(+)-(10R)-Germacrene A synthase from goldenrod,Solidago canadensis; cDNA isolation,bacterial expression and functional analysis

Profiling of sesquiterpene hydrocarbons in extracts of goldenrod, Solidago canadensis, by GC-MS revealed the presence of both enantiomers of germacrene D and lesser amounts of germacrene A, alpha-humulene, and beta-caryophyllene. A similarity-based cloning strategy using degenerate oligonucleotide primers, based on conserved amino acid sequences in known plant sesquiterpene synthases and RT-PCR, resulted in the isolation of a full length sesquiterpene synthase cDNA. Functional expression of the cDNA in E. coli, as an N-terminal thioredoxin fusion protein using the pET32b vector yielded an enzyme that was readily purified by nickel-chelate affinity chromatography. Chiral GC-MS analysis of products from of (3)H- and (2)H-labelled farnesyl diphosphate identified the enzyme as (+)-(10R)-germacrene A synthase. Sequence analysis and molecular modelling was used to compare this enzyme with the mechanistically related epi-aristolochene synthase from tobacco.     

Pumpkin malate synthase. Cloning and sequencing of the cDNA and northern blot analysis.

A cDNA clone encoding the glyoxysomal malate synthase (EC 4.1.3.2) was identified by immunoscreening of a cDNA expression library constructed from poly(A)-rich RNA of etiolated pumpkin cotyledons. Determination of the DNA sequence of the 1979-nucleotide cDNA revealed a 1698-nucleotide open reading frame that encodes a polypeptide of 64632 Da. The identification of the cDNA for malate synthase was confirmed by matching three sequences obtained by peptide-sequence analyses of fragments generated by acid treatment of the purified enzyme. Northern blot analysis revealed that the probe hybridized to a single 2.3-kb species of mRNA species from etiolated pumpkin cotyledons which was not present in green pumpkin cotyledons. In a comparison of deduced amino acid sequences, pumpkin malate synthase was found to exhibit 83% and 48% similarity to the malate synthases from rape and Escherichia coli, respectively. Based on the amino acid sequence similarity and the hydropathy profiles of these three malate synthases, the signal for targeting the enzyme to microbodies is discussed.     

Cloning, expression, purification and characterization of recombinant (+)-germacrene D synthase from Zingiber officinale

A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.     

Isoprenoid biosynthesis in Artemisia annua: cloning and heterologous expression of a germacrene A synthase from a glandular trichome cDNA library

Artemisia annua (Asteraceae) is the source of the anti-malarial compound artemisinin. To elucidate the biosynthetic pathway and to isolate and characterize genes involved in the biosynthesis of terpenoids including artemisinin in A. annua, glandular trichomes were used as an enriched source for biochemical and molecular biological studies. The sequencing of 900 randomly selected clones from a glandular trichome plasmid cDNA library revealed the presence of many ESTs involved in isoprenoid biosynthesis such as enzymes from the methylerythritol phosphate pathway and the mevalonate pathway, amorpha-4,11-diene synthase and other sesquiterpene synthases, monoterpene synthases and two cDNAs showing high similarity to germacrene A synthases. Full-length sequencing of the latter two ESTs resulted in a 1686-bp ORF encoding a protein of 562 aa. Upon expression in Escherichia coli, the recombinant protein was inactive with geranyl diphosphate, but catalyzed the cyclization of farnesyl diphosphate to germacrene A. These results demonstrate the potential of the use of A. annua glandular trichomes as a starting material for studying isoprenoid biosynthesis in this plant species.     

Photomodulation of strigolactone biosynthesis and accumulation during sunflower seedling growth

Present investigations report the presence of strigolactones (SLs) and photomodulation of their biosynthesis in sunflower seedlings (roots, cotyledons and first pair of leaves) during early phase of seedling development. Qualitative analyses and characterization by HPLC, ESI-MS and FT-IR revealed the presence of more than one type of SLs. Orobanchyl acetate was detected both in roots and leaves. Five-deoxystrigol, sorgolactone and orobanchol were exclusively detected in seedling roots. Sorgomol was detectable only in leaves. HPLC eluted fraction from seedling roots and leaves co-chromatographing with GR24 (a synthetic SL) could also bring about germination in Orobanche cernua (a weed) seeds, which are established to exhibit SL – mediated germination, thereby indicating the SL identity of the eluates using this bioassay. SLs accumulation was always more in the roots of light-grown seedlings, it being maximum at 4 d stage. Although significant activity of carotenoid cleavage dioxygenase (CCD, the enzyme critical for SL biosynthesis) was detected in 2 d old seedling roots, SLs remained undetectable in cotyledons at all stages of development and also in the roots of 2 d old light and dark-grown seedlings. Roots of light-grown seedlings showed maximum CCD activity during early (2 d) stage of development, thereby confirming photomodulation of enzyme activity. These observations indicate the migration of a probable light-sensitized signaling molecule (yet to be identified) or a SL precursor from light exposed aerial parts to the seedling roots maintained in dark. Thus, a photomodulation and migration of SL precursor/s is evident from the present work.     

Genetic control and evolution of sesquiterpene biosynthesis in Lycopersicon esculentum and L. hirsutum

Segregation analysis between Lysopersicon esculentum (cultivated tomato) and L. hirsutum (wild form) in conjunction with positional verification by using near-isogenic lines demonstrated that biosynthesis of two structurally different classes of sesquiterpenes in these species is controlled by loci on two different chromosomes. A locus on chromosome 6, Sesquiterpene synthase1 (Sst1), was identified for which the L. esculentum allele is associated with the biosynthesis of beta-caryophyllene and alpha-humulene. At this same locus, the L. hirsutum allele is associated with biosynthesis of germacrene B, germacrene D, and an unidentified sesquiterpene. Genomic mapping, cDNA isolation, and heterologous expression of putative sesquiterpene synthases from both L. esculentum and L. hirsutum revealed that Sst1 is composed of two gene clusters 24 centimorgans apart, Sst1-A and Sst1-B, and that only the genes in the Sst1-A cluster are responsible for accumulation of chromosome 6-associated sesquiterpenes. At a second locus, Sst2, on chromosome 8, the L. hirsutum allele specified accumulation of alpha-santalene, alpha-bergamotene, and beta-bergamotene. Surprisingly, the L. esculentum allele for Sst2 is not associated with the expression of any sesquiterpenes, which suggests that cultivated tomato may have a nonfunctional allele. Sesquiterpene synthase cDNA clones on chromosome 6 do not cross-hybridize on genomic DNA gel blots with putative sesquiterpene synthases on chromosome 8, an indication that the genes in Sst1 and Sst2 are highly diverged, each being responsible for the biosynthesis of structurally different sets of sesquiterpenes.     

The diverse sesquiterpene profile of patchouli, Pogostemon cablin, is correlated with a limited number of sesquiterpene synthases

Pogostemon cablin (patchouli), like many plants within the Lamiaceae, accumulates large amounts of essential oil. Patchouli oil is unique because it consists of over 24 different sesquiterpenes, rather than a blend of different mono-, sesqui- and di-terpene compounds. To determine if this complex mixture of sesquiterpenes arises from an equal number of unique sesquiterpene synthases, we developed a RT-PCR strategy to isolate and functionally characterize the respective patchouli oil synthase genes. Unexpectedly, only five terpene synthase cDNA genes were isolated. Four of the cDNAs encode for synthases catalyzing the biosynthesis of one major sesquiterpene, including a gamma-curcumene synthase, two germacrene D synthases, and a germacrene A synthase. The fifth cDNA encodes for a patchoulol synthase, which catalyzes the conversion of FPP to patchoulol plus at least 13 additional sesquiterpene products. Equally intriguing, the yield of the different in vitro reaction products resembles quantitatively and qualitatively the profile of sesquiterpenes found in patchouli oil extracted from plants, suggesting that a single terpene synthase is responsible for the bulk and diversity of terpene products produced in planta.     

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

cDNA cloning and expression of rat leukotriene C(4) synthase: elevated expression in rat basophilic leukemia-1 cells after treatment with retinoic acid

Leukotriene C(4) synthase (LTC(4) S) is considered a pivotal enzyme for generation of potent proinflammatory mediators, cysteinyl-leukotrienes (cysLTs). LTC(4) S cDNA was cloned in rat basophilic leukemia-1 (RBL-1) cells, and exhibited 84.8% and 94.5% identity with the reported human and mouse LTC(4) S cDNA sequences, respectively. Homology between the rat LTC(4) S amino acid sequence and the corresponding sequences from the other species was 86.5% and 95.3% with human and mouse sequences, respectively. Rat LTC(4) S thus showed extensive homology with both mouse and human cDNA sequences. The active enzyme as assessed by LTC(4) S activity was expressed in COS-7 cells. While RBL-1 cells after the culture for 48 h in the presence of 0.1 microg/ml all trans -retinoic acid (RA) exhibited 27 times higher LTC(4) S activity than control cells, Northern-blot analysis of RA-treated cells showed upregulation of LTC(4) S mRNA. Polyclonal antibody was raised against the synthesized peptide deduced from the nucleotide sequence. Thus, Western-blot analysis of RBL-1 cells treated with RA and COS-7 cells transfected with pcDNA-LTC(4) S commonly showed a band at approximately 18 kDa in each solubilized enzyme solution, but either control cells did not. This cDNA probe and antibody may be useful for investigating the roles of cysLTs in various experimental models of rats.     

Combination of microbial and chemical synthesis for the sustainable production of β-elemene,a promising plant-extracted anticancer compound

Beta-elemene, a class of sesquiterpene derived from the Chinese medicinal herb Curcuma wenyujin, is widely used in clinical medicine due to its broad-spectrum antitumor activity. However, the unsustainable plant extraction prompted the search for environmentally friendly strategies for β-elemene production. In this study, we designed a Yarrowia lipolytica cell factory that can continuously produce germacrene A, which is further converted into β-elemene with 100% yield through a Cope rearrangement reaction by shifting the temperature to 250°C. First, the productivity of four plant-derived germacrene A synthases was evaluated. After that, the metabolic flux of the precursor to germacrene A was maximized by optimizing the endogenous mevalonate pathway, inhibiting the competing squalene pathway, and expressing germacrene A synthase gene in multiple copies. Finally, the most promising strain achieved the highest β-elemene titer reported to date with 5.08 g/L. This sustainable and green method has the potential for industrial β-elemene production.     

Identification of sesquiterpene synthases from Nostoc punctiforme PCC 73102 and Nostoc sp. strain PCC 7120

Cyanobacteria are a rich source of natural products and are known to produce terpenoids. These bacteria are the major source of the musty-smelling terpenes geosmin and 2-methylisoborneol, which are found in many natural water supplies; however, no terpene synthases have been characterized from these organisms to date. Here, we describe the characterization of three sesquiterpene synthases identified in Nostoc sp. strain PCC 7120 (terpene synthase NS1) and Nostoc punctiforme PCC 73102 (terpene synthases NP1 and NP2). The second terpene synthase in N. punctiforme (NP2) is homologous to fusion-type sesquiterpene synthases from Streptomyces spp. shown to produce geosmin via an intermediate germacradienol. The enzymes were functionally expressed in Escherichia coli, and their terpene products were structurally identified as germacrene A (from NS1), the eudesmadiene 8a-epi-α-selinene (from NP1), and germacradienol (from NP2). The product of NP1, 8a-epi-α-selinene, so far has been isolated only from termites, in which it functions as a defense compound. Terpene synthases NP1 and NS1 are part of an apparent minicluster that includes a P450 and a putative hybrid two-component protein located downstream of the terpene synthases. Coexpression of P450 genes with their adjacent located terpene synthase genes in E. coli demonstrates that the P450 from Nostoc sp. can be functionally expressed in E. coli when coexpressed with a ferredoxin gene and a ferredoxin reductase gene from Nostoc and that the enzyme oxygenates the NS1 terpene product germacrene A. This represents to the best of our knowledge the first example of functional expression of a cyanobacterial P450 in E. coli.     

Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of germacrene A,a precursor of beta-elemene

Beta-elemene, a sesquiterpene and the major component of the medicinal herb Curcuma wenyujin, has antitumor activity against various types of cancer and could potentially serve as a potent antineoplastic drug. However, its current mode of production through extraction from plants has been inefficient and suffers from limited natural resources. Here, we engineered a yeast cell factory for the sustainable production of germacrene A, which can be transformed to beta-elemene by a one-step chemical reaction in vitro. Two heterologous germacrene A synthases (GASs) converting farnesyl pyrophosphate (FPP) to germacrene A were evaluated in yeast for their ability to produce germacrene A. Thereafter, several metabolic engineering strategies were used to improve the production level. Overexpression of truncated 3-hydroxyl-3-methylglutaryl-CoA reductase and fusion of FPP synthase with GAS, led to a sixfold increase in germacrene A production in shake-flask culture. Finally, 190.7 mg/l of germacrene A was achieved. The results reported in this study represent the highest titer of germacrene A reported to date. These results provide a basis for creating an efficient route for further industrial application re-placing the traditional extraction of beta-elemene from plant sources.     

Expression of a chimeric nitrate reductase gene in transgenic lettuce reduces nitrate in leaves

 Transgenic plants of four glasshouse-grown lettuce cultivars ('Cortina', 'Evola', 'Flora' and 'Luxor') were obtained by co-cultivating excised cotyledons with Agrobacterium tumefaciens. The Agrobacterium strain LBA4404 contained the binary vector pBCSL16, which carried a nitrate reductase (nia) cDNA linked to CaMV promoter and terminator sequences, and the neomycin phosphotransferase II (nptII) gene. Transformed shoots were selected by their ability to root on medium containing kanamycin sulphate, by a positive NPTII assay and by PCR analysis. The presence of the nia cDNA in transgenic lettuce was confirmed by nitrate reductase (NR) enzymatic assay, a reduction in the nitrate content of leaves and by Southern hybridisation. PCR analysis of cDNA fragments from transgenic plants confirmed that both nia and nptII genes were expressed in first seed-generation (T1) lettuce plants. The commercial importance of reduced nitrate concentrations in lettuce is discussed. Received: 7 January 1998 / Revision received: 24 February 1998 / Accepted: 22 March 1999     

Diversity of sesquiterpene synthases in the basidiomycete Coprinus cinereus

Fungi are a rich source of bioactive secondary metabolites, and mushroom-forming fungi ( Agaricomycetes ) are especially known for the synthesis of numerous bioactive and often cytotoxic sesquiterpenoid secondary metabolites. Compared with the large number of sesquiterpene synthases identified in plants, less than a handful of unique sesquiterpene synthases have been described from fungi. Here we describe the functional characterization of six sesquiterpene synthases (Cop1 to Cop6) and two terpene-oxidizing cytochrome P450 monooxygenases (Cox1 and Cox2) from Coprinus cinereus. The genes were cloned and, except for cop5 , functionally expressed in Escherichia coli and/or Saccharomyces cerevisiae . Cop1 and Cop2 each synthesize germacrene A as the major product. Cop3 was identified as an α-muurolene synthase, an enzyme that has not been described previously, while Cop4 synthesizes δ-cadinene as its major product. Cop6 was originally annotated as a trichodiene synthase homologue but instead was found to catalyse the highly specific synthesis of α-cuprenene. Coexpression of cop6 and the two monooxygenase genes next to it yields oxygenated α-cuprenene derivatives, including cuparophenol, suggesting that these genes encode the enzymes for the biosynthesis of antimicrobial quinone sesquiterpenoids (known as lagopodins) that were previously isolated from C. cinereus and other Coprinus species.     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

Stored mRNA in cotyledons of Vigna unguiculata seeds: nucleotide sequence of cloned cDNA for a stored mRNA and induction of its synthesis by precocious germination

By differential hybridization screening, we previously selected a class of cDNA clones from a gt10 cDNA library that was constructed from the total poly(A)⁺ RNA of mature cowpea cotyledons (Plant Cell Physiol 31: 39–44, 1990). pSAS10, a clone of this class, hybridized with a cDNA probe complementary to poly(A)⁺ RNA from cotyledons collected 1 day after the onset of imbibition (DAI), but not with the cDNA probe from cotyledons at development stage II (13 to 15 days after flowering, DAF). pSAS10 mRNA was detectable only in cotyledons at development stage III (17 to 19 DAF) or later, and its level began to decline when seeds germinated. We have suggested that pSAS10 mRNA is likely to belong to the class of stored mRNA or the mRNA that is formed at the late stage of seed maturation, is conserved in quiescent seeds and becomes functional at the early stage of germination. We determined the nucleotide sequence of pSAS10 cDNA consisting of 459 bp and an approximately 36 bp poly(A) tract, and deduced the amino acid sequence of its product, a 10-kDa cysteine-rich polypeptide. Synthesis of pSAS10 mRNA was induced just before germination began, not only in mature seeds but also in immature seeds even at stages I (9 to 11 DAF) and II (13 to 15 DAF) if they were placed under conditions suitable for germination.