Carotenoid Biosynthesis and Biotechnological Application

Archives of Biochemistry and Biophysics Vol. 385, No. 1, January 1, pp. 4 –12, 2001 doi:10.1006/abbi.2000.2170, available online at http://www.idealibrary.com on

MINIREVIEW Carotenoid Biosynthesis and Biotechnological Application Gerhard Sandmann 1 Biosynthesis Group, Botanisches Institut, J. W. Goethe Universita¨t Frankfurt, P.O. Box 111932, 60054 Frankfurt, Germany

Received August 8, 2000; published online December 8, 2000

REACTION SEQUENCE TO CYCLIC CAROTENOIDS A survey is given on the carotenoid biosynthetic pathway leading to ␤-carotene and its oxidation products in bacteria and plants. This includes the synthesis of prenyl pyrophosphates via the mevalonate or the 1-deoxyxylulose-5-phosphate pathways as well as the reaction sequences of carotenoid formation and interconversion together with the properties of the enzymes involved. Biotechnological application of this knowledge resulted in the development of heterologous carotenoid production systems using bacteria and fungi with metabolic engineered precursor supply and crop plants with manipulated carotenoid biosynthesis. The recent developments in engineering crops with increased carotenoid contents are covered. © 2001 Academic Press

Carotenoids are synthesized in bacteria, algae, fungi, and green plants. In addition to very few bacterial carotenoids with 30, 45, or 50 carbon atoms, C 40carotenoids represent the majority of the more than 600 known structures. Especially bacterial carotenoids are most diverse. Hydroxy groups at the ionone ring may be glycosilated or carry a glycoside fatty acid ester moiety. Furthermore, carotenoids with aromatic rings or acyclic structures with different polyene chains and typically 1-methoxy groups can be found (Fig. 1A). Typical fungal carotenoids possess 4-keto groups, may be monocyclic, or possess 13 conjugated double bonds (Fig. 1B). 3-Hydroxy ␣- and ␤- as well as 5,6-epoxy ␤-carotene derivatives (Fig. 1C) are abundant in chloroplast of some algal groups and green plants (see Ref. (1) for an extensive compilation).

The mevalonate pathway to prenyl pyrophosphates. In fungi, carotenoids are derived via the mevalonate biosynthetic pathway. The reaction sequence is shown in Fig. 2A. The metabolite mevalonate is formed from acetyl-CoA via hydroxymethylglutaryl-CoA. The subsequent steps involve two kinase reactions yielding mevalonic acid pyrophosphate which then is decarboxylated to isopentenyl pyrophosphate (IPP) 2 (see Ref. 1 for details of this pathway). IPP is the building unit for the synthesis of terpenoids. The 1-deoxyxylulose-5-phosphate pathway to prenyl pyrophosphates. In bacteria and plastids of plants, formation of prenyl pyrophosphates which are the precursors of carotenoids proceed via an alternative pathway (2, 3). This novel pathway is referred to either as the 1-deoxyxylulose-5-phosphate pathway since this is the first intermediate or as the 2-C-methyl-D-erythritol-4-phosphate pathway since this is the first product which is converted to prenyl pyrophosphate exclusively. Not all of the reaction steps are elucidated to date. Initially, a C2unit from pyruvate is condensed to glyceraldehyde by a thiamin-dependent 1-deoxyxylulose-5-phosphate synthase (Fig. 2B). Then, the product is converted to 2-Cmethyl-D-erythritol by 1-deoxyxylulose-5-phosphate reductoisomerase. The following reactions involve the formation of 4-diphosphocytidyl-2-C-methylerythritol in a CTP-dependent reaction (4) and phosphorylation at position 2 by ATP (5). Subsequently, CMP is released and a 2-C-methyl-D-erythritol 2,4-cyclodiphosphate is formed (6). This product may be the branching point for independent routes to IPP and dimethylallyl pyrophosphate (DMAPP). For example, cleavage of phosphate from C-2 may result either in a 1-keto or 3-keto pyrophospate 2

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To whom correspondence should be addressed.

Abbreviations used: IPP, isopentenyl pyrophosphate; DMAPP, dimethylallyl pyrophosphate; GGPP, geranyl geranyl pyrophosphate; CCS, capsanthin– capsorubin synthase. 0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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FIG. 1.

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Carotenoids typical for bacteria (A), fungi (B), and plants including green algae (C).

intermediate. However, the final steps to IPP and DMAPP are still open. Evidence is accumulating that the pathways of prenyl pyrophosphate synthesis differ in prokaryotes and eukaryotes. In Synechocystis the formation of IPP and DMAPP must be parallel as indicated in Fig. 3A since no IPP isomerase gene exists in this species and IPP isomerase activity is absent (7). The situation is similar to Escherichia coli where a parallel synthesis of IPP and DMAPP has been established (8). Nevertheless, an IPP isomerase is present in E. coli and its overexpression leads to increased formation of carotenoids in the corresponding transformant (see Genetic Manipulation of Carotenoid Synthesis). Since IPP isomerase is present in plant plastids and its activity is the limiting step of carotenoid synthesis in etioplasts (9), it may be concluded that IPP is the major or only product of the 1-deoxyxylulose-5-phosphate pathway. Then DMAPP should originate from isomerization of IPP. Since the possibility of parallel formation of DMAPP cannot be excluded yet, this reaction is indicated in Fig. 3A with a question mark. Formation and interconversion of carotenoids. From prenyl pyrophosphates of different chain lengths, specific routes branch off into various terpenoid end products. The prenyl pyrophosphates are formed by different prenyl transferases after isomerisation of IPP to DMAPP by successive 1⬘– 4 condensations with IPP molecules. The carbon bond is formed between the C-4 of IPP and the C-1 of the allylic co-substrate. In the case of geranylgeranyl pyrophosphate (GGPP) synthesis (Fig. 3B) this is dimethylallyl pyrophosphate in

higher plants (10) or farnesyl pyrophosphate in many bacteria. In all prenyl transferase reactions the mechanism of this head-to-tail joining is basically the same. Phytoene synthesis is the first committed step in C 40-carotenoid biosynthesis (Fig. 4). It proceeds as a head-to-head condensation of two molecules of GGPP forming a cyclopropylcarbonyl between carbon 1 of the first and carbons 2 and 3 of the second GGPP molecule yielding prephytoene pyrophosphate as an intermediate. In plants, 15-cis phytoene is then formed by elimination of the pyrophposphate group and stereospecific proton abstraction. Subsequently, four dehydrogenation steps are carried out in the conversion of phytoene to lycopene which is the plant carotene with the highest number of conjugated double bonds. Two completely unrelated types of phytoene desaturases exist (11). The enzyme found in bacteria (except cyanobacteria) and in fungi catalyzes the entire four-step desaturation process of phytoene to lycopene (12). The plant-type phytoene desaturase from cyanobacteria, algae, and plants carries out a two-step desaturation reaction with different ␨-carotene stereoisomers as reaction products (13). Lycopene is the regular substrate for cyclization reactions. Two types of ionone rings are formed: the ␤-ring as an end group of both sides of ␤-carotene and the ⑀-ring at one side of ␣-carotene in addition to a ␤-ring. The mechanisms of cyclization which involve proton attack at C-2 and C-2⬘ of lycopene and addition of the C-1 carbocation to the C-5,6 double bond is substantially supported by D 2 O-labeling ex-

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FIG. 2. Synthesis of isopentenyl and dimethylallyl pyrophosphate via mevalonate (A) or via 1-deoxyxylulose-5-phosphate (B).

periments followed by determination of the position of the deuteriated carbon at the ␤- or ⑀-ionone ring. The resulting carbonium ion intermediate is stabilized by loss of a proton either from C-1 or C-4 to yield a ␤- or ⑀-ring, respectively (14). Formation of the ␤- or ⑀-ring is under different gene control. Consequently, we can assume that formation of ␤-carotene is catalyzed by a single enzyme, lycopene cyclase b. In case of ␣-carotene, two different enzymes, lycopene cyclase b and lycopene cyclase ⑀, are involved. Genetic complementation analysis demonstrated that the ⑀-cyclase forms only one ring whereas the ␤-cyclase introduces two rings into lycopene or an additional ring into ␦-carotene which is a lycopene molecule with one ⑀-end group (15).

Xanthophylls are enzymatically formed oxidation products of ␣- and ␤-carotene (Fig. 1). Typical oxo groups are hydroxy at C-3 of the ␤- and ⑀-ionone ring, epoxy at the 5,6 position of the ␤-ionone ring, and keto groups at position 4. BIOSYNTHESIS AND ENZYMATIC REACTIONS

In the past 10 years, the biochemistry of carotenogenesis and especially the cloning of carotenogenic genes has made considerable progress. To date, many genes for different carotenogenic reactions are known. Detailed information on their cloning, their structure, and their function can be found in several reviews (11, 16 –18). The availability of these genes was a great

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FIG. 3. Sequential or parallel formation of isopentenyl and dimethylallyl pyrophosphate in different organisms (A) and their conversion to geranylgeranyl pyrophosphate (B).

help in the characterization of enzymes of carotenogenic pathways and their regulation. In return, biochemical insights facilitated the cloning of novel carotenogenic genes. Enzymes involved in carotenoid biosynthesis are all membrane-associated or integrated into membranes. This feature together with their sensitivity against detergents which have to be used for solubilization and their very low abundance makes the isolation and purification rather difficult (11). This is the major reason why only a few have been purified from plant tissue. Many others have been overexpressed in a heterologous host in a way that highpressure cell breaking resulted in a soluble and enzymatically active form (19). Furthermore, gene ex-

pression allows the extension of the coding region by an affinity group, e.g., a 6xHis tag which facilitates purification of the expressed enzymes considerably. Phytoene synthase. This enzyme is encoded by the closely related bacterial crtB and eukaryotic psy genes. The crtB gene encoding phytoene synthase from the eubacterium Erwinia uredovora was overexpressed in E. coli (20). The phytoene formed in the enzymatic reaction was present in both a 15-cis and all-trans isomeric configuration. The essential cofactors required were ATP in combinations with either Mn 2⫹ or Mg 2⫹. Phytoene synthesis was inhibited by phosphate ions and squalestatin. In tomato, enzymatic studies of wild-type and mutant fruit in the green and ripening stage demon-

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FIG. 4. Biosynthesis of ␤-carotene and its oxidation products. The enzymes catalyzing the individual reactions were assigned according to the corresponding gene: CrtB, bacterial phytoene synthase; Psy, eukaryotic phytoene synthase; CrtI, bacterial phytoene desaturase; CrtP, cyanobacterial phytoene desaturase; Pds, eukaryotic phytoene desaturase; CrtQ, CrtI-related ␨-carotene desaturase; CrtQb, CrtPrelated ␨-carotene desaturase; Zds, CrtQ, eukaryotic ␨-carotene desaturase; CrtY, eubacterial lycopene cyclase; CrtYb and CrtYc, protein components of the heterodimeric lycopene cyclase from gram positive bacteria; Lcy-␤ eukaryotic lycopene cyclase-b; CrtZ, bacterial ␤-carotene hydroxylase; Bhy, eukaryotic ␤-carotene hydroxylase; Zep, zeaxanthin epoxydase; CrtW bacterial ␤-carotene ketolase; Bkt, eukaryotic ␤-carotene ketolase.

strated the existence of two different phytoene synthases, Psy-1 (21), encoded by the formerly called pTOM5 cDNA, and Psy-2 (22), which both mediate the formation of 15-cis phytoene. These enzymes are expressed organelle-specific. Psy-2 predominates in green tissue and is responsible for carotenoid synthesis in chloroplasts whereas Psy-1 is expressed during fruit ripening and contributes to carotenoid tomato fruit chromoplasts. The chromoplast enzyme has been isolated from ripe Capsicum annuum fruit (23). In addition, the phytoene synthase from Narcissus pseudonarcissus has been obtained after heterologous expression of the cDNA (24). The most detailed characterization was carried out with a purified preparation from green tomato fruit of

Psy-2 which provides information not only about its biochemical properties but also on its interaction with other carotenogenic enzymes (25). This work revealed the existence of a very close association between phytoene synthase and other proteins, particularly those involved in the synthesis of other terpenoids. This association may be important for the effective functioning of the enzymes in vivo, by enabling the channeling of biosynthetic precursors. Phytoene desaturase. Different phytoene desaturases are known. They differ in the number of desaturation steps and in their structures. Among them is the bacterial/fungal type encoded by crtI and the cyanobacterial/algal/plant type encoded by crtP or pds. Bacterial type phytoene desaturases. Phytoene desaturase from E. uredovora was expressed in E. coli (12). Purification was achieved after solubilization of the protein sequestered in inclusion bodies by urea treatment. Reactivation was possible by removal of the denaturant and dilution of the sample in the presence of dithiothreitol. It could be shown that FAD was involved as cofactor in the desaturation reaction of this bacterial type of phytoene desaturase. Plant type phytoene desaturase. In cyanobacteria and plants desaturation of phytoene to lycopene proceeds in two steps which are catalyzed by two individual enzymes, phytoene desaturase and ␨-carotene desaturase (11). Phytoene desaturase converts phytoene to ␨-carotene with phytofluene as an intermediate. The enyzme from Synechococcus was expressed and purified (26). The resulting 53-kDa membrane protein could be reactivated after lipid replenishment. Inhibition was observed by several bleaching herbicides. Substrates in addition to phytoene were phytofluene and 1,2-epoxy phytoene which were converted to ␨-carotene and the corresponding 1,2-epoxyde. The reaction was stimulated by NAD, NADP, and oxygen. The phytoene desaturase from C. annuum chromoplasts has been solubilized by detergent and purified to a polypeptide of 52 kDa with a reported FAD prosthetic group (27). The cofactor for the desaturation reaction remains obscure. The phytoene desaturase from N. pseudonarcissus chromoplasts is dependent on oxidized quinones as hydrogen acceptor (28). Regeneration of reduced quinone occurs with oxidation with molecular oxygen by an oxidoreductase, a homodimeric enzyme with a molecular mass of the subunit of 23 kDa (29). In leaves, a plastidic alternative oxidase involved in phytoene desaturation exists as exemplified for Arabidopsis thaliana (30, 31). A model has been proposed on the participation of this alternative oxidase and of the photosynthetic electron transport chain in different developmental and physiological stages. ␨-Carotene desaturases. Since the phytoene desaturases encode by crtP and pds synthesize ␨-carotene as

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end product, a second enzyme must exist for the formation of lycopene. This ␨-carotene desaturase catalyzes the final two desaturation steps to lycopene by introducing two double bonds at positions 7,8 of ␨-carotene and 7⬘,8⬘ of neurosporene. From cyanobacteria two structurally unrelated gens for ␨-carotene desaturase were cloned. One crtQ (formerly called zds) from Anabaena is related to the bacterial crtI gene, whereas the second crtQb (also called crtQ-2) from Synechocystis is quite similar to crtP. This gene is very closely related to the plant zds gene (32). ␨-Carotene desaturase CrtQ. The ␨-carotene desaturase CrtQ from Anabaena was expressed in E. coli, solubilized from the membranes by Chaps, and purified to homogeneity (33). Substrates for this enzyme apart from ␨-carotene are those carotenes which partially resemble the latter, like neurosporene and ␤-zeacarotene yielding lycopene and ␥-carotene, respectively, as reaction products. Also cis isomers like pro-␨-carotene were converted to the corresponding products. ␨-Carotene desaturase CrtQ-2/Zds. The desaturase Zds from the plant C. annuum was expressed in E. coli, purified, and biochemically characterized (34). Product analysis showed that different lycopene isomers are formed including substantial amounts of the all-trans form together with 7,7⬘,9,9⬘-tetracis prolycopene via the corresponding neurosporene isomers. Application of different geometric isomers as substrates revealed that the ␨-carotene desaturase has no preference for certain isomers and also revealed the nature of the isomers formed during catalysis. The hydrogen acceptors for the dehydrogenation of ␨-carotene and neurosporene are lipophilic quinones including plastoquinone. The authors proposed that similar to phytoene desaturation the plastidic alternative oxidase may be the terminal electron acceptor in ␨-carotene desaturation, accepting electrons from the plastoquinone cofactor. Lycopene cyclases. Two completely unrelated types exist in bacteria, fungi, and plants. One encodes the classical monomeric bacterial ␤-cyclase gene, crtY, which may be an ancestor of crtL, the gene for a ␤-cyclase in cyanobacteria. From crtL the plant ␤- and ⑀-cyclase genes developed (18). In gram positive bacteria two genes crtYc and crtYd are present (35, 36). They encode two proteins which have to interact as a heterodimer for lycopene ␤-cyclization. From this type of lycopene cyclase genes the fungal lycopene cyclase/ phytoene synthase fusion gene crtYB evolved (37). Lycopene ␤-cyclase CrtY from E. uredovora has been purified to homogeneity in an active state after expression in E. coli (38). In addition to its two-step reaction in which both sides of the lycopene molecule are cyclized to ␤-ionone rings with the monocyclic ␥-carotene

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as an intermediate, neurosporene as well as 1-hydroxylycopene were cyclized to ␤-zeacarotene and hydroxy␥-carotene, respectively. The cofactors involved in the reaction were either NADH or NADPH. Also the lycopene ␤-cyclase Lcy-b was expressed in E. coli, purified, and subjected to enzyme kinetic studies (39). Complementation studies with the ␤-lycopene cyclase from C. annuum demonstrated that this enzyme, like the ␤-cyclase from E. uredovora, is not specific for the cyclization of the ␺-end group as found in lycopene and at one end of neurosporene, but also is able to convert the 7,8-dihydro-␺-ends to 7,8-dihydro␤-end groups yielding 7,8,-dihydro-␤-carotene in addition to ␤-zeacarotene from neurosporene (40). In C. annuum fruit another enzyme with homology to lycopene ␤-cyclase was found. This enzyme also exhibits lycopene ␤-cyclase activity but its major catalytic activity is the formation of capsanthin and capsorubin from violaxanthin or antheraxanthin, respectively. This capsanthin– capsorubin synthase (CCS) is the only enzyme from a biosynthetic pathway leading to secondary carotenoids which has been purified (41). ␤-Carotene hydroxylase. Bacterial ␤-carotene hydroxylases, CrtZ, were characterized with respect to substrate specificity and reaction mechanism after expression in E. coli (42). The reactions depend on oxygen and are stimmulated by 2-oxoglutarate, ascorbate, and Fe 2⫹, which is typical for a dioxygenase reaction. Similar mechanistic properties were also found for bacterial ketolases encoded by crtW. Of the two distinct plant carotene hydroxylases which introduce a hydroxy group at position 3 of either an ⑀-ionone or a ␤-ionone end group, only the ␤-carotene hydroxylase has been purified (43). The enzyme originated from expression of a C. annuum cDNA in E. coli. The conversion of ␤-carotene via ␤-cryptoxanthin to zeaxanthin was strictly dependent on the presence of molecular oxygen. In addition, the catalytic activity was strictly dependent upon reduced ferredoxin which could not be replaced by NADPH. Treatment of purified hydroxylase with the iron chelators o-phenanthroline and 8-hydroxyquinoline strongly inhibit activity, indicating the presence of iron as a functional group. Zeaxanthin epoxidase. This enzyme introduces 5,6epoxy groups into a 3-hydroxy-␤-ionone end group. It was expressed in E. coli from a C. annuum cDNA (44). The recombinant protein acts specifically on ␤-cryptoxanthin, zeaxanthin, and antheraxanthin. The epoxidation reaction involved oxygen and reduced ferredoxin as reductant. Neoxanthin synthase. A cDNA encoding neoxanthin synthase has been cloned very recently from tomato (45). Based on mechanistic similarities between the neoxanthin synthase and capsanthin– capsorubin synthase reactions forming a transient carbocation

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during the opening of the cyclohexyl 5,6,-epoxides, oligonucleotides against CCS cDNA sequences were used to screen a neoxanthin synthase cDNA from a tomato library. This cDNA which encodes a 56-kDa plastidtargeted protein was expressed in E. coli. The resulting transformant was functional in converting violaxanthin into neoxanthin. GENETIC MANIPULATION OF CAROTENOID SYNTHESIS

The availability of a large number of carotenogenic genes makes it possible to modify and engineer the carotenoid biosynthetic pathways in microorganisms and plants. These molecular biology approaches aimed for carotenoid production in heterologous microorganisms, plants resistant to bleaching herbicides, and plants with enhanced nutritional value. Carotenoid production in noncarotenogenic hosts: Fungi. Carotenoids have been successfully synthesized in noncarotenogenic fungi. The first attempts have been made with Saccharomyces cerevisiae which was transformed with the carotenogenic genes from E. uredovora for the production of lycopene (46). Since then, Candida utilis has been developed systematically as a production host for lycopene, ␤-carotene, and astaxanthin (49). The exogenous carotenoid biosynthesis genes originating from the eubacterium E. uredovora and the marine bacterium Agrobacterium aurantiacum were modified based on the codon usage of C. utilis and expressed under the control of constitutive promoters derived from this host. The resultant yeast strains accumulated lycopene, ␤-carotene, and astaxanthin by directing the carbon flow into carotenoid production at the expense of ergosterol biosynthesis. Carotenoid formation could be further increased by metabolic engineering of the mevalonate pathway (48). Expression of the catalytic domain of hydroxymethylglutaryl-CoA reductase gene together with the disruption of the endogenous erg9 gene encoding squalene synthase yielded lycopene concentrations of 7.8 mg/g dry weight. E. coli. E. coli is a very convenient host for heterologous carotenoid production. Most of the carotenogenic genes from bacteria, fungi, and higher plants can be functionally expressed in this bacterium. Furthermore, plasmids belonging to different incompatibility groups with different antibiotic resistance markers are available. They can all be introduced simultaneously and combined in E. coli for carotenoid synthesis, allowing combinations of individual genes. By combination of genes from different organisms with different carotenoid biosynthetic branches, novel carotenoids not found in any other pathway can be synthesized. The potential of E. coli as a carotenoid production system has been reviewed recently (49). Carotenoid production

of E. coli can be increased by metabolic engineering of the supply of prenyl pyrophosphates as a precursor for carotenoid production. Transformation with the genes for overexpression of 1-deoxy-D-xylulose 5-phosphate synthase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase, and isopentenyl pyrophosphate isomerase stimulated carotenogenesis up to 3.5-fold to a final yield of 1.5 mg/g dry weight (50). Engineering of resistance to bleaching herbicides. The typical bleaching herbicides directly interact with phytoene desaturase of plants as the target enzyme. Resistance to these herbicides was engineered in tobacco by introducing the crtI gene from E. uredovora (51). Enzyme kinetic studies of the encoded phytoene desaturase showed that this enzyme is naturally resistant to bleaching herbicides. Since the endogenous tobacco phytoene desaturase is located in the thylakoid membrane, the 5⬘ region was fused to the sequence for a transit peptide and put under the control of the cauliflower mosaic virus 35S promoter. After transformation of tobacco, expression and processing of the corresponding protein could be demonstrated. The resulting tobacco plants were resistant not only to bleaching herbicides interfering with phytoene desaturase but also to inhibitors which affect ␨-carotene desaturase (52). Carotenoids in tomato fruit. In several crops the carotenoid content was manipulated by transfer of foreign carotenogenic genes. During lycopene deposition in tomato fruit ripening, phytoene synthase is the controlling enzyme (53). Therefore, this enzyme was the first target for the genetic manipulation of the carotenoid composition of tomato fruit (54). The constitutive high level expression of Psy1 in tomato plants resulted in carotenoid rich seed coats, cotyledons, and hypocotyls. However, hemizygous and homozygous plants were reduced in stature due to changes in the giberellic acid and abscisic acid contents. Manipulation of the desaturation activity in tomato (55) was achieved with the same plasmid containing the crtI gene from E. uredovora which was used for the generation of herbicide resistant tobacco (51). Expression of the gene did not change the amount of total carotenoids in transgenic tomato fruit but resulted in a threefold increase of the ␤-carotene content. The authors could demonstrate increased transcript levels of especially the ␨-carotene desaturase and the lycopene ␤-cyclase genes. Rapeseed. Overexpression of a phytoene synthase gene from E. uredovora extended with a plastid targeting sequence under the seed-specific napin promoter was a successful attempt to increase the carotenoid content of mature rapeseed up to 50-fold (56). In the transformant the embryos were bright orange compared to the green embryos in the control. In the trans-

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genic seeds, carotenoid concentrations of more than 1 mg/g fresh weight accumulated, consisting mainly of ␣and ␤-carotene. Concurrent to the enhanced carotenoid production, the chlorophyll and ␣-tocopherol contents decreased in the transformant. Rice endosperm. Rice was transformed with a plasmid containing plant phytoene synthase gene and a bacterial phytoene desaturase gene which should mediate the synthesis of lycopene from GGPP (57). Both reading frames were extended with transit sequences for the targeting of the endosperm plastids. They were under the control of the endosperm-specific glutelin and the constitutive cauliflower mosaic virus 35S promoter, respectively. Surprisingly, the accumulated carotenoids were lutein and zeaxanthin ␣- and ␤-carotene instead of lycopene. Thus, it was assumed that the lycopene ␤- and ⑀-cyclases and the ␤- and ⑀-hydroxylases must be induced in the transformant or constitutively expressed. Cotransformation with a lycopene ␤-cyclase containing plasmid increased the ␤-carotene content. Even then, the total amount of 1.6 ␮g/g carotenoid in the rice endosperm is only 1 to 2% of the carotenoid concentration in the transgenic rapeseed (56). CONCLUSION

The cloning of a great number of different carotenogenic genes in the past 10 years boosted the biochemical investigations of carotenogenic pathways and individual reactions in microorganisms and plants. In most cases, enzymes were characterized after their heterologous expression. This facilitated enzyme kinetic studies including substrate and product specificity and led to a better understanding of controlling steps in individual pathways and of their regulation. Based on this knowledge, targets could be selected for genetic manipulations of carotenoid biosynthetic pathways. This included microbial carotenoid production systems, transgenic plants conferring resistance against herbicides which affect carotenogenesis, and genetically modified crops with increased carotenoid accumulation. REFERENCES 1. Goodwin, T. W. (1980) The Biochemistry of the Carotenoids, Vol. I, Plants. 2nd ed., Chapman and Hall, London. 2. Rohmer, M. (1999) in Comprehensive Natural Product Chemistry, Isoprenoids Including Steroids and Carotenoids (Cane, D. E., Ed.), Vol. 2, pp. 45– 68, Pergamon Press, Oxford. 3. Lichtenthaler, H. K. (1999) Annu. Rev. Plant Physiol. Mol. Biol. 50, 47– 65. 4. Lu¨ttgen, H., Rohdich, F., Herz, S., Wungsintaweekul, J., Hecht, S., Schuhr, C. A., Fellermeier, M., Sagner, S., Zenk, M. H., Bacher, A., and Eisenreich, W. (2000) Proc. Natl. Acad. Sci. 97, 1062–1067.

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