Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Cane, David E.; Sohng, Jae Kyung; Lamberson, Carol R.; Rudnicki, Suzanne M.; Wu, Zezhou; Lloyd, Matthew D.; Oliver, John S.; Hubbard, Brian

doi:10.1021/bi00185a024

Cited by 147 publications

(130 citation statements)

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“…3). In contrast to these eukaryotic isoprenoid cyclases, only limited studies on prokaryotic isoprenoid cyclases have been reported so far [10,62]. Therefore, studies were conducted to investigate the biosynthetic genes and enzymes of isoprenoid antibiotics produced by actinomycetes, particularly focusing on the cloning and characterization of the genes of the mevalonate pathway and the genes of isoprenoid cyclase.…”

Section: Biosynthesis Of Isoprenoidsmentioning

confidence: 99%

“…On the other hand, actinomycete strains produce several isoprenoid compounds such as 2-methylisoborneol, geosmin, squalene-hopene etc., and frontier studies on pentalenene synthase, a sesquiterpene synthase from an actinomycete strain, demonstrated that the enzyme is not membrane-bound and is expressed with relative ease as a recombinant enzyme in E. coli [10]. This enabled us to investigate the properties of biosynthetic enzymes in detail.…”

Section: Introductionmentioning

confidence: 99%

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Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Dairi

2005

J Antibiot

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Most Streptomyces strains are equipped with only the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the formation of isopentenyl diphosphate, a common precursor of isoprenoids. In addition to this pathway, some Streptomyces strains possess the mevalonate (MV) pathway via which isoprenoid antibiotics are produced. We have recently cloned and analyzed the MV pathway gene clusters and their flanking regions from terpentecin, BE-40644, and furaquinocin A producers. All these clusters contained genes coding for mevalonate kinase, mevalonate diphosphate decarboxylase, phosphomevalonate kinase, type 2 IPP isomerase, HMGCoA reductase, and HMG-CoA synthase. The order of each of the open reading frames (ORFs) is also the same, and the respective homologous ORFs show more than 70% amino acid identity with each other. In contrast to these conservative gene organizations, the biosynthetic genes of terpentecin, BE-40644, and furaquinocin A were located just upstream and/or downstream of the MV pathway gene cluster. These facts suggested that all the actinomycete strains possessing both the MV and MEP pathways produce isoprenoid compounds and the biosynthetic genes of one of these isoprenoids usually exist adjacent to the MV pathway gene cluster. Therefore, when the presence of the MV cluster is detected by molecular genetic techniques, isoprenoids may be produced by the cultivation of these actinomycete strains. During the course of these studies, we identified diterpene cyclases possessing unique primary structures that differ from those of eukaryotes and catalyze unique reactions.Keywords isoprenoid, mevalonate pathway, biosynthesis, cyclase, Actinomycetes IntroductionActinomycete strains usually produce a number of secondary metabolites that often possess pharmaceutical activities. Therefore, for a long time, the strains have been used for many screenings to find novel, medically useful compounds. Consequently, more than 60 percent of the known antibiotics, including not only antibacterial antibiotics but also bioactive microbial compounds, have been reported to be produced by actinomycetes [1]. Presently, such pharmaceutically important, novel compounds can be found in the culture broth of actinomycete strains by screening rare actinomycetes, developing new screening strategies, employing sensitive assay methods for the detection of low concentrations of antibiotics, etc. However, sometimes, "semi-new antibiotics"-derivatives of known antibiotics-were isolated; discovering novel antibiotics every year became very difficult.In the past two decades, recombinant DNA technology has come into play in this field. Gene clusters encoding many natural products have been cloned and characterized. Moreover, whole-genome sequencing has uncovered hundreds of candidates for secondary metabolic pathways. Among them, the biosynthetic machinery of nonribosomal peptide and polyketide natural products has been extensively investigated [2ϳ7]. Recent progress in the application of combinatorial biosynthesis methods to these bi...

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Section: Biosynthesis Of Isoprenoidsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Dairi

2005

J Antibiot

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“…Germacradienol/geosmin synthase is atypical in that it consists of two terpene cyclase domains. Typical singledomain SCs characterized from Streptomyces and Nostoc include those involved in the synthesis of pentalenene, epi-isozizaene, avermitilol, germacrene A, 8a-epi-␣-selinene, (Ϫ)-␦-cadinene, and (ϩ)-T-muurolol (7)(8)(9)(10)(11)(12)(13). However, recent genome sequencing projects have revealed a large number of potential bacterial SC genes.…”

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confidence: 99%

Characterization of a Novel Sesquiterpene Cyclase Involved in (+)-Caryolan-1-ol Biosynthesis in Streptomyces griseus

Nakano

Horinouchi²,

Ohnishi

2011

Journal of Biological Chemistry

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Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (؉)-caryolan-1-ol using spectroscopic analyses. (؉)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (؉)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (؉)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (؉)-␤-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (؉)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (؉)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (؉)-␤-caryophyllene. Several ␤-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.Terpenoids are the largest family of natural products, including over 24,000 known compounds. They have various biological functions and act as flavorings, pigments, antibiotics, hormones, and antitumor agents. Most terpenoids have been isolated from plants and fungi and only a few from bacteria. All terpenoids are derived from the C 5 precursors isopentenyl diphosphate and dimethylallyl diphosphate. The successive condensation of isopentenyl diphosphate and dimethylallyl diphosphate gives rise to linear isoprenyl diphosphate precursors of various chain lengths, such as geranyl diphosphate (C 10 ), farnesyl diphosphate (FPP, 2 C 15 ), and geranylgeranyl diphosphate (C 20 ). These linear substrates are generally cyclized by terpenoid cyclases to produce the parent skeletons of monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), and other homologs. Terpenoid cyclases are therefore the first key enzymes in pathways leading to the diverse range of terpenoids. A large number of terpenoid cyclases have been reported from eukaryotes, and these have been studied extensively (1, 2). In contrast, at present there are only a small number of reports of bacterial terpenoid cyclases. Most of the bacterial terpenoid cyclases reported are sesquiterpene cyclases (SCs). Bacterial SCs show no significant amino acid sequence identity with eukaryotic S...

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“…The pentalenene synthase gene from Streptomyces sp. UC5319 encodes a protein of M D 38 kDa, which has been cloned and expressed at high levels in Escherichia coli (11).…”

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confidence: 99%

Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis

Cane

Watt²

2003

Proc. Natl. Acad. Sci. U.S.A.

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147

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The PCR has been used to amplify a 2,181-bp ORF from Streptomyces coelicolor A3(2), designated SC9B1.20 ‫؍(‬ SCO6073), encoding a protein of 726 amino acids and showing significant sequence similarity at the deduced amino acid level in both the N-terminal and C-terminal halves to the known sesquiterpene synthase pentalenene synthase. The full-length recombinant protein was expressed at high levels in Escherichia coli and shown to catalyze the Mg 2؉ -dependent conversion of farnesyl diphosphate to the sesquiterpene alcohol (4S, 7R)-germacra-1 (10)E, 5E-diene-11-ol. The enzymatic cyclization had a k cat of 6.2 ؎ 0.5 ؋ 10 ؊3 s ؊1 and a Km for farnesyl diphosphate of 62 ؎ 8 nM. Expression of the Nterminal (366 amino acids) domain of the SC9B1.20 protein also gave a fully functional cyclase which converted farnesyl diphosphate to the identical sesquiterpene alcohol with a slightly lower k cat of 3.2 ؎ 0.4 ؋ 10 ؊3 s ؊1 and a twofold greater km of 115 ؎ 14 nM. By contrast, the expressed C-terminal domain of SC9B1.20 had no farnesyl diphosphate cyclase activity. The formation of the germacradienol seems to be the committed step in the formation of geosmin, the characteristic odoriferous constituent of Streptomyces species.secondary metabolism ͉ sesquiterpene synthase ͉ Streptomyces gene

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Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Cited by 147 publications

References 51 publications

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Studies on Biosynthetic Genes and Enzymes of Isoprenoids Produced by Actinomycetes

Characterization of a Novel Sesquiterpene Cyclase Involved in (+)-Caryolan-1-ol Biosynthesis in Streptomyces griseus

Expression and mechanistic analysis of a germacradienol synthase from Streptomyces coelicolor implicated in geosmin biosynthesis

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