Defining paxilline biosynthesis in Penicillium paxilli: functional characterization of two cytochrome P450 monooxygenases

Indole diterpenes are a large, structurally and functionally diverse group of secondary metabolites produced by filamentous fungi. Biosynthetic schemes have been proposed for these metabolites but until recently none of the proposed steps had been validated by biochemical or genetic studies. Using Penicillium paxilli as a model experimental system to study indole diterpene biosynthesis we previously showed by deletion analysis that a cluster of seven genes is required for paxilline biosynthesis. Two of these pax genes, paxP and paxQ (encoding cytochrome P450 monooxygenases), are required in the later steps in this pathway. Here, we describe the function of paxP and paxQ gene products by feeding proposed paxilline intermediates to strains lacking the pax cluster but containing ectopically integrated copies of paxP or paxQ. Transformants containing paxP converted paspaline into 13-desoxypaxilline as the major product and beta-PC-M6 as the minor product. beta-PC-M6, but not alpha-PC-M6, was also a substrate for PaxP and was converted to 13-desoxypaxilline. paxQ-containing transformants converted 13-desoxypaxilline into paxilline. These results confirm that paspaline, beta-PC-M6, and 13-desoxypaxilline are paxilline intermediates and that paspaline and beta-PC-M6 are substrates for PaxP, and 13-desoxypaxilline is a substrate for PaxQ. PaxP and PaxQ also utilized beta-paxitriol and alpha-PC-M6 as substrates converting them to paxilline and alpha-paxitriol, respectively. These findings have allowed us to delineate clearly the biosynthetic pathway for paxilline for the first time.     

Functional analysis and subcellular localization of two geranylgeranyl diphosphate synthases from <Emphasis Type="Italic">Penicillium paxilli</Emphasis>

The filamentous fungus Penicillium paxilli contains two distinct geranylgeranyl diphosphate (GGPP) synthases, GgsA and GgsB (PaxG). PaxG and its homologues in Neotyphodium lolii and Fusarium fujikuroi are associated with diterpene secondary metabolite gene clusters. The genomes of other filamentous fungi including Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae and Fusarium graminearum also contain two or more copies of GGPP synthase genes, although the diterpene metabolite capability of these fungi is not known. The objective of this study was to understand the biological significance of the presence of two copies of GGPP synthases in P. paxilli by investigating their subcellular localization. Using a carotenoid complementation assay and gene deletion analysis, we show that P. paxilli GgsA and PaxG have GGPP synthase activities and that paxG is required for paxilline biosynthesis, respectively. In the ΔpaxG mutant background ggsA was unable to complement paxilline biosynthesis. A GgsA-EGFP fusion protein was localized to punctuate organelles and the EGFP-GRV fusion protein, containing the C-terminus tripeptide GRV of PaxG, was localized to peroxisomes. A truncated PaxG mutant lacking the C-terminus tripeptide GRV was unable to complement a ΔpaxG mutant demonstrating that the tripeptide is functionally important for paxilline biosynthesis. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Four gene products are required for the fungal synthesis of the indole-diterpene, paspaline

Paspaline belongs to a large, structurally and functionally diverse group of indole-diterpenes synthesized by filamentous fungi. However, the identity of the gene products required for the biosynthesis of paspaline, a key intermediate for the synthesis of paxilline and other indole-diterpenes, is not known. Transfer of constructs containing different pax gene combinations into a paxilline negative deletion derivative of Penicillium paxilli demonstrated that just four proteins, PaxG, a geranylgeranyl diphosphate synthase, PaxM, a FAD-dependent monooxygenase, PaxB, a putative membrane protein, and PaxC, a prenyl transferase, are required for the biosynthesis of paspaline.     

An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in taxol biosynthesis, we cloned, characterized and functionally expressed the GGPP synthase gene from Taxus media. A 3743-bp genomic sequence of T. media was isolated by genome walking strategy which contained an 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed high similarity to other plant GGPPSs. Subsequently the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene and its deduced polypeptide contained all the five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, angiosperm and gymnosperm classes, which might have evolved in parallel from the same ancestor. To our knowledge this was the first report that the geranylgeranyl diphosphate synthase genes were free of intron and evolved in parallel between angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed in yeast mutant (SFNY368) lacking of GGPP synthase activity through functional complementation, and the transgenic yeast showed to have activity of GGPP synthase. This was also the first time to use SFNY368 to identify the function of plant-derived GGPPSs. Furthermore, investigation of the impact of methyl jasmonate (MeJA) on the expression of TmGGPPS revealed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.     

Functional analysis of a prenyltransferase gene (paxD) in the paxilline biosynthetic gene cluster

Paxilline is an indole-diterpene produced by Penicillium paxilli. Six genes (paxB, C, G, M, P, and Q) in paxilline biosynthetic gene cluster were previously shown to be responsible for paxilline biosynthesis. In this study, we have characterized paxD, which is located next to paxQ and has weak similarities to fungal dimethylallyl tryptophan synthase genes. PaxD was overexpressed in Escherichia coli and the purified enzyme was used for in vitro analysis. When paxilline and dimethylallyl diphosphate were used as substrates, one major and one minor product, which were identified as di-prenyl paxilline and mono-prenyl paxilline by liquid chromatography–mass spectrometry analysis, were formed. The structure of the major product was determined to be 21,22-diprenylated paxilline, showing that PaxD catalyzed the successive di-prenylation. Traces of both products were detected in culture broth of P. paxilli by liquid chromatography–mass spectrometry analysis. The enzyme is likely to be a dimer and required no divalent cations. The optimum pH and temperature were 8.0 and 37 °C, respectively. The Km values were calculated as 106.4?±?5.4 μM for paxilline and 0.57?±?0.02 μM for DMAPP with a kcat of 0.97?±?0.01/s.     

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.     

Molecular analysis of two cytochrome P450 monooxygenase genes required for paxilline biosynthesis in Penicillium paxilli,and effects of paxilline intermediates on mammalian maxi-K ion channels

The gene cluster required for paxilline biosynthesis in Penicillium paxilli contains two cytochrome P450 monooxygenase genes, paxP and paxQ. The primary sequences of both proteins are very similar to those of proposed cytochrome P450 monooxygenases from other filamentous fungi, and contain several conserved motifs, including that for a haem-binding site. Alignment of these sequences with mammalian and bacterial P450 enzymes of known 3-D structure predicts that there is also considerable conservation at the level of secondary structure. Deletion of paxP and paxQ results in mutant strains that accumulate paspaline and 13-desoxypaxilline, respectively. These results confirm that paxP and paxQ are essential for paxilline biosynthesis and that paspaline and 13-desoxypaxilline are the most likely substrates for the corresponding enzymes. Chemical complementation of paxilline biosynthesis in paxG (geranygeranyl diphosphate synthase) and paxP, but not paxQ, mutants by the external addition of 13-desoxypaxilline confirms that PaxG and PaxP precede PaxQ, and are functionally part of the same biosynthetic pathway. A pathway for the biosynthesis of paxilline is proposed on the basis of these and earlier results. Electrophysiological experiments demonstrated that 13-desoxypaxilline is a weak inhibitor of mammalian maxi-K channels (K_i=730 nM) compared to paxilline (K_i=30 nM), indicating that the C-13 OH group of paxilline is crucial for the biological activity of this tremorgenic mycotoxin. Paspaline is essentially inactive as a channel blocker, causing only slight inhibition at concentrations up to 1 M.Communicated by E. Cerdà-Olmedo     

Regulation of Carotenogenesis and Secondary Metabolism by Nitrogen in Wild-Type Fusarium fujikuroi and Carotenoid-Overproducing Mutants

The fungus Fusarium fujikuroi (Gibberella fujikuroi MP-C) produces metabolites of biotechnological interest, such as gibberellins, bikaverins, and carotenoids. Gibberellin and bikaverin productions are induced upon nitrogen exhaustion, while carotenoid accumulation is stimulated by light. We evaluated the effect of nitrogen availability on carotenogenesis in comparison with bikaverin and gibberellin production in the wild type and in carotenoid-overproducing mutants (carS). Nitrogen starvation increased carotenoid accumulation in all strains tested. In carS strains, gibberellin and bikaverin biosynthesis patterns differed from those of the wild type and paralleled the expression of key genes for both pathways, coding for geranylgeranyl pyrophosphate (GGPP) and kaurene synthases for the former and a polyketide synthase for the latter. These results suggest regulatory connections between carotenoid biosynthesis and nitrogen-controlled biosynthetic pathways in this fungus. Expression of gene ggs1, which encodes a second GGPP synthase, was also derepressed in the carS mutants, suggesting the participation of Ggs1 in carotenoid biosynthesis. The carS mutations did not affect genes for earlier steps of the terpenoid pathway, such as fppS or hmgR. Light induced carotenoid biosynthesis in the wild type and carRA and carB levels in the wild-type and carS strains irrespective of nitrogen availability.     

Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from <Emphasis Type="Italic">Coleus forskohlii</Emphasis>Briq

Background  

Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis. Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin. Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq.     

A complex gene cluster for indole-diterpene biosynthesis in the grass endophyte Neotyphodium lolii

Lolitrems are a structurally diverse group of indole-diterpene mycotoxins synthesized by Epichloë/Neotyphodium endophytes in association with Pooid grasses. Using suppression subtractive hybridization combined with chromosome walking, two clusters of genes for lolitrem biosynthesis were isolated from Neotyphodium lolii, a mutualistic endophyte of perennial ryegrass. The first cluster contains five genes, ltmP, ltmQ, ltmF, ltmC, and ltmB, four of which appear to be orthologues of functionally characterized genes from Penicillium paxilli. The second cluster contains two genes, ltmE and ltmJ, that appear to be unique to lolitrem biosynthesis. The two clusters are separated by a 16 kb AT-rich sequence that includes two imperfect direct repeats. A previously isolated ltm cluster composed of ltmG, ltmM, and ltmK, is linked to these two new clusters by 35 kb of AT-rich retrotransposon relic sequence. All 10 genes at this complex LTM locus were highly expressed in planta but expression was very low or undetectable in mycelia. ltmM and ltmC were shown to be functional orthologues of P. paxilli paxM and paxC, respectively. This work provides a genetic foundation for elucidating the metabolic grid responsible for the diversity of indole-diterpenes synthesized by N. lolii.     

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.     

Geranylgeranyl pyrophosphate synthase encoded by the newly isolated gene GGPS6 from Arabidopsis thaliana is localized in mitochondria

We have cloned a new geranylgeranyl pyrophosphate (GGPP) synthase gene, designated GGPS6/, from Arabidopsis thaliana genomic DNA. Nucleotide sequence analysis revealed that the GGPS6 gene contains an open reading frame coding for a protein of 343 amino acid residues with a calculated molecular mass of 37 507 Da. Also, the gene is not interrupted by an intron. The predicted amino acid sequence of the GGPS6 gene shows significant homology (34.0–57.7%) with other GGPP synthases from Arabidopsis. The GGPS6 protein contains a N-terminal signal peptide which is thought to function as an organelle targeting sequence. In fact, the GGPS6-GFP fusion protein was found to be localized exclusively in mitochondria when expressed in tobacco BY-2 cells. In vitro analysis of the enzyme activity as well as genetic complementation analysis with Erwinia uredovora crt gene cluster expressed in Escherichia coli showed that the GGPS6 gene most certainly encodes a GGPP synthase catalyzing the conversion of farnesyl pyrophosphate to GGPP.     

Quantitative analysis of isoprenoid diphosphate intermediates in recombinant and wild-type Escherichia coli strains

In biotechnology, the heterologous biosynthesis of isoprenoid compounds in Escherichia coli is a field of great interest and growth. In order to achieve higher isoprenoid yields in heterologous E. coli strains, it is necessary to quantify the pathway intermediates and adjust gene expression. In this study, we developed a precise and sensitive nonradioactive method for the simultaneous quantification of the isoprenoid precursors farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) in recombinant and wild-type E. coli cells. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–mass spectrometry. The integration of a geranylgeranyl diphosphate synthase gene into the E. coli chromosome leads to the accumulation of GGPP, generating quantities as high as those achieved with a multicopy expression vector. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. T. Vallon and S. Ghanegaonkar contributed equally to this work.     

The genetic basis for indole-diterpene chemical diversity in filamentous fungi

Indole-diterpenes are a structurally diverse group of secondary metabolites with a common cyclic diterpene backbone derived from geranylgeranyl diphosphate and an indole group derived from indole-3-glycerol phosphate. Different types and patterns of ring substitutions and ring stereochemistry generate this structural diversity. This group of compounds is best known for their neurotoxic effects in mammals, causing syndromes such as ‘ryegrass staggers’ in sheep and cattle. Because many of the fungi that synthesise these compounds form symbiotic relationships with plants, insects, and other fungi, the synthesis of these compounds may confer an ecological advantage to these associations. Considerable recent progress has been made on understanding indole-diterpene biosynthesis in filamentous fungi, principally through the cloning and characterisation of the genes and gene products for paxilline biosynthesis in Penicillium paxilli. Important insights into how the indole-diterpene backbone is synthesised and decorated have been obtained using P. paxilli mutants in this pathway. This review provides an overview of these recent developments.     

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.