Geosmin Biosynthesis in Streptomyces avermitilis. Molecular Cloning, Expression, and Mechanistic Study of the Germacradienol/Geosmin Synthase

Cane, David E.; He, Xiaofei; Kobayashi, Seiji; Ōmura, Satoshi; Ikeda, Haruo

doi:10.1038/ja.2006.66

Cited by 119 publications

(109 citation statements)

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“…Conditions for seed culture and avermectin production medium were described previously (34). Streptomycin, cephamycin C, and pladienolide production media of the original strains are described in refs.…”

Section: Methodsmentioning

confidence: 99%

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Komatsu

Uchiyama

Ōmura

et al. 2010

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

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463

380

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To construct a versatile model host for heterologous expression of genes encoding secondary metabolite biosynthesis, the genome of the industrial microorganism Streptomyces avermitilis was systematically deleted to remove nonessential genes. A region of more than 1.4 Mb was deleted stepwise from the 9.02-Mb S. avermitilis linear chromosome to generate a series of defined deletion mutants, corresponding to 83.12-81.46% of the wild-type chromosome, that did not produce any of the major endogenous secondary metabolites found in the parent strain. The suitability of the mutants as hosts for efficient production of foreign metabolites was shown by heterologous expression of three different exogenous biosynthetic gene clusters encoding the biosynthesis of streptomycin (from S. griseus Institute for Fermentation, Osaka [IFO] 13350), cephamycin C (from S. clavuligerus American type culture collection (ATCC) 27064), and pladienolide (from S. platensis Mer-11107). Both streptomycin and cephamycin C were efficiently produced by individual transformants at levels higher than those of the native-producing species. Although pladienolide was not produced by a deletion mutant transformed with the corresponding intact biosynthetic gene cluster, production of the macrolide was enabled by introduction of an extra copy of the regulatory gene pldR expressed under control of an alternative promoter. Another mutant optimized for terpenoid production efficiently produced the plant terpenoid intermediate, amorpha-4,11-diene, by introduction of a synthetic gene optimized for Streptomyces codon usage. These findings highlight the strength and flexibility of engineered S. avermitilis as a model host for heterologous gene expression, resulting in the production of exogenous natural and unnatural metabolites.genome engineering | host development | natural products A prominent property of members of the genus Streptomyces is the ability to produce numerous secondary metabolites, including antibiotics and other biologically active compounds of proven value in human and veterinary medicine and agriculture; they are also useful as biochemical tools. These structurally diverse metabolites collectively express not only antibacterial, antifungal, antiviral, and antitumor activities but also antihypertensive and immunosuppressant properties. Streptomyces have been a rich source of pharmaceutical compounds in which common cellular intermediates, including amino acids, sugars, fatty acids, and terpenes, are combined to give more complex structures by defined biochemical pathways. Genomic analysis of three species of Streptomyces, S. avermitilis (1, 2), S. coelicolor A3(2) (3), and S. griseus (4), has revealed that these microorganisms each have large linear chromosomes that harbor over 20 secondary metabolic gene clusters encoding the biosynthesis of polyketides by polyketide synthases (PKSs), peptides by nonribosomal peptide synthetases (NRPSs), bacteriocins, terpenoids, shikimate-derived metabolites, aminoglycosides, and other natural products (5).T...

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Section: Methodsmentioning

confidence: 99%

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Komatsu

Uchiyama

Ōmura

et al. 2010

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

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463

380

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“…Moreover, expression in Escherichia coli of heterologous bacterial terpene synthases frequently results in the formation of insoluble inclusion bodies. Thus, although recombinant S. avermitilis pentalenene synthase (SAV_2998) could be expressed in E. coli in soluble form, the three remaining S. avermitilis terpene synthases [geosmin synthase (SAV_2163) (42), epi-isozizaene synthase (SAV_3032) (43), and avermitilol synthase (SAV_76) (44)] were all obtained as inclusion bodies when expressed in E. coli. In these cases, the geosmin and epi-isozizaene synthases could fortunately be refolded to obtain soluble proteins that retained the requisite terpene synthase activity, whereas soluble avermitilol synthase could be expressed in E. coli using a synthetic gene with codons optimized for expression in E. coli.…”

Section: Heterologous Expression Of Genes Encoding Bacterial Terpenementioning

confidence: 99%

Terpene synthases are widely distributed in bacteria

Yamada

Kuzuyama

Komatsu

et al. 2014

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

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Odoriferous terpene metabolites of bacterial origin have been known for many years. In genome-sequenced Streptomycetaceae microorganisms, the vast majority produces the degraded sesquiterpene alcohol geosmin. Two minor groups of bacteria do not produce geosmin, with one of these groups instead producing other sesquiterpene alcohols, whereas members of the remaining group do not produce any detectable terpenoid metabolites. Because bacterial terpene synthases typically show no significant overall sequence similarity to any other known fungal or plant terpene synthases and usually exhibit relatively low levels of mutual sequence similarity with other bacterial synthases, simple correlation of protein sequence data with the structure of the cyclized terpene product has been precluded. We have previously described a powerful search method based on the use of hidden Markov models (HMMs) and protein families database (Pfam) search that has allowed the discovery of monoterpene synthases of bacterial origin. Using an enhanced set of HMM parameters generated using a training set of 140 previously identified bacterial terpene synthase sequences, a Pfam search of 8,759,463 predicted bacterial proteins from public databases and in-house draft genome data has now revealed 262 presumptive terpene synthases. The biochemical function of a considerable number of these presumptive terpene synthase genes could be determined by expression in a specially engineered heterologous Streptomyces host and spectroscopic identification of the resulting terpene products. In addition to a wide variety of terpenes that had been previously reported from fungal or plant sources, we have isolated and determined the complete structures of 13 previously unidentified cyclic sesquiterpenes and diterpenes.terpene synthase | bacteria | heterologous expression

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“…96 They inquired whether I would be interested in collaborating to assign the actual biochemical function of the four putative terpene synthase genes that were harbored in the S. avermitilis genome. In fact, it was evident that one of these S. avermitilis terpene synthase genes was a geosmin synthase, 66 while a second corresponded to epi-isozizaene synthase whose function in S. coelicolor we had just established. 97,98 A third gene was subsequently shown by Dr Wayne Chou in my laboratory to control the cyclization of FPP to a previously unknown sesquiterpene alcohol that we named avermitilol.…”

Section: Enzymology and Molecular Genetics Of Natural Product Biosyntmentioning

confidence: 95%

“…My laboratory has published more than a dozen papers in Journal of Antibiotics, covering our studies of both terpenoid and polyketide biosynthesis. [63][64][65][66][67] SABBATICAL AT THE UNIVERSITY OF CAMBRIDGE AND THE JOHN INNES CENTRE In 1989, after 6 years as Chair of the Brown Chemistry Department, I spent a 1-year sabbatical leave at the University of Cambridge. My host was Dr Chris Abell, who had been a postdoc in my laboratory in 1982-1983 before returning to a faculty position in the Department of Chemistry in Cambridge.…”

Section: Visiting Japanmentioning

confidence: 99%

Nature as organic chemist

Cane

2016

J Antibiot

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Figure 15 Stereospecific reduction of (2R)-2-methyl-3-ketopentanoyl-ACP by recombinant ketoreductase (KR) domains: EryKR6, from module 6 of the 6dEB synthase (DEBS); TylKR1, from module 1 of the tylactone synthase; EryKR1, from DEBS module 1; RifKR7, from module 7 of the rifamycin PKS. The (2R)-2-methyl-3-ketopentanoyl-ACP substrate is generated in situ by a reconstituted mixture of dissected PKS domains, Ery[KS6][AT6] (ketosynthase-acyl transferase didomain) and EryACP6 are both from DEBS module 6. Editorial

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Geosmin Biosynthesis in Streptomyces avermitilis. Molecular Cloning, Expression, and Mechanistic Study of the Germacradienol/Geosmin Synthase

Cited by 119 publications

References 27 publications

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

Terpene synthases are widely distributed in bacteria

Nature as organic chemist

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