Regulation of carotenoid biosynthesis in plants: evidence for a key role of hydroxymethylbutenyl diphosphate reductase in controlling the supply of plastidial isoprenoid precursors

Carotenoids are isoprenoid pigments that function as photoprotectors, precursors of the hormone abscisic acid (ABA), colorants and nutraceuticals. A major problem for the metabolic engineering of high carotenoid levels in plants is the limited supply of their isoprenoid precursor geranylgeranyl diphosphate (GGPP), formed by condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) units usually synthesized by the methylerythritol phosphate (MEP) pathway in plastids. Our earlier work with three of the seven MEP pathway enzymes suggested that the first reaction of the pathway catalyzed by deoxyxylulose 5-phosphate synthase (DXS) is limiting for carotenoid biosynthesis during tomato (Lycopersicon esculentum) fruit ripening. Here we investigate the contribution of the enzyme hydroxymethylbutenyl diphosphate reductase (HDR), which simultaneously synthesizes IPP and DMAPP in the last step of the pathway. A strong upregulation of HDR gene expression was observed in correlation with carotenoid production during both tomato fruit ripening and Arabidopsis thaliana seedling deetiolation. Constitutive overexpression of the tomato cDNA encoding HDR in Arabidopsis did not increase carotenoid levels in etioplasts. By contrast, light-grown transgenic plants showed higher carotenoid levels and an enhanced seed dormancy phenotype suggestive of increased ABA levels. The analysis of double transgenic Arabidopsis plants overproducing both the enzyme taxadiene synthase (which catalyzes the production of the non-native isoprenoid taxadiene from GGPP) and either HDR or DXS showed a twofold stronger effect of HDR in increasing taxadiene levels. Together, the data support a major role for HDR in controlling the production of MEP-derived precursors for plastid isoprenoid biosynthesis.     

Cloning,expression and characterization of a functional cDNA clone encoding geranylgeranyl diphosphate synthase of Hevea brasiliensis

Geranylgeranyl diphosphate (GGPP) synthase catalyzes the condensation of isopentenyl diphosphate (IPP) with allylic diphosphates to give (all-E)-GGPP. GGPP is one of the key precursors in the biosynthesis of biologically significant isoprenoid compounds. In order to examine possible participation of the GGPP synthase in the enzymatic prenyl chain elongation in natural rubber biosynthesis, we cloned, overexpressed and characterized the cDNA clone encoding GGPP synthase from cDNA libraries of leaf and latex of Hevea brasiliensis. The amino acid sequence of the clone contains all conserved regions of trans-prenyl chain elongating enzymes. This cDNA was expressed in Escherichia coli cells as Trx-His-tagged fusion protein, which showed a distinct GGPP synthase activity. The apparent K(m) values for isopentenyl-, farnesyl-, geranyl- and dimethylallyl diphosphates of the GGPP synthase purified with Ni(2+)-affinity column were 24.1, 6.8, 2.3, and 11.5 microM, respectively. The enzyme shows optimum activity at approximately 40 degrees C and pH 8.5. The mRNA expression of the GGPP synthase was detected in all tissues examined, showing higher in flower and leaf than petiole and latex, where a large quantity of natural rubber is produced. On the other hand, expression levels of the Hevea farnesyl diphosphate synthase were significant in latex as well as in flower.     

Identification of the GGPS1 genes encoding geranylgeranyl diphosphate synthases from mouse and human

E,E,E-Geranylgeranyl diphosphate (GGPP) is an important precursor of carotenoids and geranylgeranylated proteins such as small G proteins. In this study, we have identified mouse and human GGPP synthase genes. Sequence analysis showed that mouse and human GGPP synthases share a high level of amino acid identity (94%) with each other, and share a high level of similarity (45-50%) with GGPP synthases of lower eukaryotes, but only weak similarity (22-31%) to plant and prokaryotic GGPP synthases. Both of the newly identified GGPP synthase genes from mouse and human were expressed in Escherichia coli, and their gene products displayed GGPP synthase activity when isopentenyl diphosphate and farnesyl diphosphate were used as substrates. The GGPP synthase activity of these genes was also confirmed by demonstrating carotenoid synthesis after co-transformation of E. coli with a plasmid expressing the crt genes derived from Erwinia uredovora, and a plasmid expressing either the mouse or human GGPS1 gene. Southern blot analysis suggests that the human GGPS1 gene is a single copy gene.     

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.     

Bacterial phytoene synthase: molecular cloning,expression, and characterization of Erwinia herbicola phytoene synthase

Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene. These reactions constitute the first pathway specific step in carotenoid biosynthesis. The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system. Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein. To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column. Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE. Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography. The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent. The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM. The sole product of the reaction was 15,15'-Z-phytoene.     

Membrane-bound geranylgeranyl diphosphate phosphatases: purification and characterization from Croton stellatopilosus leaves

Geranylgeranyl diphosphate phosphatase is an enzyme catalyzing the dephosphorylation of geranylgeranyl diphosphate (GGPP) to form geranylgeraniol (GGOH). The enzyme activity of GGPP phosphatase was detected in leaves of Croton stellatopilosus, a Thai medicinal plant containing plaunotol, a commercial anti-peptic acyclic diterpenoid. Enzymological studies of GGPP phosphatase in C. stellatopilosis leaves revealed that the enzyme is a membrane-bound protein that could be removed from 20,000g pellet by 0.1% Triton X-100 without significant loss of enzyme activity. The solubilized enzyme preparation was separated into two activity peaks, PI and PII, by BioGel A gel filtration chromatography. PI and PII were both partially purified and characterized. PI appeared to be a tetrameric enzyme with its native molecular mass of 232kDa and subunit size of 58kDa, whereas PII was a monomeric enzyme with a molecular mass of 30-34kDa. Both phosphatases utilized GGPP as the preferred substrate over farnesyl and geranyl diphosphates. The apparent K(m) values for GGPP of PI and PII appeared to be 0.2 and 0.1mM, respectively. Both activities were Mg(2+) independent and exhibited slightly acidic pH optima, 6.0-6.5 for PI and 6.5-7.0 for PII. The catalytic activities of PII was strongly inhibited by 1.0mM of Zn(2+), Mn(2+) and Co(2+), whereas that of PI was not affected. Both enzyme preparations were very stable upon storage at -20 degrees C for 45 days without significant loss of phosphatase activity. The presence of GGPP phosphatase enzymes in C. stellatopilosus is consistent with its putative involvement in the biosynthetic pathway of plaunotol although whether PI or PII is the actual enzyme involved in the pathway remains to be clarified.     

Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins

The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.     

Emission of ent-kaurene, a diterpenoid hydrocarbon precursor for gibberellins, into the headspace from plants

ent-Kaurene is a tetracyclic hydrocarbon precursor for gibberellins (GAs) in plants and fungi. To address whether fungal GA biosynthesis enzymes function in plants, we generated transgenic Arabidopsis plants overexpressing ent-kaurene synthase (GfCPS/KS) from a GA-producing fungus Gibberella fujikuroi. GfCPS/KS catalyzes a two-step reaction corresponding to ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS) activities in plants. When GfCPS/KS was overexpressed and targeted to plastids, a range of GA-deficient phenotypes of the ga1-3 and ga2-1 mutants (defective in CPS and KS, respectively) were restored to wild type. Unexpectedly, the transgenic lines overproducing GfCPS/KS emitted the GA precursor ent-kaurene into the headspace besides its accumulation in the plant body. When co-cultivated with the ent-kaurene overproducers in a closed environment, the airborne ent-kaurene was able to fully complement the dwarf phenotype of ga1-3 and ga2-1 mutants, but not that of the ga3-1 mutant (defective in ent-kaurene oxidase). These results suggest that ent-kaurene may be efficiently metabolized into bioactive GAs in Arabidopsis when supplied as a volatile. We also provide evidence that ent-kaurene is released in the headspace of wild-type Chamaecyparis obtusa and Cryptomeria japonica plants, suggesting the occurrence of this hydrocarbon GA precursor as a volatile in nature.     

An overview of gibberellin metabolism enzyme genes and their related mutants in rice

To enhance our understanding of GA metabolism in rice (Oryza sativa), we intensively screened and identified 29 candidate genes encoding the following GA metabolic enzymes using all available rice DNA databases: ent-copalyl diphosphate synthase (CPS), ent-kaurene synthase (KS), ent-kaurene oxidase (KO), ent-kaurenoic acid oxidase (KAO), GA 20-oxidase (GA20ox), GA 3-oxidase (GA3ox), and GA 2-oxidase (GA2ox). In contrast to the Arabidopsis genome, multiple CPS-like, KS-like, and KO-like genes were identified in the rice genome, most of which are contiguously arranged. We also identified 18 GA-deficient rice mutants at six different loci from rice mutant collections. Based on the mutant and expression analyses, we demonstrated that the enzymes catalyzing the early steps in the GA biosynthetic pathway (i.e. CPS, KS, KO, and KAO) are mainly encoded by single genes, while those for later steps (i.e. GA20ox, GA3ox, and GA2ox) are encoded by gene families. The remaining CPS-like, KS-like, and KO-like genes were likely to be involved in the biosynthesis of diterpene phytoalexins rather than GAs because the expression of two CPS-like and three KS-like genes (OsCPS2, OsCPS4, OsKS4, OsKS7, and OsKS8) were increased by UV irradiation, and four of these genes (OsCPS2, OsCPS4, OsKS4, and OsKS7) were also induced by an elicitor treatment.     

Engineered isoprenoid pathway enhances astaxanthin production in Escherichia coli

The isoprenoid pathway is a versatile biosynthetic network leading to over 23,000 compounds. Similar to other biosynthetic pathways, the production of isoprenoids in microorganisms is controlled by the supply of precursors, among other factors. To engineer a host that has the capability to supply geranylgeranyl diphosphate (GGPP), a common precursor of isoprenoids, we cloned and overexpressed isopentenyl diphosphate (IPP) isomerase (encoded by idi) from Escherichia coli and GGPP synthase (encoded by gps) from the archaebacterium Archaeoglobus fulgidus. The latter was shown to be a multifunctional enzyme converting dimethylallyl diphosphate (DMAPP) to GGPP. These two genes and the gene cluster (crtBIYZW) of the marine bacterium Agrobacterium aurantiacum were introduced into E. coli to produce astaxanthin, an orange pigment and antioxidant. This metabolically engineered strain produces astaxanthin 50 times higher than values reported before. To determine the rate-controlling steps in GGPP production, the IDI-GPS pathway was compared with another construct containing idi, ispA (encoding farnesyl diphosphate (FPP) synthase in E. coli), and crtE (encoding GGPP synthase from Erwinia uredovora). Results show that the conversion from FPP to GGPP is the first bottleneck, followed sequentially by IPP isomerization and FPP synthesis. Removal of these bottlenecks results in an E. coli strain providing sufficient precursors for in vivo synthesis of isoprenoids.     

Incorporation of phytol precursors into chlorophylls of tobacco cell cultures

The incorporation of [1-³H] geranylgeranyl diphosphate (GGPP), [1-³H] geranylgeranyl monophosphate (GGMP) and [U-¹⁴C] phytyl diphosphate (PhPP) into chlorophylls a and b in growing tobacco cell cultures was investigated. The substrates were effectively incorporated into chlorophylls a and b, 3.2% of the total activity of applied GGPP or GGMP and 12.4% of the total activity of applied PhPP being found in chlorophylls a and b after 24 h incubation. The radioactivity was found in phytyl chlorophyllide through-out which means effective hydrogenation of the alcohol moiety in the case of GGPP and GGMP. With increasing substrate concentration, the specific radioactivity of chlorophyll increased up to a saturation level which was reached either at 20–40 M PhPP or at 60 M GGPP and GGMP. The specific radioactivity of the chlorophyll formed during the 24-h incubation period was the same as that of the applied substrate at saturating substrate concentration. The specific radioactivity of chlorophyll a was higher than that of chlorophyll b only in the case of PhPP.Abbreviations Chlide chlorophyllide a - Chl_Ph phytyl chloro-phyllide - Chl_GG geranylgeranyl chlorophyllide a - GGPP geranylgeranyl diphosphate - GGMP geranylgeranyl monophosphate - HPLC high-performance liquid chromatography - PhPP phytyl diphosphate Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday     

The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.

The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.     

Ethylene-mediated enhancement of apical hook formation in etiolated Arabidopsis thaliana seedlings is gibberellin dependent

Dark-grown Arabidopsis seedlings develop an apical hook by differential elongation and division of hypocotyl cells. This allows the curved hypocotyl to gently drag the apex, which is protected by the cotyledons, upwards through the soil. Several plant hormones are known to be involved in hook development, including ethylene, which causes exaggeration of the hook. We show that gibberellins (GAs) are also involved in this process. Inhibition of GA biosynthesis with paclobutrazol (PAC) prevented hook formation in wild-type (WT) seedlings and in constitutive ethylene response (ctr)1-1, a mutant that exhibits a constitutive ethylene response. In addition, a GA-deficient mutant (ga1-3) did not form an apical hook in the presence of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC). Analysis of transgenic Arabidopsis seedlings expressing a green fluorescent protein (GFP)-repressor of ga1-3 (RGA) fusion protein suggested that ACC inhibits cell elongation in the apical hook by inhibition of GA signaling. A decreased feedback of GA possibly causes an induction of GA biosynthesis based upon the expression of genes encoding copalyl diphosphate synthase (CPS; GA1) and GA 2-oxidase (AtGA2ox1). Furthermore, expression of GASA1, a GA-response gene, suggests that differential cell elongation in the apical hook might be a result of differential GA-sensitivity.     

Novel Medium-Chain Prenyl Diphosphate Synthase from the Thermoacidophilic Archaeon Sulfolobus solfataricus

Two open reading frames which encode the homologues of (all-E) prenyl diphosphate synthase are found in the whole-genome sequence of Sulfolobus solfataricus, a thermoacidophilic archaeon. It has been suggested that one is a geranylgeranyl diphosphate synthase gene, but the specificity and biological significance of the enzyme encoded by the other have remained unclear. Thus, we isolated the latter by the PCR method, expressed the enzyme in Escherichia coli cells, purified it, and characterized it. The archaeal enzyme, 281 amino acids long, is highly thermostable and requires Mg(2+) and Triton X-100 for full activity. It catalyzes consecutive E-type condensations of isopentenyl diphosphate with an allylic substrate such as geranylgeranyl diphosphate and yields the medium-chain product hexaprenyl diphosphate. Despite such product specificity, phylogenetic analysis revealed that the archaeal medium-chain prenyl diphosphate synthase is distantly related to the other medium- and long-chain enzymes but is closely related to eucaryal short-chain enzymes.     

Chain length distribution of the products formed in solanesyl diphosphate synthase reaction.

Factors that affect the termination of isoprenoid chain elongation catalyzed by prenyltransferase were investigated. The chain-length distribution of reaction products of solanesyl diphosphate synthase [EC 2.5.1.11] homogeneously purified from Micrococcus luteus changed dramatically according to the concentration of the complex formed between isopentenyl diphosphate and Mg2+ (IPP-Mg) in the reaction mixture. However, the concentration of the complex between farnesyl diphosphate and Mg2+ (FPP-Mg), the priming substrate for this synthase, did not affect the product distribution, provided that the concentration of IPP-Mg was maintained at a certain level. Thus, the level of IPP-Mg is decisive in affecting the chain length distribution of the products of the prenyltransferase reaction, and the Mg(2+)-dependent variability of product specificity so far observed can now be understood in terms of the effect of IPP-Mg concentration.     

The effects of direct inhibition of geranylgeranyl pyrophosphate synthase on osteoblast differentiation

Statins, drugs commonly used to lower serum cholesterol, have been shown to stimulate osteoblast differentiation and bone formation. These effects have been attributed to the depletion of geranylgeranyl pyrophosphate (GGPP). In this study, we tested whether specific inhibition of GGPP synthase (GGPPS) with digeranyl bisphosphonate (DGBP) would similarly lead to increased osteoblast differentiation. DGBP concentration dependently decreased intracellular GGPP levels in MC3T3‐E1 pre‐osteoblasts and primary rat calvarial osteoblasts, leading to impaired Rap1a geranylgeranylation. In contrast to our hypothesis, 1 µM DGBP inhibited matrix mineralization in the MC3T3‐E1 pre‐osteoblasts. Consistent with this, DGBP inhibited the expression of alkaline phosphatase and osteocalcin in primary osteoblasts. By inhibiting GGPPS, DGBP caused an accumulation of the GGPPS substrate farnesyl pyrophosphate (FPP). This effect was observed throughout the time course of MC3T3‐E1 pre‐osteoblast differentiation. Interestingly, DGBP treatment led to activation of the glucocorticoid receptor in MC3T3‐E1 pre‐osteoblast cells, consistent with recent findings that FPP activates nuclear hormone receptors. These findings demonstrate that direct inhibition of GGPPS, and the resulting specific depletion of GGPP, does not stimulate osteoblast differentiation. This suggests that in addition to depletion of GGPP, statin‐stimulated osteoblast differentiation may depend on the depletion of upstream isoprenoids, including FPP. J. Cell. Biochem. 112: 1506–1513, 2011. © 2011 Wiley‐Liss, Inc.     

Directed evolution of metabolically engineered Escherichia coli for carotenoid production

We have previously introduced a reconstructed isoprenoid pathway into Escherichia coli that exhibits amplified biosynthetic flux to geranylgeranyl diphosphate (GGPP), a common isoprenoid precursor. It was shown that GGPP synthase is an important rate-controlling enzyme in this reconstructed isoprenoid pathway. In this investigation, we applied directed evolution to GGPP synthase from Archaeoglobus fulgidus to enable the enhanced production of carotenoids in metabolically engineered E. coli. Eight mutants were isolated, and the best one increased lycopene production by 100%. Among the mutants that were isolated, mutation points were clustered in four "hot regions". The "hottest" region is located in the sequence upstream of the coding region, which presumably improves the expression level of the enzyme. The other three are within the coding sequence and are believed to improve the enzyme-specific activity in E coli. These results demonstrate that modulating both enzymatic expression and specific activity are important for optimizing the metabolic flux distribution.     

Glycogen synthase kinase-3 inhibition by lithium and beryllium suggests the presence of two magnesium binding sites.

Lithium inhibits (Li(+)) glycogen synthase kinase-3 (GSK-3) by competition for magnesium (Mg(2+)), but not ATP or substrate. Here, we show that the group II metal ion beryllium (Be(2+)) is a potent inhibitor of GSK-3 and competes for both Mg(2+) and ATP. Be(2+) also inhibits the related protein kinase cdc2 at similar potency, but not MAP kinase 2. To compare the actions of Li(+) and Be(2+) on GSK-3, we have devised a novel dual inhibition analysis. When Be(2+) and ADP are present together each interferes with the action of the other, indicating that both agents inhibit GSK-3 at the ATP binding site. In contrast, Li(+) exerts no interference with ADP inhibition or vice versa. We find, however, that Li(+) and Be(2+) do interfere with each other. These results suggest that Be(2+) competes for two distinct Mg(2+) binding sites: one is Li(+)-sensitive and the other, which is Li(+)-insensitive, binds the Mg:ATP complex.