Komatsu Mamoru, Tsuda Muneya, Omura Satoshi, Oikawa Hideaki, Ikeda Haruo
Kitasato Institute for Life Sciences, Kitasato University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228-8555, Japan.
Proc Natl Acad Sci U S A. 2008 May 27;105(21):7422-7. doi: 10.1073/pnas.0802312105. Epub 2008 May 20.
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.
为了鉴定产生异味萜类醇2-甲基异冰片（2-MIB）生物合成的基因，通过结合隐马尔可夫模型、蛋白质家族搜索及其基因产物的序列比对，在细菌基因组数据库中定位了编码单萜环化酶的关键基因。预测的萜类环化酶分为三组：倍半萜、二萜和其他萜类环化酶。在属于放线菌的七种微生物的基因组数据中发现了与编码S-腺苷-L-甲硫氨酸（SAM）依赖性甲基转移酶的基因形成操纵子的萜类环化酶组基因，这些微生物包括浅青紫链霉菌ISP5053、天蓝色链霉菌A3(2)、灰色链霉菌IFO13350、拉萨链霉菌NRRL3382R、疮痂链霉菌87.22、红色糖多孢菌NRRL2338和橄榄孢小单孢菌KY11048。在所测试的六种微生物中，浅青紫链霉菌、天蓝色链霉菌A3(2)、灰色链霉菌和拉萨链霉菌产生2-MIB，但橄榄孢小单孢菌产生的是2-亚甲基冰片（2-MB）。分别从浅青紫链霉菌、拉萨链霉菌和红色糖多孢菌中通过PCR扩增出包含单萜环化酶和甲基转移酶基因的区域，并将其基因在阿维链霉菌中进行异源表达，阿维链霉菌因插入和缺失而天然缺乏2-MIB生物合成。阿维链霉菌的所有外接合子都产生了2-MIB。拉萨链霉菌的全长重组蛋白单萜环化酶和甲基转移酶在大肠杆菌中高水平表达。重组甲基转移酶在SAM存在的情况下催化香叶基二磷酸（GPP）的C2位甲基化。通过与GPP、SAM、重组甲基转移酶和萜类环化酶一起孵育生成了2-MIB。我们得出结论，生物合成途径涉及GPP甲基转移酶对GPP的甲基化及其随后由单萜环化酶环化为2-MIB。
