Identification and functional analysis of genes controlling biosynthesis of 2-methylisoborneol

Abstract

To identify the genes for biosynthesis of the off-flavor terpenoid alcohol, 2-methylisoborneol (2-MIB), the key genes encoding monoterpene cyclase were located in bacterial genome databases by using a combination of hidden Markov models, protein-family search, and the sequence alignment of their gene products. Predicted terpene cyclases were classified into three groups: sesquiterpene, diterpene, and other terpene cyclases. Genes of the terpene cyclase group that form an operon with a gene encoding S-adenosyl-L-methionine (SAM)-dependent methyltransferase were found in genome data of seven microorganisms belonging to actinomycetes, Streptomyces ambofaciens ISP5053, Streptomyces coelicolor A3(2), Streptomyces griseus IFO13350, Streptomyces lasaliensis NRRL3382R, Streptomyces scabies 87.22, Saccharopolyspora erythraea NRRL2338, and Micromonospora olivasterospora KY11048. Among six microorganisms tested, S. ambofaciens, S. coelicolor A3(2), S. griseus, and S. lasaliensis produced 2-MIB but M. olivasterospora produced 2-methylenebornane (2-MB) instead. The regions containing monoterpene cyclase and methyltransferase genes were amplified by PCR from S. ambofaciens, S. lasaliensis, and Saccharopolyspora erythraea, respectively, and their genes were heterologously expressed in Streptomyces avermitilis, which was naturally deficient of 2-MIB biosynthesis by insertion and deletion. All exoconjugants of S. avermitilis produced 2-MIB. Full-length recombinant proteins, monoterpene cyclase and methyltransferase of S. lasaliensis were expressed at high level in Escherichia coli. The recombinant methyltransferase catalyzed methylation at the C2 position of geranyl diphosphate (GPP) in the presence of SAM. 2-MIB was generated by incubation with GPP, SAM, recombinant methyltransferase, and terpene cyclase. We concluded that the biosynthetic pathway involves the methylation of GPP by GPP methyltransferase and its subsequent cyclization by monoterpene cyclase to 2-MIB.genome mining ͉ methyltransferase ͉ monoterpene cyclase T erpenoid metabolites, monoterpenes, sesquiterpenes, and diterpenes, have been isolated from terrestrial and marine plants or from fungi, with only a relatively minor fraction from prokaryotes. Their compounds are used as antibiotics, hormones, flavor or odor constituents, and pigments. Some of them possess other physiologically or commercially important properties (1, 2). Three terpenoid compounds (Fig. 1), 2-methylisoborneol (2-MIB), geosmin, and albaflavenone are known as odorous and volatile microbial metabolites. The former two terpenoid alcohols are the most frequently found secondary metabolites of actinomycetes (3-5), filamentous cyanobacteria (6-8), myxobacteria (9), and fungi (10, 11), and account for many odor problems encountered with freshwater or with fish (7,(12)(13)(14). Geosmin is also known to contribute to the characteristic earthy red beet flavor (15). 2-MIB is related to the musty-earthy notes in Brie and Camembert cheese flavor (16). An ␣,␤-unsaturated sesquiterpene ketone, albaflavenone, ...

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

“…We have recently examined heterologous expression of actinomycete monoterpene synthase genes in S. avermitilis (26). We have now extended these observations to the expression plant terpene synthase genes in S. avermitilis.…”

“…S8), were for gene disruption of the Streptomyces genome by allelic replacement. Cloning, transformation, conjugation, and Escherichia coli strains used were described previously (26,32). The mutant loxP sequence (mut-loxP) (33) was used for the deletion cassettes.…”

Terpene synthases are widely distributed in bacteria

“…Application of the first generation HMM (PF03936) to 1,922,990 bacterial proteins had originally revealed 41 predicted terpene synthases that were classified by phylogenetic analysis into three main groups consisting of monoterpene, sesquiterpene, and diterpene synthases (27). By contrast, the phylogenetic tree derived from the newly identified 262 bacterial terpene synthases uncovered by the third generation HMM model is considerably more complex, exhibiting a much higher degree of branching, thus indicating that bacterial terpene synthases are far more structurally diverse than previously indicated.…”

“…The resultant mutant host has been shown to be particularly suited for the production of a wide variety of natural products (27,38,45,46). This versatile engineered host system, which has been stripped of all of its native terpene synthase genes and is, thus, unable to produce endogenous terpenoid metabolites that might otherwise interfere with production and analysis of heterologously expressed terpenes and their derivatives, is ideally suited to the generation of exogenous terpenoid metabolites in quantities sufficient for their rigorous spectroscopic analysis.…”

“…To address this challenge, we recently described the successful application of an alternative genome mining strategy for the discovery of previously unidentified bacterial terpene synthases based on the use of hidden Markov models (HMMs) and protein families database (Pfam) searching methods (26). These initial efforts identified a large number of previously unrecognized bacterial terpene synthase candidates, including the discovery of the previously unidentified synthase for the methylated monoterpene 2-methylisoborneol, and led to the heterologous expression of the relevant genes that produce 2-methylisoborneol and 2-methylenebornane from 2-methylgeranyl diphosphate (27). We subsequently refined and expanded the set of HMM parameters using as a reference set exclusively the group of newly predicted bacterial terpene synthases in distinction to the original HMM model (PF03936), which had been based on plant terpene synthases.…”