Yu Zhang1, Shiquan Bian1, Xiaofeng Liu1, Ning Fang1, Chunkai Wang1, Yanhua Liu1, Yongmei Du1, Michael P Timko2, Zhongfeng Zhang3, Hongbo Zhang4. 1. TRI of CAAS-UVA Joint Laboratory of Synthetic Biology, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 266101, China. 2. Department of Biology, University of Virginia, 485 McCormick Road, Charlottesville, VA, 22904, USA. 3. TRI of CAAS-UVA Joint Laboratory of Synthetic Biology, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 266101, China. zhangzhongfeng@caas.cn. 4. TRI of CAAS-UVA Joint Laboratory of Synthetic Biology, Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao, 266101, China. zhanghongbo@caas.cn.
Abstract
BACKGROUND: Cembranoids are one kind of diterpenoids with multiple biological activities. The tobacco cembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. RESULTS: We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. CONCLUSIONS: We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.
BACKGROUND:Cembranoids are one kind of diterpenoids with multiple biological activities. The tobaccocembratriene-ol (CBT-ol) and cembratriene-diol (CBT-diol) have high anti-insect and anti-fungal activities, which is attracting great attentions for their potential usage in sustainable agriculture. Cembranoids were supposed to be formed through the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway, yet the involvement of mevalonate (MVA) pathway in their synthesis remains unclear. Exploring the roles of MVA pathway in cembranoid synthesis could contribute not only to the technical approach but also to the molecular mechanism for cembranoid biosynthesis. RESULTS: We constructed vectors to express cembratriene-ol synthase (CBTS1) and its fusion protein (AD-CBTS1) containing an N-terminal GAL4 AD domain as a translation leader in yeast. Eventually, the modified enzyme AD-CBTS1 was successfully expressed, which further resulted in the production of CBT-ol in the yeast strain BY-T20 with enhanced MVA pathway for geranylgeranyl diphosphate (GGPP) production but not in other yeast strains with low GGPP supply. Subsequently, CBT-diol was also synthesized by co-expression of the modified enzyme AD-CBTS1 and BD-CYP450 in the yeast strain BY-T20. CONCLUSIONS: We demonstrated that yeast is insensitive to the tobacco anti-fungal compound CBT-ol or CBT-diol and could be applied to their biosynthesis. This study further established a feasibility for cembranoid production via the MVA pathway and provided an alternative bio-approach for cembranoid biosynthesis in microbes.
Authors: Farrukh Aqil; Maryam Zahin; Khalid A El Sayed; Iqbal Ahmad; Khaled Y Orabi; Jamal M Arif Journal: Drug Chem Toxicol Date: 2011-04 Impact factor: 3.356
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