Significance Terpenes are generally considered to be plant or fungal metabolites, although a small number of odoriferous terpenes of bacterial origin have been known for many years. Recently, ...extensive bacterial genome sequencing and bioinformatic analysis of deduced bacterial proteins using a profile based on a hidden Markov model have revealed 262 distinct predicted terpene synthases. Although many of these presumptive terpene synthase genes seem to be silent in their parent microorganisms, controlled expression of these genes in an engineered heterologous Streptomyces host has made it possible to identify the biochemical function of the encoded terpene synthases. Genes encoding such terpene synthases have been shown to be widely distributed in bacteria and represent a fertile source for discovery of new natural products.
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.
Five pairs of bacterial type-A and type-B diterpene synthases have been characterized: BAD86798/BAD86797, AHK61133/AHK61132, BAB39207/BAB39206, CldD/CldB and RmnD/RmnB, and are involved in the ...formation of pimara-9(11),15-diene, terpente-3,13,15-triene and labda-8(17),12(E),14-triene. Mining of bacterial genome data revealed an additional four pairs of type-A and type-B diterpene synthases: Sros_3191/Sros_3192 of Streptosporangium roseum DSM 43021, Sare_1287/Sare_1288 of Salinispora arenicola CNS-205, SCLAV_5671/SCLAV_5672 and SCLAV_p0491/SCLAV_p0490 of Streptomyces clavuligerus ATCC 27064. Since SCLAV_p0491/SCLAV_p0490 is similar to the labdane-type diterpene synthase pairs, CldD/CldB and RmnD/RmnB based on the alignment of the deduced amino acid sequences and phylogenetic analyses of the aligned sequences, these predicted diterpene synthases were characterized by an enzymatic reaction using a pair of recombinant type-A and type-B diterpene synthases prepared in Escherichia coli and the heterologous expression of two genes encoding type-A and type-B diterpene synthases in an engineered Streptomyces host. The generation of labda-8(17),12(E),14-triene (1) by CldB and CldD was reconfirmed by enzymatic synthesis. Furthermore, labda-8(17),13(16),14-triene (2) was generated by SCLAV_p0491 and CldB, and ladba-7,12(E),14-triene (3) by CldD and SCLAV_p0490. SCLAV_p0491 and SCLAV_p0490 catalyzed the generation of the novel diterpene hydrocarbon, labda-7,13(16),14-triene (4).
An industrial microorganism, Streptomyces avermitilis, which is a producer of anthelmintic macrocyclic lactones, avermectins, has been constructed as a versatile model host for heterologous ...expression of genes encoding secondary metabolite biosynthesis. Twenty of the entire biosynthetic gene clusters for secondary metabolites were successively cloned and introduced into a versatile model host S. avermitilis SUKA17 or 22. Almost all S. avermitilis transformants carrying the entire gene cluster produced metabolites as a result of the expression of biosynthetic gene clusters introduced. A few transformants were unable to produce metabolites, but their production was restored by the expression of biosynthetic genes using an alternative promoter or the expression of a regulatory gene in the gene cluster that controls the expression of biosynthetic genes in the cluster using an alternative promoter. Production of metabolites in some transformants of the versatile host was higher than that of the original producers, and cryptic biosynthetic gene clusters in the original producer were also expressed in a versatile host.
Mining of bacterial genome data has revealed numerous presumptive terpene synthases. Heterologous expression of several putative terpene synthase genes in an engineered Streptomyces host has revealed ...13 newly discovered terpenes whose GC-MS and NMR data did not match with any known compounds in spectroscopic databases. Each of the genes encoding the corresponding terpene synthases were silent in their parent microorganisms. Heterologous expression and detailed NMR spectroscopic analysis allowed assignment of the structures of 13 new cyclic terpenes. Among these newly identified compounds, two were found to be linear triquinane sesquiterpenes that have never previously been isolated from bacteria or any other source. The remaining 11 new compounds were shown to be diterpene hydrocarbons and alcohol, including hydropyrene (1), hydropyrenol (2), tsukubadiene (11) and odyverdienes A (12) and B (13) each displaying a novel diterpene skeleton that had not previously been reported.
In the Yodo River system, where various ayu stocks are released, it is not clear which forms of ayu, amphidromous or landlocked, are established and contribute to reproduction. Microsatellite DNA ...polymorphism analysis was used to determine the phylogeny of spawning stocks, estuarine and coastal larvae and juveniles, and upstream migrants in the Yodo River system to elucidate their reproductive contribution and presence. In all the living seasons, we confirmed that there were mainly individuals belonging to the amphidromous form, but we also confirmed individuals from the landlocked form, and it became clear that there was a high possibility that ayu from the landlocked form also reproduced.
Terpenoid compounds are generally considered to be plant or fungal metabolites, although a small number of odorous terpenoid metabolites of bacterial origin have been known for many years. Recently, ...extensive bacterial genome sequencing and bioinformatic analysis of deduced bacterial proteins using a profile hidden Markov model have revealed more than a hundred distinct predicted terpene synthase genes. Although some of these synthase genes might be silent in the parent microorganisms under normal laboratory culture conditions, the controlled overexpression of these genes in a versatile heterologous host has made it possible to identify the biochemical function of cryptic genes and isolate new terpenoid metabolites.
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