additive, such as a color improver, and as a safe natural food additives.
  • carotenoids are useful as supplements, particularly vitamin supplements, as vegetable oil based food products and food ingredients, and as colorants.
  • phytoene finds use in treating skin disorders. See, for example, U.S. Pat. No. 4,642,318. Lycopene and carotene are used as food coloring agents. Consumption of carotene and lycopene has also been implicated as having preventative effects against certain kinds of cancers. In addition, lutein consumption has been associated with prevention of macular degeneration of the eye.
  • Naturally occurring carotenoids are natural pigments that are responsible for many of the yellow, orange, and red colors seen in living organisms. Although carotenoids are integral constituents of the protein-pigment complexes of the light-harvesting antennae in photosynthetic organisms, they are also important components of the photosynthetic reaction centers. Most of the total carotenoids are located in the light harvesting complex II (Bassi et al., Eur J Biochem 212: 297-302 (1993)). Accordingly, transgenic plants that comprise artificial carotenoid pathways as described herein can be produced such that the plants exhibit novel color variations in their flowers, leaves, fruit, and the like.
  • novel flower and plant colors can be attained.
  • transformation will be accomplished using Agrobacterium. Transformation of mediated by co-cultivation using disarmed Agrobacterium tumefaciens containing transformation vectors comprising the particular genes to be used. Transformation is accomplished in vitro, using sterile plant tissue and or plant tissue culture.
  • an in vivo transformation system is also used where the infected plants are allowed to set seed and transgenic seed selected in vitro.
  • Two genes, crtM and crtN are engineered into standard expression cassettes (e.g. CaMV35S; see NLRD 163/2002) and a binary vector- Agrobacterium system used to introduce T-DNA carrying the expression cassettes into plants. Transformed plants with novel and/or desired flower color will be selected.
  • Examples of the plant types that can be used include, for example, genus Arabidopsis.
  • A. thaliana is commonly used as a generic/model experimental plant because of its size and the ease of growing it in culture in the laboratory.
  • P. x hybrida is an ornamental, typically found in flowerbeds. The cultivated rose, Rosa X hybrida can be used (e.g., R. rubiginosa and R anina).
  • carotenoids are lipophyllic and therefore not soluble in water in useful quantities.
  • the therapeutic and diagnostic potential of the carotenoids of the invention can find use in their ability to be transported in the bloodstream in conjunction with low- density lipoproteins.
  • purified means altered “by the hand of man” from its natural state; i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polypeptide naturally present in a living animal, a biological sample, or an environmental sample in its natural state is not “purified”, but the same polypeptide separated from the coexisting materials of its natural state is “purified”, as the term is employed herein.
  • the term “purified” does not necessarily mean 100% purified from other materials, but rather means that some substantial degree of purification has taken place.
  • a substantially purified polypeptide includes a polypeptide that is 20% or more free of material of which it is naturally associated.
  • Polypeptide refers to any polymer of two or more individual amino acids linked via a peptide bond.
  • the term “polypeptide” is understood to include the terms “protein” and “peptide” (which, at times, maybe used interchangeably herein).
  • polypeptides comprising multiple polypeptide subunits (e.g., DNA polymerase III, RNA polymerase II) or other components (for example, an RNA molecule, as occurs in telomerase) will also be understood to be included within the meaning of "polypeptide” as used herein.
  • fragments of polypeptides are also within the scope of the invention.
  • amino acid is a molecule having a central carbon atom (the ⁇ -carbon atom) linked to a hydrogen atom, a carboxylic acid group (the carbon atom of which is referred to herein as a “carboxyl carbon atom”), an amino group (the nitrogen atom of which is referred to herein as an "amino nitrogen atom"), and a side chain group, R.
  • an amino acid loses either a hydroxyl group from its carboxylic acid group or a hydrogen from the amino group in a dehydration reaction that links one amino acid to another in a peptide bond.
  • Naturally occurring amino acids include the twenty amino acids naturally occurring in proteins. These naturally occurring amino acids are the L-isomers of glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine.
  • Unnatural amino acids are any amino acid other than the twenty named above.
  • D-isomers of the twenty amino acids named above D or L isomers or racemic mixtures of selenocysteine and selenomethionine, and the D or L forms (or racemic mixtures) of, for example, nor-leucine, para-nitrophenylalanine, homophenylalanine, para- fluorophenylalanine, 3-amino-2-benzylproprionic acid, homoarginine, and the like.
  • These unnatural amino acids may be used, for example, in polypeptide synthesis for increased polypeptide stability and the like.
  • an R-group is a side-chain attached to the ⁇ -carbon of an amino acid residue.
  • the R-group is an important determinant of the overall chemical character of an amino acid.
  • a positively charged amino acid is any naturally occurring or unnatural amino acid having a side chain that is positively charged under normal physiological conditions.
  • the positively charged, naturally occurring amino acids are arginine, lysine, and histidine.
  • a negatively charged amino acid is any naturally occurring or unnatural amino acid having a side chain that is negatively charged under normal physiological conditions. Examples of negatively charged, naturally occurring amino acids are aspartic acid and glutamic acid.
  • a hydrophobic amino acid is any naturally occurring or unnatural amino acid that contains a hydrophobic R-group. Examples of naturally occurring hydrophobic amino acids are alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • An uncharged, hydrophilic amino acid is any naturally occurring or unnatural amino acid that is contains a hydrophilic R-group, but is uncharged at physiological pH.
  • Examples of naturally occurring uncharged, hydrophilic amino acids are serine, threonine, tyrosine, asparagine, glutamine, and cysteine.
  • primary structure when written from the amino-terminus to carboxy-terminus) can be determined by the nucleotide sequence of the coding portion of a mRNA, which is in turn specified by genetic information, typically genomic DNA.
  • amino acid residue of a polypeptide interacts with adjacent residues (e.g., residues that are adjacent in primary, secondary or tertiary structure of a polypeptide) and/or with ligands or substrates based, in part, on the type of R-group present.
  • adjacent residues e.g., residues that are adjacent in primary, secondary or tertiary structure of a polypeptide
  • ligands or substrates based, in part, on the type of R-group present.
  • hydrophobic amino acids are more likely to interact with other hydrophobic amino acids or hydrophobic molecules.
  • hydrophilic amino acids are more likely to interact with other hydrophilic amino acids or hydrophilic molecules.
  • a polypeptide of the invention can be produced recombinantly.
  • a polynucleotide encoding a polypeptide of the invention can be introduced into a recombinant expression vector, which can be expressed in a suitable expression host cell system using techniques well known in the art.
  • a suitable expression host cell system A variety of bacterial, yeast, plant, mammalian, and insect expression systems are available in the art and any such expression system can be used.
  • a polynucleotide encoding a polypeptide can be translated in a cell-free translation system.
  • a "polynucleotide” refers to a polymeric form of nucleotides. In some instances a polynucleotide refers to a polymer of nucleotides that are not. immediately contiguous with either of the coding sequences with which it is immediately contiguous (one on the 5' end and one on the 3' end) in the naturally occurring genome of the organism from which it is derived.
  • the term therefore includes, for example, a recombinant DNA which is inco ⁇ orated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA) independent of other sequences.
  • the nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms of either nucleotide.
  • a polynucleotides as used herein refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • isolated as used in association with a polynucleotide means altered "by the hand of man" from its natural state; i.e., if it occurs in nature, it has been changed or removed from its original environment, or both.
  • a naturally occurring polynucleotide naturally present in a living animal a biological sample or an environmental sample in its natural state is not “isolated”, but the same polynucleotide separated from the coexisting materials of its natural state is "isolated”, as the term is employed herein.
  • Polynucleotides when introduced into host cells in culture or in whole organisms, still would be isolated, as the term is used herein, because they would not be in their naturally occurring form or environment.
  • polynucleotides may occur in a composition, such as a media formulation (solutions for introduction of polynucleotides, for example, into cells or compositions or solutions for chemical or enzymatic reactions).
  • a composition such as a media formulation (solutions for introduction of polynucleotides, for example, into cells or compositions or solutions for chemical or enzymatic reactions).
  • isolated does not necessarily mean 100% isolated from other materials, but rather means that some substantial degree of isolation has taken place.
  • a substantially isolated polynucleotide includes polynucleotides that are 20% or more free of material with which it naturally associated.
  • Polynucleotides can be cloned into an appropriate vector.
  • the vector used will depend upon whether the polynucleotide (e.g., DNA) is to be expressed, amplified, sequenced etc. Cloning techniques are known in the art or can be developed by one skilled in the art, without undue experimentation. The choice of a vector will also depend on the size of the polynucleotide and the host cell to be employed in the methods of the invention.
  • a vector used in the invention may be a plasmid, a phage, a cosmid, a phagemid, a virus (e.g.
  • retroviruses parainfluenzavirus, herpesviruses, reoviruses, paramyxoviruses, and the like), or selected portions thereof.
  • cosmids and phagemids are typically used where the specific polynucleotide to be cloned is large because these vectors are able to stably propagate large polynucleotides.
  • Polynucleotides of the invention can also comprise other nucleotide sequences, such as sequences coding for linkers, signal sequences, heterologous signal sequences, TMR stop transfer sequences, transmembrane domains, or ligands useful in protein purification such as glutathione-S-transferase, histidine tag, and staphylococcal protein A. More than one polypeptide of the invention can be present in a fusion protein.
  • a vector containing a cloned polynucleotide of the invention can then be amplified by plating or transfecting a suitable host cell with the vector (e.g., a phage on an E. coli host).
  • transformation is meant a permanent (e.g., stable) or transient genetic change induced in a cell following incorporation of a polynucleotide (e.g., DNA exogenous to the cell). Where the cell is a mammalian cell, a permanent genetic change is generally achieved by introduction of the polynucleotide into the genome of the cell.
  • transformed cell or “recombinant host cell” is meant a cell (e.g., prokaryotic or eukaryotic) into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques a polynucleotide of the invention or fragment thereof.
  • a polynucleotide of the invention is cloned into a vector it can be clonally amplified by inserting each vector into a host cell and allowing the host cell to amplify the vector. This is referred to as clonal amplification.
  • Polynucleotides can be cloned into an expression vector comprising, for example, promoters, enhancers, or other regulatory elements that drive expression of the polynucleotides of the invention in recombinant host cells.
  • An expression vector can be, for example, a plasmid, such as pBR322, pUC, or ColEl, or an adenovirus vector, such as an adenovirus Type 2 vector or Type 5 vector.
  • Minichromosomes such as MC and MCI, bacteriophages, phagemids, yeast artificial chromosomes, bacterial artificial chromosomes, virus particles, virus-like particles, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under their own control in a cell) can also be used.
  • a polynucleotide is cloned into a vector at an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • a typical cloning scenario may have the DNA "blunted" with an appropriate nuclease (e.g., Mung Bean Nuclease), methylated with, for example, EcoR. I Methylase and ligated to EcoR I linkers GGAATTCC.
  • the linkers are then digested with an EcoR I Restriction Endonuclease and the DNA size fractionated (e.g., using a sucrose gradient).
  • the resulting size fractionated DNA is then ligated into a suitable vector for sequencing, screening or expression (e.g., a lambda vector and packaged using an in vitro lambda packaging extract).
  • a polynucleotide in the expression vector is operatively linked to an appropriate regulatory element (e.g., a promoter) to direct mRNA synthesis.
  • a promoter e.g., bacterial promoters
  • bacterial promoters include lad, lacZ, T3, T7, gpt, lambda PR, PL and tip.
  • Eukaryotic promoters include CMN immediate early, HSN thymidine kinase, early and late SN40, LTRs from retrovirus, and mouse metallothionein-I.
  • an expression vector typically contains one or more selectable marker genes to provide a phenotypic trait for selection of recombinant host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • a vector comprising a polynucleotide of the invention can be transformed into, for example, bacterial, plant cells, yeast, insect, or mammalian cells so that the polypeptides of the invention can be expressed from the polynucleotides and purified from cell culture or used to generate transgene organism (including plants). Any technique available in the art can be used to introduce polynucleotides into the host cells. These include, but are not limited to, transfection with naked or encapsulated polynucleotides, cellular fusion, protoplast fusion, viral infection, and electroporation, to name a few.
  • Suitable regulatory elements in an expression vector include promoters that initiate transcription only, or predominantly, in certain cell types.
  • promoters specific to vegetative tissues such as ground meristem, vascular bundle, cambium, phloem, cortex, shoot apical meristem, lateral shoot meristem, root apical meristem, lateral root meristem, leaf primordium, leaf mesophyll, or leaf epidermis can be suitable regulatory elements.
  • a promoter specific to a reproductive tissue e.g., fruit, ovule, seed, pollen, pistils, female gametophyte, egg cell, central cell, nucellus, suspensor, synergid cell, flowers, embryonic tissue, embryo, zygote, endosperm, integument, seed coat or pollen
  • a cell type or tissue-specific promoter can drive expression of operably linked sequences in tissues other than the target tissue.
  • a cell type or tissue-specific promoter is one that drives expression preferentially in the target tissue, but can also lead to some expression in other cell types or tissues as well.
  • Methods for identifying and characterizing promoter regions in plant genomic D ⁇ A include, for example, those described in the following references: ordano, et al., Plant Cell, 1 :855-866 (1989); Bustos, et al., Plant Cell, 1:839-854 (1989); Green, et al., EMBO J., 7:4035-4044 (1988); Meier, et al., Plant Cell, 3:309-316 (1991); and Zhang, et al., Plant Physio., 110:1069-1079 (1996).
  • Exemplary reproductive tissue promoters include those derived from the following seed-genes: zygote and embryo LEC1 (see, Lotan, Cell 93:1195-1205 (1998)); suspensor G564 (see, Weterings, Plant Cell 13:2409-2425 (2001)); maize MAC1 (see, Sheridan, Genetics, 142:1009-1020 (1996)); maize Cat3 (see, GenBank No. L05934; Abler, Plant Mol. Biol., 22:10131-1038 (1993)); Arabidopsis viviparous-1 (see, GenbankNo. U93215); Arabidopsis atmycl (see, Urao, Plant Mol.
  • reproductive tissue promoters include those derived from the following embryo genes: Brassica napus 2s storage protein (see, Dasgupta, Gene, 133:301-302 (1993)); Arabidopsis 2s storage protein (see, GenBank No. AL161566); soybean b-conglycinin (see, GenBank No. S44893); Brassica napus oleosin 20kD gene (see, GenBank No. M63985); soybean oleosin A (see, Genbank No. U09118); soybean oleosin B (see, GenBank No.
  • soybean lectinl see, GenBank K00821); soybean Kunitz trypsin inhibitor 3 (see, GenBank No. AF233296); soybean glycininl (see, GenBank No. X15121); Arabidopsis oleosin (see, GenBank No. Z17657); maize oleosin 18kD (see, GenBank No. J05212; Lee, Plant Mol. Biol. 26:1981-1987 (1994)); and the gene encoding low molecular weight sulfur rich protein from soybean (see, Choi, Mol. Gen. Genet., 246:266-268 (1995)).
  • reproductive tissue promoters include those derived from the following endosperm genes: Arabidopsis Fie (see, GenBank No. AF129516); Arabidopsis Mea; Arabidopsis Fis2 (see, GenBank No. AF096096); rice Glul (see, GenBank No. M28156); and rice 26 kDa globulin (see, GenBank No. D50643).
  • Yet other exemplary reproductive tissue promoters include those derived from the following genes: ovule BEL1 (see, Reiser, Cell, 83:735-742 (1995); Ray, Proc. Natl. Acad. Sci. USA, 91:5761-5765 (1994); GenBank No.
  • Suitable vegetative tissue promoters include those derived from the following genes: pea Blec4, active in epidermal tissue of vegetative and floral shoot apices of transgenic alfalfa; potato storage protein patatin gene (see, Kim, Plant Mol. Biol., 26:603-615 (1994); Martin, Plant J., 11:53-62 (1997)); root Agrobacterium rhizogenes ORF13 (see, Hansen, Mol. Gen.
  • Cell type or tissue-specific promoters derived from viruses also can be suitable regulatory elements.
  • exemplary viral promoters include: the tobamovirus subgenomic promoter (Kumagai, Proc. Natl. Acad. Sci. USA, 92:1679-1683 (1995); the phloem-specific tungro bacilliform virus (RTBN) promoter; the cassava vein mosaic virus (CVMN) promoter, expressed most strongly in vascular elements, leaf mesophyll cells, and root tips (Nerdaguer, Plant. Mol. Biol., 31:1129-1139 (1996)).
  • RTBN phloem-specific tungro bacilliform virus
  • CVMN cassava vein mosaic virus
  • a polynucleotide encoding a polypeptide of the invention can be obtained by, for example, D ⁇ A synthesis or the polymerase chain reaction (PCR).
  • PCR refers to a procedure or technique in which target nucleic acids are amplified (in some instances under conditions that allow for the incorporation of mutations).
  • PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA.
  • Various PCR methods are described, for example, in PCR Primer: A Laboratory Manual, Dieffenbach, C. & Dveksler, G., Eds., Cold Spring Harbor Laboratory Press, 1995.
  • sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified.
  • Various PCR strategies are available by which site-specific nucleotide sequence modifications can be introduced into a template nucleic acid.
  • Polynucleotides of the invention can be detected in culture or other samples by methods such as ethidium bromide staining of agarose gels, Southern or Northern blot hybridization, PCR or in situ hybridizations.
  • Hybridization typically involves Southern or Northern blotting (see, for example, sections 9.37-9.52 of Sambrook et al., 1989, "Molecular Cloning, A Laboratory Manual", 2nd Edition, Cold Spring Harbor Press, Plainview; NY). Probes should hybridize under high stringency conditions to a nucleic acid or the complement thereof.
  • High stringency conditions can include the use of low ionic strength and high temperature washes, for example 0.015 M NaCl/0.0015 M sodium citrate (0.1X SSC), 0.1% sodium dodecyl sulfate (SDS) at 65°C.
  • denaturing agents such as formamide
  • formamide can be employed during high stringency hybridization, e.g., 50% formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42°C.
  • the term "host” or "host cell” includes not only prokaryotes, such as E.
  • a host cell can be transformed or transfected with a DNA molecule (e.g., a vector) using techniques known to those of ordinary skill in this art, such as calcium phosphate or lithium acetate precipitation, electroporation, lipofection, and particle bombardment.
  • Host cells containing a vector of the invention can be used for such purposes as propagating the vector, producing a polynucleotide (e.g., DNA, RNA, antisense RNA) or expressing a polypeptide or fragments thereof.
  • eukaryotic organisms useful as host cells of the invention are plants containing an exogenous polynucleotide that encodes a polypeptide of the invention.
  • plants containing an exogenous polynucleotide that encodes a polypeptide of the invention are known in the art, and include, without limitation, Agrobacterium-mediated transformation, viral vector-mediated transformation, electroporation, and particle gun transformation, e.g., U.S. Patents 5,204,253 and 6,013,863. If a cell or tissue culture is used as the recipient tissue for transformation, plants can be regenerated from transformed cultures by techniques known to those skilled in the art.
  • Transgenic plants can be entered into a breeding program, e.g., to introduce a polynucleotide encoding a polypeptide into other lines, to transfer the polynucleotide to other species or for further selection of other desirable traits.
  • transgenic plants can be propagated vegetatively for those species amenable to such techniques.
  • Progeny includes descendants of a particular plant or plant line.
  • Progeny of an instant plant include seeds formed on Fl, F2, F3, and subsequent generation plants, or seeds formed on BC1, BC2, BC3, and subsequent generation plants. Seeds produced by a transgenic plant can be grown and then selfed (or outcrossed and selfed) to obtain seeds homozygous for the polynucleotide encoding a polypeptide of the invention.
  • a suitable group of plants with which to practice the invention include dicots, such as safflower, alfalfa, soybean, rapeseed (high erucic acid and canola), or sunflower. Also suitable are monocots such as corn, wheat, rye, barley, oat, rice, millet, amaranth or sorghum. Also suitable are vegetable crops or root crops such as potato, broccoli, peas, sweet corn, popcorn, tomato, beans (including kidney beans, lima beans, dry beans, green beans) and the like. Also suitable are fruit crops such as peach, pear, apple, cherry, orange, lemon, grapefruit, plum, mango and palm.
  • dicots such as safflower, alfalfa, soybean, rapeseed (high erucic acid and canola), or sunflower.
  • monocots such as corn, wheat, rye, barley, oat, rice, millet, amaranth or sorghum.
  • vegetable crops or root crops
  • the invention has use over a broad range of plants, including species from the genera Anacardium, Arachis, Asparagus, Atropa, Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea, Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium, Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium, Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana, Olea, Oryza, Panicum, Pannesetum, Persea, Phaseolus, Pistachia, Pisum, Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum, Sorghum, Tlieobromus, Trigonella, Triticum, Vicia, Vitis,
  • Chimeric polypeptides comprising a polypeptide of the invention can be expressed in plants in a cell- or tissue-specific manner according to the regulatory elements chosen to include in a particular nucleic acid construct present in the plant.
  • Suitable cells, tissues and organs in which to express a chimeric polypeptide of the invention include, without limitation, egg cell, central cell, synergid cell, zygote, ovule primordia, nucelfus, integuments, endothelium, female gametophyte cells, embryo, axis, cotyledons, suspensor, endosperm, seed coat, ground meristem, vascular bundle, cambium, phloem, cortex, shoot or root apical meristems, lateral shoot or root meristems, floral meristem, leaf primordia, leaf mesophyll cells, and leaf epidermal cells, e.g., epidermal cells involved in forming the cuti
  • eukaryotic organisms useful in the invention are fungi containing an exogenous polynucleotide that encodes a polypeptide or a chimeric polypeptide of the invention.
  • the invention also includes a method comprising introducing a polynucleotide construct as described herein into a fungus.
  • Techniques for introducing exogenous polynucleotides into many fungi are known in the art, e.g., U.S. Patents 5,252,726 and 5,070,020.
  • Transformed fungi can be cultured by techniques known to those skilled in the art. Such fungi can be used to introduce a nucleic acid encoding a polypeptide into other fungal strains, to transfer the nucleic acid to other species or for further selection of other desirable traits.
  • a suitable group of fungi with which to practice the invention include fission yeast and budding yeast, such as Saccharomyces cereviseae, S. pombe, S. carlsbergeris and Candida albicans. Filamentous fungi such as AspergUlus sp. and Penicillium sp. are also useful.
  • eukaryotic organisms useful in the invention include animal cells (e.g., insects such mosquitoes and flies; fish; and non-human mammals such as rodents, bovines and porcines) that contain an exogenous polynucleotide that encodes a polypeptide of the invention.
  • animal cells e.g., insects such mosquitoes and flies; fish; and non-human mammals such as rodents, bovines and porcines
  • Such techniques typically involve generating a plurality of animals whose genomes can be screened for the presence or absence of the transgene.
  • a transgene can be introduced into a non-human mammal by pronuclear microinjection (U.S. Patent No.
  • mice retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad. Sci. USA, 82:6148, 1985), gene targeting into embryonic stem cells (Thompson et al., Cell, 56:313, 1989), electroporation of embryos (Lo, Mol. Cell. Biol., 3:1803, 1983), and transformation of somatic cells in vitro followed by nuclear transplantation (Wihnut et al., Nature, 385(6619):810-813, 1997; and Wakayama et al., Nature, 394:369-374, 1998).
  • suitable genetic backgrounds for use in making founder lines include, without limitation, C57B6, SJL/J, FVB/N, 129SV, BALB/C, C3H, and hybrids thereof.
  • modulated gene expression in plants can alter seed development, seed yield, seed composition, endosperm development, embryo development, cotyledon development, seed size, flowering time, plant size, leaf size, leaf shape, plant fertility, apical dominance, floral organ identity, root development, senescence, or organ composition.
  • cell type-specific expression of chimeric polypeptides can cause somatic embryogenesis.
  • expression of a heterologous polypeptide can cause fertilization independent seed development.
  • Such plants include, for example, plants in which certain genes that are otherwise transcriptionally inactive become transcriptionally active.
  • Polypeptides and polynucleotides that are homologous to the polypeptide and/or polynucleotides of the invention can be identified using database search algorithms and genomic databases. Such algorithms can be used in combination with other algorithms that can predict protein folding and putative three-dimensional protein structures and/or putative functional action by.
  • a number of source databases are available that contain either genomic sequences and/or a deduced amino acid sequence for use with the invention in identifying or determining the activity encoded by a particular polynucleotide or to identify homologs of the polynucleotides and/or polypeptides of the invention.
  • sequence database e.g., GenBank, PFAM or ProDom
  • sequence database e.g., GenBank, PFAM or ProDom
  • the databases can be specific for a particular organism or a collection of organisms.
  • sequence alignment methods include, for example, BLAST (Altschul et al., 1990), BLITZ (MPsrch) (Sturrock & Collins, 1993), and FASTA (Person & Lipman, 1988).
  • the probe sequence (e.g., a mutated SEQ ID NO: 2) can be the full length or a length that comprises a mutated amino acid as described herein, and will be recognized as homologous based upon a threshold homology value.
  • the sequence used to probe the database can be a fragment of the full-length sequence (e.g., a fragment comprising amino acid 26-38 of SEQ ID NO:2) or the full-length sequence.
  • the percent identity is determined based upon a comparison to the full sequence and not a fragment of the sequence.
  • the threshold value may be predetermined, although this is not required.
  • the threshold value can be based upon the particular polynucleotide length. To align sequences a number of different procedures can be used. Typically, Smith- Waterman or Needleman- Wunsch algorithms are used. However, faster procedures such as BLAST, FASTA, PSI- BLAST can be used.
  • optimal alignment of sequences for aligning a comparison window may be conducted by the local homology algorithm of Smith (Smith and Waterman, Adv Appl Math, 1981; Smith and Waterman, J Theor Biol, 1981; Smith and Waterman, J Mol Biol, 1981; Smith et al., J Mol Evol, 1981), by the homology alignment algorithm of Needleman (Needleman and Wuncsch, 1970), by the search of similarity method of Pearson (Pearson and Lipman, 1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis., or the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin, Madison, Wis.), or by inspection, and the best alignment (i.e., resulting in the highest percentage of homology over the comparison window) generated by the various methods is selected.
  • the similarity of the two sequence i.e., the probe sequence and the
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences (i.e., probe sequences) are compared.
  • probe and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the probe sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions (e.g., nucleotides or amino acids in a polynucleotide or polypeptide, respectively) selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a probe sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the comparison window can be the full-length sequence of a polypeptide or polynucleotide of the invention.
  • One example of a useful algorithm is BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc.
  • the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0).
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. ⁇ atl. Acad. Sci. USA 90:5873, 1993).
  • One measure of similarity provided by BLAST algorithm is the smallest sum probability (P( ⁇ )), which provides an indication of the probability by which a match between two nucleotide sequences would occur by chance.
  • P( ⁇ ) the smallest sum probability
  • a probe sequence is considered similar to a references sequence if the smallest sum probability in a comparison of the probe sequence to the reference sequence is less than about 0.2, typically less than about 0.01, and more commonly less than about 0.001.
  • Sequence homology or identity means that two polynucleotide sequences are homologous (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison.
  • a percentage of sequence identity or homology is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid bases (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence homology.
  • substantial homology denotes a characteristic of a polynucleotide sequence, wherein the polynucleotide comprises a sequence having at least 60 percent sequence homology, typically at least 70 percent homology, often 80 to 90 percent sequence homology, and most commonly at least 99 percent sequence homology as compared to a reference sequence of a comparison window of at least 25-50 nucleotides, wherein the percentage of sequence homology is calculated by comparing the reference sequence to the polynucleotide sequence which may include deletions or additions which total 20 percent or less of the reference sequence over the window of comparison.
  • Sequences having sufficient homology can the be further identified by any annotations contained in the database from which a reference sequence is derived, including, for example, species and activity information.
  • crtE GGPP synthase
  • crtB phytoene synthase
  • crtl phytoene desaturase
  • crtY ⁇ -end lycopene cyclase from Erwinia uredovora
  • Carotene synthases (crtM and crtB) were flanked by Xbal-Xbol sites, carotene desaturases (crtN and crtl) by XhoI-EcoRI sites, and crtE by EcoRI-NcoI sites.
  • cyclic carotenoids For production of cyclic carotenoids a two-plasmid expression system was used. Each operon that produces acyclic carotenoids was transferred from the pUC vector into pACYC184. To do this, an entire operon using a pair of primers (5'- AGCTGGGTCGACAGGTTTCCCGACTGGAAAGCG-3' (SEQ ID NO:28)) and (5'- ACCATAGTCGACGTGAAATACCGCACAGATGCG-3' (SEQ ID NO:29)) targeted outside the promoter and multicloning sites were used to amplify the segment.
  • primers 5'- AGCTGGGTCGACAGGTTTCCCGACTGGAAAGCG-3' (SEQ ID NO:28)
  • ACCATAGTCGACGTGAAATACCGCACAGATGCG-3' SEQ ID NO:29
  • PCR product was then digested and cloned into the Sail site of pACYC184, resulting in pAC- crtM-crtN-crtE.
  • pAC-crtM-crtl-crtE and others were constructed.
  • Carotene cyclase genes (Erwinia crtY and lettuce dy4) were subcloned into the EcoRI-NcoI sites of pUC, resulting in pUC-crtT and p ⁇ C-dy4.
  • the upper phase containing the carotenoids was dried with anhydrous MgSO 4 and concentrated in a rotary evaporator.
  • An aliquot of the extract was passed through a Spherisorb ODS2 column (250 x 4.6 mm, 5 mm Waters, Milford, MA) and eluted with an acetonitrile-isopropanol mixture (85:15 or 80: 20 v/v) at a flow rate of 1 ml/min using an Alliance-HPLC system (Waters) equipped with a photodiode array detector.
  • Mass spectra were obtained using a Series 1100 LC/MSD (Hewlett-Packard/Agilent, Palo Alto, CA) coupled with an atmospheric pressure chemical ionization (APCI) interface.
  • APCI atmospheric pressure chemical ionization
  • flanking crtN were designed to amplify the 1.6-kb gene by PCR under mutagemc conditions: 5 U AmpliTaq (100 ml total volume); 20 ng template (pUC-crtM-crtN-crtE); 50 pmol each primer; 0.2 mM each dNTP; 5.5 mM MgCl 2 .
  • mutagenic libraries were made using three different MnCl 2 concentrations: 0.1 mM, 0.05 mM, and 0.02 mM.
  • the temperature cycling scheme was 95 °C for 4 minutes followed by 30 cycles of (95 °C (30 sec), 52 °C (30 sec), 72 °C (2 min)), followed by a final stage of 72 °C for 10 minutes.
  • PCR yields for the 1.6-kb amplified fragment were 5 mg, corresponding to an amplification factor of ca. 500 or 9 effective cycles.
  • the PCR product from each library was purified using a Zymoclean gel-purification kit (Zymo Research, Orange, CA) followed by digestion with Xhol and EcoRI.
  • PCR products were purified, digested, and ligated as described above into the desaturase site of pUC-crtM-crtl-crtE, resulting in four pUC-crt - crtI -crtE libraries.
  • the ligation mixtures were transformed into E. coli XLl-Blue cells. Colonies were grown on LB-carbenicillin plates at 37 °C for 16 hours. Colonies were lifted onto white nitrocellulose membranes (Pall, Port Washington, NY) and visually screened for color variants after an additional 12-24 hours at room temperature. Selected colonies were picked and cultured overnight in 96-deep well plates, each well containing 0.5 ml liquid LB medium supplemented with carbenicillin (50 mg/ml). For the selected variants, the entire operons containing promoter region were subcloned into pACYC vector.
  • E. coli XL1 transformed with pUC-crtM-crtN show typical yellow color due to C 30 carotenoid production.
  • XL1 cells were transformed with pUC-crt - crtN-crtE, where crtE (GGPP synthase) from Erwinia uredovora and the gene products expressed, an intense red color was observed (Fig. 3A).
  • the spectrum of the acetone extract has a shoulder at 525nm, indicating the presence of a polyene made of 13 conjugated double bonds. Because the C 30 backbone only accommodates 11 conjugated double bonds, it appeared the cells were producing a >C 30 carotenoid.
  • CrtN is functional to some extent on C 40 carotenoids, but both in vitro and in vivo experiments showed that it is not more than a three- step desaturase in a C 0 pathway. Indeed, XL1 harboring pUC-crtB-crtN-crtE (with the C 40 synthase) are yellow (Fig. 3 A) and solely accumulate neurosporene. Therefore, the source of the red hue of XL1 cells transformed with pUC-crtM-crtN-crtE was believed to be a novel carotenoid with a non-C 30 /non-C 4 o backbone. [00147] CrtM produces 4-diapophytoene in the presence of GGPP.
  • E. coli cells harboring pXJC-crtE-crtB exclusively produced phytoene (C 40 ) (3, Fig. 4C), while E. coli carrying pUC-crtR accumulated undetectable amounts of carotenoids.
  • crtB appears to be a specific C 40 synthase.
  • E. coli cells transformed with pUC-crt produced only diapophytoene (C 30 ) (1, Fig. 4B).
  • HB101 was the best producer of 2, both in its production level (220-350 nmol (100-170 mg) C 35 carotenoid/g cell, dry mass) and in its proportion to the total carotenoids ( ⁇ 55%).
  • BL21 and BL21Gold also showed similarly good C 35 production, while XL1, DH5a, XLlOGold, and TOP10 cells accumulated 2 at a slightly lower level (35-45 mg/g cell, ca. 40% of total carotenoids).
  • JM109, JM101, and BL21(D ⁇ 3) turned out to be very poor and unstable producers of C 35 carotenoids.
  • the production of 2 was reproducible and insensitive to changes in other parameters.
  • 5B and 5C show the HPLC analysis of the extracted pigments from XL1 transformed with pVC-crtM-crtN-crtE or pUC-crtM-crtl-crtE, respectively. Both cells accumulated two carotenoids, 6 and 7, not found in cells without CrtE (Fig. 5A). Elution profiles, UN- visible spectra, and mass spectra confirmed that peak 6 is the fully-conjugated C 35 carotenoid, 4-apo-3'4'-didehydrolycopene, while peak 7 corresponds to a C 35 carotenoid with 11 conjugated double bonds. Because C 5 carotenoids are asymmetric, there are two possible C 35 structures, 4-apolycopene and 4-apo-3',4'- didehydro-7,8-dihydrolycopene (see Fig. 2).
  • the apparent step number of Crtl was slightly greater than that of CrtN in the C 35 pathway, and XL1 cells carrying pOC-crtM-crtl-crtE accumulated 6 as the main product.
  • Apparent in vivo activity of these desaturases in the C 5 pathway was high enough so that unconverted substrate (2) and products with lower desaturation step number did not accumulate.
  • C 30 desaturase CrtN showed higher (4-5) apparent step number in the C 35 pathway than in its native C 30 pathway (3-4 steps).
  • cells with carotenoid biosynthetic enzymes CrtM and CrtN develop intense red color in the presence of GGPP, due to the production of highly desaturated C 35 carotenoids (Fig. 3A).
  • Both CrtN and Crtl are 4-5 step desaturases in the C 35 pathway and therefore accumulate acyclic C 35 carotenoids with either 11 (for 6) or 13 (for 7) conjugated double bonds.
  • directed evolution was used to alter the step number of these desaturases in the pathway.
  • Each desaturation step extends the chromophore by two double bonds, providing a large bathochromic shift and a basis for product-based color screening of desaturase variants with altered step number.
  • This approach has been used to alter the step number of Crtl from Erwinia and Rhodobacter (34), as well as CrtN from Staphylococcus in their respective natural pathways.
  • XL1 cells harboring pAC-crtM-N6A-crtE accumulated 15 at high level (Fig. 5D). When cyclases were coexpressed with this, 8 was efficiently converted into 12 (by crtY, Fig. 5H) and 14 (by Dy4, Fig. 5K), respectively.
  • XL1 harboring pAC-crtM-Il 3-crtE accumulates a high level of 7 (Fig. 5F), the direct precursor for 11 and 13.
  • CrtY and Dy4 11 (by crtY, Fig. 51) and 13 (by Dy4, Fig. 4L) were produced at high proportion, respectively.
  • C 30 pathway against the production of larger carotenoids is achieved by limiting the precursor pool (environment) and not by the synthase itself.
  • Asymmetry of carotenoid structures ensures greater molecular diversity in the C 35 pathway. Because C 35 carotenoids have asymmetrical backbone structures, each desaturation step along the backbone yields more than one product. Thus, acyclic C 35 carotenoids with 3, 5, 7, 9, 11, and 13 conjugated double bonds can take 1, 2, 3, 3, 2, and 1 (total 12) possible structures, respectively (Fig. 2), while C 40 has 9 possible structures for 6 different chromophore sizes.
  • Cyclization and other modifications of the backbone can further increase the number of possible C 35 carotenoids compared to symmetrical (natural) carotenoids.
  • the C 35 pathway is inherently more complex and explores a much larger 'structure space' than the symmetrical C 30 and C 40 pathways.
  • C 35 carotenoid-modifying enzymes are expected to be functional in the C 35 pathway, as were the four enzymes disclosed herein (CrtN, Crtl, CrtY, and Dy4).
  • C 35 carotenoid pathway products can be metabolized by additional C o or C 30 enzymes, which will further expand the diversity of novel carotenoids that can be generated.
  • the invention demonstrates that matching components enhances the discovery of novel chemicals. Due to their promiscuity, secondary metabolic enzymes can be expressed combinatorially to produce a very large number of chemical structures. The invention demonstrates that addition of a single enzyme, GGPP synthase, creates a whole new pathway to C 35 carotenoids.
  • carotenoid backbone structure Once a carotenoid backbone structure is created, downstream enzymes, either natural or engineered, can accept the new substrate, and whole series of novel carotenoids can be produced. With the action of carotenoid-modifying enzymes, including desaturases, cyclases, hydroxylases, and cleavage enzymes, on these new extended backbones, it should be possible to double or even triple the diversity of the carotenoid kingdom.
  • the invention further examined engineering the carotenoid synthase to accept longer diphosphate substrates. Using random mutagenesis and a functional complementation screen for C 40 synthase activity, however, single amino-acid substitutions in the C 30 synthase CrtM (F26L or F26S) were identified that confer the C 40 function. By repeating this experiment with a random mutant library that was free from mutation at F26, two more amino acid substitutions, W38C and E180G, were found that confer the same phenotype.
  • the invention demonstrates that the specificity of carotenoid synthase CrtM is controlled at the second (rearrangement) step of its two-step reaction.
  • the engineered synthase variants make previously unknown C 45 and C 50 carotenoid backbones (mono- and di-isopentenylphytoenes) from the appropriate C 20 and C 25 isoprenyldiphosphate precursors. With this ability to produce the new backbones in Escherichia coli comes the potential to generate whole series of novel carotenoids upon addition of carotenoid-modifying enzymes to the engineered pathway.
  • crtE GGDP synthase
  • crtB phytoene synthase
  • crtl phytoene desaturase
  • CrtM diapophytoene synthase
  • crtN diapophytoene desaturase
  • Bacillus stearothermophilus farnesyldiphosphate synthase was PCR-cloned from genomic DNA (ATCC #12980).
  • genes and promoters (lacP-crtN and lacP -crtE-crtl, respectively) were PCR- amplified and subcloned into the Sail site of pACYCl 84, resulting in pAC-crtN and pAC- crtl-crtE, respectively.
  • Carotene synthase genes (crtB and crtM) were cloned into the Xba VXho I site in pUC18 ⁇ mod, resulting in pUC-crtR and pUC-crt .
  • Plasmid pUC-BsFPSysiA was constructed by subcloning the Y81A mutant of BsFPS (followed by RBS) into EcoRI/Nc I site of pUCmod. CrtB or crtM were subcloned into the XbaUXh ⁇ l site of this, resulting in pXJC-crtB-BsFPS Y81 A and p ⁇ JC-crtM-BsFPS Y8 ⁇ A -
  • PCR-based site saturation/substitution mutagenesis was performed on F26 (TTT), W38 (TGG), and ⁇ 180 (GAA) using the ExSite method (Stratagene). Some site- directed mutants were obtained from the saturation mutagenesis library, but the majority were synthesized using individual primers with the appropriate codon at the targeted site. Double and triple mutants were constructed by repeated site-directed mutagenesis. Selected mutants were subcloned into to make (square bracket represents a crtM mutant).
  • Colonies were picked and cultured overnight in 96-well deep-well plates, each well containing 0.5 ml liquid LB medium supplemented with carbenicillin and chloramphenicol (50 ⁇ g/ml each), and were shaken for 12 h at 37°C. A portion (20 ⁇ L) from each preculture was inoculated into 2 ml of fresh TB culture. After being shaken for 36 h at 30°C, cells were harvested and extracted with acetone (1 ml). The highest peak (475 nm) in each " UN/vis spectrum was used to score C 40 activity, while 470 nm was used for C 30 activity. The values were determined from the average of 6 independent experiments.
  • Plasmids were transformed into HB 101 cells and grown on agar plates (LB) with carbenicillin (50 ⁇ g/ml) for 14-16 h. Fresh colonies were picked and inoculated into TB medium and shaken for 12 h at 37 °C. 0.5 ml of this preculture was inoculated into 150 ml TB medium (in 750 ml tissue culture flask, Falcon), and shaken at 30°C for 36-40 h. Wet cells were harvested from the culture and extracted with 20 ml acetone, transferred to 10 ml hexane, dried with anhydrous MgSO 4 , and concentrated in a rotary evaporator.
  • the molar quantities of each carotenoids were determined by comparing HPLC chromatogram peak area (at 286 nm) to that of a ⁇ -carotene standard (at 450 nm) and then multiplying by ⁇ b- car o t ene (138,900 cm ⁇ M '1 at 450 nm)/ --phytoene (49,800 at 286 nm) and then by their molecular weight. The molar quantities of carotenoids were then normalized to the dry cell mass of each culture.
  • Pigmentation level was determined from the peak height (at 475 nm) of the acetone extract.
  • C 30 synthase performance was evaluated from the pigmentation level of cells transformed with the genes for CrtM and S. aureus C 30 desaturase Crt ⁇ .
  • Functional CrtM variants lead to production of 4,4' -diapophytoene, which was quantified (470 nm) in the acetone extract. As shown in Fig.
  • CrtM generates 4,4'-diapophytoene (1) in two distinct steps: (i) abstraction of a diphosphate group from a prenyl donor, followed by 1-1 ' condensation of the donor and acceptor molecules, and (ii) rearrangement of the cyclic intermediate, followed by removal of a second diphosphate and a final carbocation quenching process (Fig. 8a).
  • This mechanism is virtually identical to that of squalene synthase (SqS), the first committing enzyme in cholesterol biosynthesis. Indeed, when deprived of NADPH, SqS produces 1 as the main product.
  • Carotene synthases are similar to SqS in sequence and predicted secondary structure Fig.
  • CrtM variants can also generate longer (C 45 and C5 0 ) carotenoid backbones when supplied with (C 25 DP) FGDP.
  • Isoprenyldiphosphates are ubiquitous building units for thousands of natural products and cell components. Different isoprenyldiphosphate synthases catalyze the consecutive condensation of C 5 units to produce a wide range of isoprenyldiphosphates ( o to C ⁇ 2o,ooo)- Isoprenyldiphosphate synthases with different product size distributions are known, and the molecular basis of their product size determination is well understood.
  • BsFDS Bacillus stearothermophilus
  • coli HBIOI cells harboring pUC-crtM wr BSFDS YSIA were observed to produce almost no carotenoids (Fig. 9b), while those harboring pUC-crtM wr BsFDS wt produced 1 at a high level (1.1 mg/g DCW, Fig. 9a). That no C 30 carotenoids are observed indicates that FDP is not supplied for C 30 carotenoid production, which can be attributed to its redirection toward the longer isoprenyldiphosphates, catalyzed by BsFDS.
  • MAW MX Y stands for CrtM variant with amino acid X and Y in position 26 and 38, respectively
  • MAW produced the highest levels of 4 (ca. 130 ⁇ g/g dry cell weight, DCW) and 5 (78 ⁇ g/g DCW) (Fig. 10).
  • Combining mutations at positions 26 and 38 sometimes decreased the total carotenoid production.
  • HB101 cells harboring PUC-MA A -BSFPS YS produced less carotenoids than cells with PUC-M A W-BSFPSYSIA-
  • the extent of decrease was negligible for 4 and 5, while it was significant for 2 and 3.
  • E 180 in CrtM is positioned outside the reaction pocket, closer to where the first half-reaction occurs (Fig. 7b). At this position, Gly was the only amino acid that allowed CrtM to exhibit C 0 synthase activity.
  • addition of E180G positively affected performance in both the C 30 and C 40 contexts (Fig. 11). Because E180G increases overall activity (Fig. 11), and because it is far from F26 or W38 (Fig. 8b), it is anticipated that introduction of E180G to M AA would enhance carotenoid production without altering the preference for larger (C 4 5/C 50 ) structures. Indeed, the highest production of 4 (215 ⁇ g/g DCW) and 5 (147 ⁇ g/g DCW) was attained with HB101 cells harboring pUC-M ⁇ -e- BsFPS ⁇ 81 A (Fig. 10).
  • crtE GGDP synthase
  • crtB phytoene synthase
  • crtl phytoene desaturase
  • CrtM diapophytoene synthase
  • crtN diapophytoene desaturase
  • Bacillus stearothermophilus farnesylpyrophosphate synthase was PCR cloned from genomic DNA according to the literature.
  • AmpliTaq polymerase (Perkin-Elmer, Boston, MA) was used for mutagenic PCR, while Vent polymerase (New England Biolabs, Beverly, MA) was used for cloning PCR. All chemicals and reagents were of the highest available grade.
  • CrtN were subcloned into EcoRI/Nc ⁇ l site of pUCmodll, resulting in the pUC- crtN.
  • CrtB was removed from previously constructed pUC-crtE-crtB-crtl, resulting in the p ⁇ C-crtE-crtl.
  • genes and promoters la P-crtN and lacP-crtE-crtl, respectively) were PCR-amplified and subcloned into S ⁇ K site of pACYC184, resulting in pAC-crtNor pAC-crtl-crtE, respectively.
  • Carotene synthase genes (crtB and crtM) were cloned into the Xba VXho I site in pUC18 ⁇ mod, resulting in the pUC-crtR and pUC-crtM, respectively.
  • pUb-crtM was constructed by removing BsaXl site (locating upstream of lac promoter) from pUC-crtM.
  • PCR mutagenesis of crtM gene except F26 was carried out as follows.
  • a pair of primers, 5'-GAACGTGTTTTTGTGGATAAGAGG-3' (S ⁇ Q ID NO:33) and 5'- GATGAACGTGTTTTTTTGCGCAGACCG-3' (S ⁇ Q ID NO:36), flanking crtN were designed to amplify the 0.9kb gene by PCR under mutagenic conditions: 5 U AmpliTaq (100 ⁇ l total volume); 15 ng template ( ⁇ b-crtM ins , 3.4kb); 50 pmol each primer; 0.2 mM each d ⁇ TP; 5.5 mM MgCl 2 .
  • mutagenic libraries were made using four different MhCl 2 concentrations: 0.2mM, 0.1 mM, 0.05 mM, and 0.02 mM.
  • the cycling scheme was 95 °C for 2 minutes followed by 30 cycles of (95 °C (30 sec), 52 °C (30 sec), 72 °C (1 min)), followed 72 °C for 10 minutes. This yielded 5 ⁇ g of the 0.9kb fragment, corresponding to an amplification factor of ca. 1,000 or 10 effective cycles.
  • the PCR product from each library was purified using a Zymoclean gel-purification kit (Zymo Research, Orange, CA) followed by digestion with.-Y7.oI andXbal.
  • the fragments were ligated into theXhol-Xba site of pUb- crtMi ns resulting in pUC- [crtMi ns ] libraries (square brackets indicate the randomly- mutagenized gene).
  • the ligation mixtures were transformed into XLlOGold (Stratagene) to yield 11,000-29,000 colony-forming units (cfu).
  • Super-coiled library plasmid DNA was obtained by overnight culture of transformation and miniprep (ca. 5ug). These plasmid mixtures were digested with BsaXl (8 units) for 5h at 37 °C. After purification, this reaction mixture was then subjected to intra-plasmid ligation.
  • the reaction mixtures were directly transformed into E. coli XLl -Blue cells harboring pOC-crtE-crtl for screening C 0 synthase function.
  • PCR-based site saturation/substitution mutagenesis was conducted for each of F26 (TTT), W38 (TGG), and ⁇ 180 (GAA).
  • TTT F26
  • W38 TGG
  • GAA ⁇ 180
  • Some are obtained from this saturation library, but majority of site-directed mutants were synthesized using individual primers with appropriate codon at the targeted sites.
  • Double mutants and triple mutants were constructed by repeated site-directed mutagenesis.
  • CrtM and its variants in pUCNm were transformed into XLl cells harboring pAC-crtE-crtl.
  • C 30 synthase activity was evaluated by transforming pUC-crt (or pUC- [crtM]) into XLl harboring pAC-crtN Colonies were lifted onto white nitrocellulose membranes (Pall, Port Washington, NY) and placed at room temperature for an additional 12-24 hours. Colonies were picked and cultured overnight in 96-deep well plates, each well containing 0.5 ml liquid LB medium supplemented with carbenicillin and chloramphenicol (50 ⁇ g/ ml each) and were shaken for 12h at 37 °C. A portion (20 ⁇ L) from each preculture were then inoculated in 2ml of fresh media.
  • Plasmids were transformed in HB101 and grown on agar plate (LB) with carvenicillin (50 ⁇ g/ml) for 14-16h. Fresh colonies were picked and inoculated in TB media and shaken for 12h at 37 °C. 0.5ml of this preculture was inoculated into 150ml TB media (in 175cm 2 culture flask, Falcon), and shaken at 30 °C for 36h. Wet cells were harvested from the culture and extracted with 20 ml acetone, transferred to 10 ml hexane, dried with anhydrous MgSO 4 , and concentrated in a rotary evaporator.
  • E. coli cells By expressing Erwinia CrtE, CrtB, and Crtl, E. coli cells exhibit pink color due to the production of lycopene, an acyclic C 40 carotenoid. Similarly, expression of Staphylococcus CrtM and CrtN results in characteristic yellow pigmentation by accumulating C 30 carotenoids (4,4'-diapolycopene and 4,4'-diaponeurosporene). This allowed for the screening of synthase activity in the context of both the C 40 and C 30 pathways in a high- throughput manner.
  • PCR mutagenesis was conducted in four different conditions, resulting in pUC-/crtM7- nS A-D- F° r eacn .
  • supercoiled plasmid library DNA was obtained by subcloning into pUb vector, followed by transformation into XL10 Gold cell.
  • a fraction of the supercoiled plasmid library DNA was subj ected to the BsaXI treatment to remove the insert, resulting in the three base 3 '-sticky ends (TTC) at both sides.
  • the reading frame of crtM was recovered by intra-molecular ligation process.
  • variants with mutation(s) at F26 are selectively removed from the pool due to the specificity of T4 DNA ligase (Fig. 12a).
  • the ligation mixture was gel purified and further treated withAfHl. Because insert has a unique site for this restriction enzyme, possible undigested DNA can be further removed here.
  • the resulting mixture, A ⁇ D was used for screening for C 40 activity.
  • E180X library showed pink (weak) color, indicating only a few amino acids (probably glycine alone) can positively contribute to the C 0 synthase activity.
  • E180G, E180A, E180N A clone containing CrtM variant with mutation E180G performed similarly to C ⁇ , further confirming that E180G, not N156G, is the mutation that provides C 40 function.
  • E180A and El 80N were not different from wild-type CrtM.
  • the phenylalanine at position 26 was substituted with 11 different amino acids without charge groups by PCR-based site-directed mutagenesis.
  • each of these variants was independently tested for performance in the C 30 and C 40 contexts.
  • each mutant was transformed into XLl harboring pAC-crtE-crtl and grown in TB media (2mL) at 30 °C.
  • Pigmentation level was determined by the peak height (at 475nm) of the acetone extract.
  • C 0 synthase performance was evaluated by the pigmentation level, which was defined in XLl harboring pAC-crtN (at 470nm).
  • Figure 3 a shows the relationship between C 40 synthase activity and the van der Waals volume of the amino acid at position 26.
  • SqS and CrtM catalyze the 1-1' condensation of two FDP units by an identical mechanism, except that SqS yields the reduced form of the condensation product (squalene) in the presence of NADPH.
  • SqS produces mainly 4,4' -diapolycopene (dehydrosqualene) with other related byproducts.
  • Most of the structurally characterized isoprenoid enzymes, including farnesyldiphosphate synthase, squalene synthase, and terpene cyclases share the same fold.
  • the condensation reaction catalyzed by SqS proceeds in two distinct steps.
  • the first half- reaction generates the stable intermediate presqualene pyrophosphate (PSPP), which forms upon abstraction of a pyrophosphate group from a prenyl donor, followed by 1- 1 ' condensation of the donor and acceptor molecules.
  • PSPP stable intermediate presqualene pyrophosphate
  • the intermediate undergoes a complex rearrangement followed by a second removal of pyrophosphate and a final carbocation quenching process.
  • Squalene synthases catalyze the additional reduction of the central double bond of dehydrosqualene by NADPH to form squalene, a reaction not performed by carotene synthases, but produce the same product in its absence.
  • the second half-reaction is largely prevented from going to completion by bulky residues (such as Phe at position 26 and Trp at position 38) which sterically inhibit intermediate rearrangement.
  • bulky residues such as Phe at position 26 and Trp at position 38
  • the second half reaction is permitted to proceed, and phytoene is produced.

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Abstract

L'invention concerne de nouvelles voies de caroténoïde, de nouveaux caroténoïdes et des procédés permettant de les fabriquer et de les utiliser. Les caroténoïdes, les voies et les procédés servent à générer une variété de couleurs et de pigments à usage industriel in vitro et aux fins de variations de couleurs in vivo dans les plantes.
PCT/US2003/017775 2002-06-04 2003-06-04 Synthese du carotenoide Ceased WO2003101184A2 (fr)

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US7863030B2 (en) 2003-06-17 2011-01-04 The California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US8026085B2 (en) 2006-08-04 2011-09-27 California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US8252559B2 (en) 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US8802401B2 (en) 2007-06-18 2014-08-12 The California Institute Of Technology Methods and compositions for preparation of selectively protected carbohydrates
EP2977462A1 (fr) * 2014-07-25 2016-01-27 Phycosource Production in vivo d'un complexe caroténoïde-protéine recombinant
US9540700B2 (en) 2006-05-12 2017-01-10 Monsanto Technology Llc Methods and compositions for obtaining marker-free transgenic plants
KR101860648B1 (ko) 2016-12-14 2018-05-23 전남대학교산학협력단 C35 카로티노이드 생산능을 갖는 코리네박테리움 글루타미컴 재조합 균주 및 이을 이용한 c35 카로티노이드 생산방법
EP3498836A4 (fr) * 2016-08-10 2020-04-29 Ajinomoto Co., Inc. Procédé de production d'acide l-aminé
WO2021091862A1 (fr) * 2019-11-06 2021-05-14 L.E.A.F. Holdings Group Llc Compositions de caroténoïdes et leurs utilisations
WO2021207676A1 (fr) * 2020-04-09 2021-10-14 L.E.A.F. Holdings Group Llc Procédés de synthèse de caroténoïdes
CN116286899A (zh) * 2023-05-12 2023-06-23 昆明理工大学 一种NADH激酶基因RkNADHK1及其应用
US12458597B2 (en) 2018-05-03 2025-11-04 L.E.A.F. Holdings Group Llc Carotenoid compositions and uses thereof

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US5429939A (en) * 1989-04-21 1995-07-04 Kirin Beer Kabushiki Kaisha DNA sequences useful for the synthesis of carotenoids
JP3376838B2 (ja) * 1996-11-05 2003-02-10 トヨタ自動車株式会社 プレニル二リン酸合成酵素

Cited By (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7863030B2 (en) 2003-06-17 2011-01-04 The California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US8343744B2 (en) 2003-06-17 2013-01-01 The California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US8741616B2 (en) 2003-06-17 2014-06-03 California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US9145549B2 (en) 2003-06-17 2015-09-29 The California Institute Of Technology Regio- and enantioselective alkane hydroxylation with modified cytochrome P450
US9540700B2 (en) 2006-05-12 2017-01-10 Monsanto Technology Llc Methods and compositions for obtaining marker-free transgenic plants
US11629357B2 (en) 2006-05-12 2023-04-18 Monsanto Technology, Llc DNA constructs for obtaining marker-free transgenic plants
US10240165B2 (en) 2006-05-12 2019-03-26 Monsanto Technology Llc Methods and compositions for obtaining marker-free transgenic plants
US8026085B2 (en) 2006-08-04 2011-09-27 California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US8252559B2 (en) 2006-08-04 2012-08-28 The California Institute Of Technology Methods and systems for selective fluorination of organic molecules
US8802401B2 (en) 2007-06-18 2014-08-12 The California Institute Of Technology Methods and compositions for preparation of selectively protected carbohydrates
EP2977462A1 (fr) * 2014-07-25 2016-01-27 Phycosource Production in vivo d'un complexe caroténoïde-protéine recombinant
WO2016012618A1 (fr) * 2014-07-25 2016-01-28 Phycosource Production in vivo d'un complexe caroténoïde-protéine de recombinaison
US11198894B2 (en) 2016-08-10 2021-12-14 Ajinomoto Co., Inc. Method of producing an l-amino acid involving a carotenoid biosynthesis enzyme
EP3498836A4 (fr) * 2016-08-10 2020-04-29 Ajinomoto Co., Inc. Procédé de production d'acide l-aminé
KR101860648B1 (ko) 2016-12-14 2018-05-23 전남대학교산학협력단 C35 카로티노이드 생산능을 갖는 코리네박테리움 글루타미컴 재조합 균주 및 이을 이용한 c35 카로티노이드 생산방법
US12458597B2 (en) 2018-05-03 2025-11-04 L.E.A.F. Holdings Group Llc Carotenoid compositions and uses thereof
WO2021091862A1 (fr) * 2019-11-06 2021-05-14 L.E.A.F. Holdings Group Llc Compositions de caroténoïdes et leurs utilisations
WO2021207676A1 (fr) * 2020-04-09 2021-10-14 L.E.A.F. Holdings Group Llc Procédés de synthèse de caroténoïdes
CN116286899A (zh) * 2023-05-12 2023-06-23 昆明理工大学 一种NADH激酶基因RkNADHK1及其应用
CN116286899B (zh) * 2023-05-12 2024-03-15 昆明理工大学 一种NADH激酶基因RkNADHK1及其应用

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