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. 2008 May 27;105(21):7422-7.
doi: 10.1073/pnas.0802312105. Epub 2008 May 20.

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

Mamoru Komatsu  1 , Muneya Tsuda, Satoshi Omura, Hideaki Oikawa, Haruo Ikeda
Affiliations

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

Mamoru Komatsu et al. Proc Natl Acad Sci U S A. .

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.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Structures of microbial volatile terpenoid metabolites, 2-MIB (Left; 1,2,7,7-tetramethyl-exo-bicycloheptan-2-ol), geosmin (Center; 4,8a-dimethyl-octahydro-naphthalen-4a-ol) and albaflavenone (Right; 2,6,7,7-tetramethyltricyclo [6.2.1.01,5]undec-5-en-4-one).
Fig. 2.
Fig. 2.
Phylogenetic analysis of terpene cyclases from bacterial databases. Abbreviations: alr4685 (322 aa; NP_488725), Nostoc sp. PCC 7120; Ava_1982 (322 aa; YP_322499), Anabaena variabilis ATCC 29413; BURPS1106A_A1634 (370 aa; YP_001075668), B. pseudomallei 1106a; BURPS1710b_A0219 (394 aa; YP_335378), B. pseudomallei 1710b; BURPS668_A0947 (339 aa; YP_001061946) and BURPS668_A1721 (370 aa; YP_001062716), B. pseudomallei 668; FRAAL_1336 (338 aa; YP_711586) and FRAAL_6507 (758 aa; YP_716636), Frankia alni ACN14a; Francci3_4231 (751 aa; YP_483306), Frankia sp. CcI3; Franean1_5559 (750 aa; YP_001509819), Frankia sp. EAN1pec; MOL (400 aa), M. olivasterospora KY11048; MXAN_6247 (755 aa; YP_634376), Myxococcus xanthus DK 1622; Pfl_1841 (335 aa; YP_347573), Pseudomonas fluorescens PfO-1; Rcas_0622 (326 aa; YP_001430766), Roseiflexus castenholzii DSM 13941; RoseRS_3509 (326 aa; YP_001277817), Roseiflexus sp. RS-1; Rxyl_0493 (324 aa; YP_643279), R. xylanophilus DSM 9941; SACE_3187 (758 aa; YP_001105388), SACE_3722 (354 aa; YP_001105919), SACE_3977 (732 aa; YP_001106173) and SACE_4907 (763 aa; YP_001107098), Sa. erythraea NRRL2338; SAML0357 (440 aa; CAJ89344) and SAMR0831 (343 aa; CAJ88540), S. ambofaciens ISP5053; SAV76 (335 aa; NP_821250), SAV2163 (725 aa; NP_823339), SAV2998 (336 aa; NP_824174) and SAV3032 (363 aa; NP_824208), S. avermitilis MA-4680; SCAB20121 (735 aa), SCAB5041 (440 aa), SCAB73741 (338 aa) and SCAB82161 (349 aa), S. scabies 87.22; SCO5222 (361 aa; NP_629369), SCO6073 (726 aa; NP_630182) and SCO7700 (440 aa; NP_733742), S. coelicolor A3(2); SGR1269 (437 aa), SGR2079 (335 aa), SGR6065 (339 aa) and SGR6839 (737 aa), S. griseus IF013350; SLA (481 aa), S. lasaliensis NRRL3382R; BAB39207 (311 aa; BAB39207), Kitasatospora griseola diterpene cyclase; Q55012 (337 aa; Q55012), Streptomyces sp. UC5319 pentalenene synthase; ZP_01648856 (298 aa; YP_001536181), Salinispora arenicola CNS-205 terpene synthase.
Fig. 3.
Fig. 3.
Alignment of amino acid sequences of bacterial terpene cyclases with predicted cyclases. Shadow boxes indicate metal (Mg2+)-binding motif of terpene cyclase. The strain name of each protein is described in Fig. 2. SAV indicates deduced amino acid sequence of the truncated terpene cyclase gene of S. avermitilis. SCO5222 and SAV3032, Q55012 and SAV2998, SCO6073 and SAV2163, and BAB39207 were characterized as epi-isozizaene synthases, pentalenene synthases, germacradienol/geosmin synthases, and diterpene cyclase for terpentecin biosynthesis, respectively.
Fig. 4.
Fig. 4.
Organization of genes encoding predicted monoterpene cyclases and flanking genes. All predicted monoterpene cyclase genes are located in the chromosomes, but the gene of S. lasaliensis resided in a giant linear plasmid pKSL. The grayed, filled, and oblique-lined arrows (or boxes) indicate cyclic nucleotide-binding protein, monoterpene cyclase, and methyltransferase genes, respectively. The opened arrows are hypothetical or other function protein-coding genes. S. scabies 87.22(1) and -(2) indicate regions around SCAB5041 and SCAB82161, respectively.
Fig. 5.
Fig. 5.
Volatile monoterpenoid metabolites, 2-MIB (A) and 2-MB (B), and sesquiterpenoid metabolite, geosmin (C) produced by actinomycete strains carrying predicted monoterpene cyclase and methyltransferase genes. All microorganisms were grown on SFM (left half from dashed line) and M4YE (right half from dashed line) agar plates. The strain names are as follows: SCO, S. coelicolor A3(2); SGR, S. griseus; SACE, Sa. erythraea; SAM, S. ambofaciens; SLA, S. lasaliensis; MOL, M. olivasterospora; SAV, S. avermitilis; U1717R, S. ambofaciens deletion mutant.
Fig. 6.
Fig. 6.
Biosynthetic mechanism from GPP to 2-MIB (or 2-MB).
See this image and copyright information in PMC

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