Molecular cloning, bacterial expression and promoter analysis of squalene synthase from Withania somnifera (L.) Dunal

Withania somnifera (ashwagandha) is a rich repository of large number of pharmacologically active secondary metabolites known as withanolides. Though the plant has been well characterized in terms of phytochemical profiles as well as pharmaceutical activities, but there is sparse information about the genes responsible for biosynthesis of these compounds. In this study, we have cloned and characterized a gene encoding squalene synthase (EC 2.5.1.21) from a withaferin A rich variety of W. somnifera, a key enzyme in the biosynthesis of isoprenoids. Squalene synthase catalyses dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for sterols and triterpenes. A full-length cDNA consisting of 1765 bp was isolated and contained a 1236 bp open reading frame (ORF) encoding a polypeptide of 411 amino acids. Recombinant C-terminus truncated squalene synthase (WsSQS) was expressed in BL21 cells (Escherichia coli) with optimum expression induced with 1mM IPTG at 37°C after 1h. Quantitative RT-PCR analysis showed that squalene synthase (WsSQS) expressed in all tested tissues including roots, stem and leaves with the highest level of expression in leaves. The promoter region of WsSQS isolated by genome walking presented several cis-acting elements in the promoter region. Biosynthesis of withanolides was up-regulated by different signalling components including methyl-jasmonate, salicylic acid and 2, 4-D, which was consistent with the predicted results of WsSQS promoter region. This work is the first report of cloning and expression of squalene synthase from W. somnifera and will be useful to understand the regulatory role of squalene synthase in the biosynthesis of withanolides.     

Dammaradiene synthase, a squalene cyclase, from Dryopteris crassirhizoma Nakai

Ferns produce a variety of cyclic triterpene hydrocarbons in large amount. Squalene cyclases (SCs) are responsible enzymes for formation of cyclic triterpene hydrocarbon skeletons. Although more than ten bacterial SCs have been cloned and four of them characterized for their enzymatic products, the only example of a fern SC is ACH, from Adiantum capillus-veneris, which produces hydroxyhopane. To obtain a deeper understanding of the molecular evolution of SCs and the origin of the structural diversity of fern triterpenes, further cloning and characterization of SCs have been pursued. In this study, a SC cDNA, DCD, was cloned from Dryopteris crassirhizoma by homology-based RT-PCR. DCD contains a 2058-bp open reading frame that encodes a 685 amino acid polypeptide exhibiting 66% identity to the previously identified fern SC, ACH, and 35-40% identity to bacterial SCs. Heterologous expression of DCD in yeast established it to be a dammaradiene synthase affording dammara-18(28),21-diene, a tetracyclic triterpene hydrocarbon. Although neither this compound nor any derived metabolites have been previously reported from D. crassirhizoma, re-investigation of the leaflets demonstrated the presence of dammara-18(28),21-diene. DCD represents the first SC that produces a tetracyclic triterpene hydrocarbon.     

Recent advances regarding diterpene cyclase genes in higher plants and fungi

Cyclic diterpenoids are commonly biosynthesized from geranylgeranyl diphosphate (GGDP) through the formation of carbon skeletons by specific cyclases and subsequent chemical modifications, such as oxidation, reduction, methylation, and glucosidation. A variety of diterpenoids are produced in higher plants and fungi. Rice produces four classes of diterpene phytoalexins, phytocassanes A to E, oryzalexins A to F, oryzalexin S, and momilactones A and B. The six diterpene cyclase genes involved in the biosynthesis of these phytoalexins were identified and characterized. Fusicoccin A was produced by the phytopathogenic Phomopsis amygdali and served as a plant H(+)-ATPase activator. A PaFS, encoding a fungal diterpene synthase responsible for fusicoccin biosynthesis, was isolated. The PaFS is an unusual chimeric diterpene synthase that possesses not only terpene cyclase activity (the formation of fusicoccadiene, a biosynthetic precursor of fusicoccin A), but also prenyltransferase activity (the formation of GGDP). Thus, we identified a unique multifunctional diterpene synthase family in fungi.     

Phylogenetic analysis of the triterpene cyclase protein family in prokaryotes and eukaryotes suggests bidirectional lateral gene transfer

Functional constraints to modifications in triterpene cyclase amino acid sequences make them good candidates for evolutionary studies on the phylogenetic relatedness of these enzymes in prokaryotes as well as in eukaryotes. In this study, we used a set of identified triterpene cyclases, a group of mainly bacterial squalene cyclases and a group of predominantly eukaryotic oxidosqualene cyclases, as seed sequences to identify 5288 putative triterpene cyclase homologues in publicly available databases. The Cluster Analysis of Sequences software was used to detect groups of sequences with increased pairwise sequence similarity. The sequences fall into two main clusters, a bacterial and a eukaryotic. The conserved, informative regions of a multiple sequence alignment of the family were used to construct a neighbour-joining phylogenetic tree using the AsaturA and maximum likelihood phylogenetic tree using the PhyML software. Both analyses showed that most of the triterpene cyclase sequences were similarly grouped to the accepted taxonomic relationships of the organism the sequences originated from, supporting the idea of vertical transfer of cyclase genes from parent to offspring as the main evolutionary driving force in this protein family. However, a small group of sequences from three bacterial species ( Stigmatella , Gemmata and Methylococcus ) grouped with an otherwise purely eukaryotic cluster of oxidosqualene cyclases, while a small group of sequences from seven fungal species and a sequence from the fern Adiantum grouped consistently with a cluster of otherwise purely bacterial squalene cyclases. This suggests that lateral gene transfer may have taken place, entailing a transfer of oxidosqualene cyclases from eukaryotes to bacteria and a transfer of squalene cyclase from bacteria to an ancestor of the group of Pezizomycotina fungi.     

Characterization and partial purification of squalene-hopene cyclase from Bacillus acidocaldarius

A membrane-bound enzyme activity from Bacillus acidocaldarius converted squalene to two pentacyclic triterpenes, hop-22(29)-ene and hopan-22-ol. The products were formed in a constant molar ratio of hopene:hopanol, 5:1, probably through parallel, and not successive, reactions. The conversion was independent of oxygen, in contrast to the biosynthesis of sterols from epoxysqualene in eukaryotes. The squalene-hopene cyclase was pufified 270-fold by extraction from B. acidocaldarius membranes at low concentrations of Triton X-100 followed by DEAE-cellulose chromatography. The enzyme showed optimal rates of squalene conversion at pH 6 and 60°C, corresponding to the intracellular pH and the optimal growth temperature of the bacterium. The apparent K_m for squalene is 3 μM. Effective inhibitors of the enzyme were some sulfhydryl reagents and the histidyl reagent diethyl pyrocarbonate. The squalene-hopene cyclase, like several eukaryotic epoxysqualene cyclases, was strongly inhibited by AMO 1618 and by high ionic strength. On the basis of these and other similarities a phylogenitic relationship between the dey enzyme of steroid and hopanoid biosynthesis was envisaged.     

Conserved tyr residues determine functions of Alicyclobacillus acidocaldarius squalene-hopene cyclase

The catalytic cavity of Alicyclobacillus acidocaldarius squalene-hopene cyclase is mainly lined by aromatic amino acids. In recombinant cyclases, three out of four tyrosine residues (Y) have been mutated to phenylalanine residues (F). The mutant cyclases Y495F and Y612F had less activity than the wild-type cyclase, but a wild-type product pattern. Mutant Y609F had wild-type activity but a drastically altered product pattern with hopene and significant amounts of bicyclic alpha-polypodatetraene and different tetracyclic triterpenes (dammaradienes and eupha-7,24-diene). The experiments demonstrated that Y495 and Y612 may be involved in the initiation of the cyclization reaction and Y609 in the stabilization and/or positioning of the intermediate carbocations.     

Genetic data indicate that proteins containing the GGDEF domain possess diguanylate cyclase activity

A conserved domain, called GGDEF (referring to a conserved central sequence pattern), is detected in many procaryotic proteins, often in various combinations with putative sensory-regulatory components. Most sequenced bacterial genomes contain several different GGDEF proteins. The function of this domain has so far not been experimentally shown. Through genetic complementation using genes from three different bacteria encoding proteins with GGDEF domains as the only element in common, we present genetic data indicating (a) that the GGDEF domain is responsible for the diguanylate cyclase activity of these proteins, and (b) that the activity of cellulose synthase in Rhizobium leguminosarum bv. trifolii and Agrobacterium tumefaciens is regulated by cyclic di-GMP as in Acetobacter xylinum.     

Identification of triterpene biosynthetic genes from Momordica charantia using RNA-seq analysis

Cucurbitaceae plants contain characteristic triterpenoids. Momordica charantia, known as a bitter melon, contains cucurbitacins and multiflorane type triterpenes, which confer bitter tasting and exhibit pharmacological activities. Their carbon skeletons are biosynthesized from 2,3-oxidosqualene by responsible oxidosqualene cyclase (OSC). In order to identify OSCs in M. charantia, RNA-seq analysis was carried out from ten different tissues. The functional analysis of the resulting four OSC genes revealed that they were cucurbitadienol synthase (McCBS), isomultiflorenol synthase (McIMS), β-amyrin synthase (McBAS) and cycloartenol synthase (McCAS), respectively. Their distinct expression patterns based on RPKM values and quantitative RT-PCR suggested how the characteristic triterpenoids were biosynthesized in each tissue. Although cucurbitacins were finally accumulated in fruits, McCBS showed highest expression in leaves indicating that the early step of cucurbitacins biosynthesis takes place in leaves, but not in fruits.

Abbreviations: OSC: oxidosqualene cyclase; RPKM: reads perkilobase of exon per million mapped reads     

Hopanoid lipids in Bradyrhizobium and other plant-associated bacteria and cloning of the Bradyrhizobium japonicum squalene-hopene cyclase gene

Hopanoid lipids have been discovered recently in a number of nitrogen-fixing soil bacteria and in Bradyrhizobium bacteria which fix nitrogen in association with legume plants. We report here an investigation of the hopanoid content in an additional number of soil bacteria capable of living in close association with plants. Of the strains investigated, hopanoids were discovered in phototrophic, nitrogen-fixing bacteria and in an extended number of Bradyrhizobium strains. Strains in which hopanoids so far have not been found belong to the following genera: Rhizobium, Sinorhizobium, Phyllobacterium, Agrobacterium, and Azoarcus. To address the function of hopanoids in Bradyrhizobium, we cloned the gene coding for a key enzyme of hopanoid biosynthesis, the squalene-hopene cyclase, and expressed the gene in E. coli. The recombinant enzyme catalyzed in vitro the cyclization of squalene to hopanoid derivatives.Abbreviations SHC squalene-hopene cyclase - shc squalene-hopene cyclase gene     

Towards the characterization of squalene synthase activity in extracts of Zymomonas mobilis

Abstract Extracts of Zymomonas mobilis in the presence of NADPH converted tritium-labelled farnesyl diphosphate (FPP) into squalene, resulting from the activity of squalene synthase, as well as diploptene and diplopterol, derived from further squalene cyclisation. An unidentified isoprenoid representing up to 70% of the conversion products of FPP and different from presqualene alcohol was also formed, even in the absence of NADPH. Addition of squalestatin 1, an inhibitor of squalene synthase, blocked biosynthesis from FPP of the three former triterpenes, in accordance with the role of squalene synthase in their formation, as well as that of the unknown compound.     

Structure of the class IV adenylyl cyclase reveals a novel fold

The crystal structure of the class IV adenylyl cyclase (AC) from Yersinia pestis (Yp) is reported at 1.9 A resolution. The class IV AC fold is distinct from the previously described folds for class II and class III ACs. The dimeric AC-IV folds into an antiparallel eight-stranded barrel whose connectivity has been seen in only three previous structures: yeast RNA triphosphatase and two proteins of unknown function from Pyrococcus furiosus and Vibrio parahaemolyticus. Eight highly conserved ionic residues E10, E12, K14, R63, K76, K111, D126, and E136 lie in the barrel core and form the likely binding sites for substrate and divalent cations. A phosphate ion is observed bound to R63, K76, K111, and R113 near the center of the conserved cluster. Unlike the AC-II and AC-III active sites that utilize two-Asp motifs for cation binding, the AC-IV active site is relatively enriched in glutamate and features an ExE motif as its most conserved element. Homologs of Y. pestis AC-IV, including human thiamine triphosphatase, span the three kingdoms of life and delineate an ancient family of phosphonucleotide processing enzymes.     

ABA activates ADPR cyclase and cADPR induces a subset of ABA-responsive genes in Arabidopsis

Cyclic ADP-ribose (cADPR) was previously shown to activate transient expression of two abscisic acid (ABA)-responsive genes in tomato cells. Here, we show that the activity of the enzyme responsible for cADPR synthesis, ADP-ribosyl (ADPR) cyclase, is rapidly induced by ABA in both wild-type (WT) and abi1-1 mutant Arabidopsis plants in the absence of protein synthesis. Furthermore, in transgenic Arabidopsis plants, induced expression of the Aplysia ADPR cyclase gene resulted in an increase in ADPR cyclase activity and cADPR levels, as well as elevated expression of ABA-responsive genes KIN2, RD22, RD29a, and COR47 (although to a lesser extent than after ABA induction). Genome-wide profiling indicated that about 28% of all ABA-responsive genes in Arabidopsis are similarly up- and downregulated by cADPR and contributed to the identification of new ABA-responsive genes. Our results suggest that activation of ADPR cyclase is an early ABA-signaling event partially insensitive to the abi1-1 mutation and that an increase in cADPR plays an important role in downstream molecular and physiological ABA responses.     

Squalene-hopene cyclase (Spterp25) from Streptomyces peucetius: sequence analysis, expression and functional characterization

Squalene-hopene cyclase, which catalyzes the complex cyclization of squalene to the pentacyclic triterpene, hopene, is a key enzyme in the biosynthesis of hopanoids. The deduced amino acid sequence of the Streptomyces peucetius gene (spterp25) had significant similarity to other prokaryotic squalene-hopene cyclases. Like other triterpene cyclases, the S. peucetius squalene-hopene cyclase contains eight so-called QW-motifs with an aspartate-rich domain. The 2,025-bp squalene-hopene cyclase-encoding gene was expressed in Escherichia coli BL21(DE3)pLySs, and the in vitro activity of the recombinant cyclase was demonstrated using purified membrane protein. The cyclization product hopene was identified by gas chromatography/mass spectrometry (GC/MS).     

Lanosterol biosynthesis in the prokaryote Methylococcus capsulatus: insight into the evolution of sterol biosynthesis

A putative operon containing homologues of essential eukaryotic sterol biosynthetic enzymes, squalene monooxygenase and oxidosqualene cyclase, has been identified in the genome of the prokaryote Methylococcus capsulatus. Expression of the squalene monooxygenase yielded a protein associated with the membrane fraction, while expression of oxidosqualene cyclase yielded a soluble protein, contrasting with the eukaryotic enzyme forms. Activity studies with purified squalene monooxygenase revealed a catalytic activity in epoxidation of 0.35 nmol oxidosqualene produced/min/nmol squalene monooxygenase, while oxidosqualene cyclase catalytic activity revealed cyclization of oxidosqualene to lanosterol with 0.6 nmol lanosterol produced/min/nmol oxidosqualene cyclase and no other products observed. The presence of prokaryotic sterol biosynthesis is still regarded as rare, and these are the first representatives of such prokaryotic enzymes to be studied, providing new insight into the evolution of sterol biosynthesis in general.     

Mono and diterpene production in Escherichia coli

Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.     

Triterpene synthases from the Okinawan mangrove tribe, Rhizophoraceae

Oleanane-type triterpene is one of the most widespread triterpenes found in plants, together with the lupane type, and these two types often occur together in the same plant. Bruguiera gymnorrhiza (L.) Lamk. and Rhizophora stylosa Griff. (Rhizophoraceae) are known to produce both types of triterpenes. Four oxidosqualene cyclase cDNAs were cloned from the leaves of B. gymnorrhiza and R. stylosa by a homology-based PCR method. The ORFs of full-length clones termed BgbAS (2280 bp, coding for 759 amino acids), BgLUS (2286 bp, coding for 761 amino acids), RsM1 (2280 bp, coding for 759 amino acids) and RsM2 (2316 bp coding for 771 amino acids) were ligated into yeast expression plasmid pYES2 under the control of the GAL1 promoter. Expression of BgbAS and BgLUS in GIL77 resulted in the production of beta-amyrin and lupeol, suggesting that these genes encode beta-amyrin and lupeol synthase (LUS), respectively. Furthermore, RsM1 produced germanicol, beta-amyrin, and lupeol in the ratio of 63 : 33 : 4, whereas RsM2 produced taraxerol, beta-amyrin, and lupeol in the proportions 70 : 17 : 13. This result indicates that these are multifunctional triterpene synthases. Phylogenetic analysis and sequence comparisons revealed that BgbAS and RsM1 demonstrated high similarities (78-93%) to beta-amyrin synthases, and were located in the same branch as beta-amyrin synthase. BgLUS formed a new branch for lupeol synthase that was closely related to the beta-amyrin synthase cluster, whereas RsM2 was found in the first branch of the multifunctional triterpene synthase evolved from lupeol to beta-amyrin synthase. Based on these sequence comparisons and product profiles, we discuss the molecular evolution of triterpene synthases and the involvement of these genes in the formation of terpenoids in mangrove leaves.