Heo Kyung Taek, Kang Sun-Young, Hong Young-Soo
Chemical Biology Research Center, Korea Research Institute of Bioscience and Biotechnology, 30 Yeongudanji-ro, Ochang-eup, Chungbuk, 363-883, Republic of Korea.
Major of Biomolecular Science, University of Science and Technology, Daejeon, Republic of Korea.
Microb Cell Fact. 2017 Feb 15;16(1):30. doi: 10.1186/s12934-017-0644-6.
Pterostilbene, a structural analog of resveratrol, has higher oral bioavailability and bioactivity than that of the parent compound; but is far less abundant in natural sources. Thus, to efficiently obtain this bioactive resveratrol analog, it is necessary to develop new bioproduction systems.
We identified a resveratrol O-methyltransferase (ROMT) function from a multifunctional caffeic acid O-methyltransferase (COMT) originating from Arabidopsis, which catalyzes the transfer of a methyl group to resveratrol resulting in pterostilbene production. In addition, we constructed a biological platform to produce pterostilbene with this ROMT gene. Pterostilbene can be synthesized from intracellular L-tyrosine, which requires the activities of four enzymes: tyrosine ammonia lyase (TAL), p-coumarate:CoA ligase (CCL), stilbene synthase (STS) and resveratrol O-methyltransferase (ROMT). For the efficient production of pterostilbene in E. coli, we used an engineered E. coli strain to increase the intracellular pool of L-tyrosine, which is the initial precursor of pterostilbene. Next, we tried to produce pterostilbene in the engineered E. coli strain using L-methionine containing media, which is used to increase the intracellular pool of S-adenosyl-L-methionine (SAM). According to this result, pterostilbene production as high as 33.6 ± 4.1 mg/L was achieved, which was about 3.6-fold higher compared with that in the parental E. coli strain harboring a plasmid for pterostilbene biosynthesis.
As a potential phytonutrient, pterostilbene was successfully produced in E. coli from a glucose medium using a single vector system, and its production titer was also significantly increased using a L-methionine containing medium in combination with a strain that had an engineered metabolic pathway for L-tyrosine. Additionally, we provide insights into the dual functions of COMT from A. thaliana which was characterized as a ROMT enzyme.
紫檀芪是白藜芦醇的结构类似物，其口服生物利用度和生物活性均高于母体化合物；但在天然来源中含量却少得多。因此，为了高效获得这种具有生物活性的白藜芦醇类似物，有必要开发新的生物生产系统。
我们从源自拟南芥的多功能咖啡酸O-甲基转移酶（COMT）中鉴定出白藜芦醇O-甲基转移酶（ROMT）功能，该酶催化甲基转移至白藜芦醇从而产生紫檀芪。此外，我们构建了一个利用该ROMT基因生产紫檀芪的生物平台。紫檀芪可由细胞内的L-酪氨酸合成，这需要四种酶的活性：酪氨酸解氨酶（TAL）、对香豆酸：辅酶A连接酶（CCL）、芪合酶（STS）和白藜芦醇O-甲基转移酶（ROMT）。为了在大肠杆菌中高效生产紫檀芪，我们使用了一种工程化大肠杆菌菌株来增加L-酪氨酸的细胞内池，L-酪氨酸是紫檀芪的初始前体。接下来，我们尝试在含有L-甲硫氨酸的培养基中利用工程化大肠杆菌菌株生产紫檀芪，L-甲硫氨酸用于增加细胞内S-腺苷-L-甲硫氨酸（SAM）池。根据这一结果，实现了高达33.6±4.1mg/L的紫檀芪产量，与携带紫檀芪生物合成质粒的亲本大肠杆菌菌株相比，产量提高了约3.6倍。
作为一种潜在的植物营养素，紫檀芪利用单一载体系统在大肠杆菌中成功地从葡萄糖培养基中生产出来，并且使用含有L-甲硫氨酸的培养基结合具有L-酪氨酸工程代谢途径的菌株，其产量也显著提高。此外，我们还深入了解了拟南芥COMT作为ROMT酶的双重功能。
