檀香烯与檀香醇的合成生物学优产研究

Sandalwood oil is one of the most precious essential oils in the world. However, it is hard to meet the strong demand of the Sandalwood oil market because of the shortage of Sandalwood supply and the irreplaceability of Sandalwood oil by chemical synthetic substitutes. The santalene synthase (SaSS) gene and cytochrome P450 monooxygenase (Sa-CYP76F39v1) gene from Santalum album were selected for codon optimization and synthesis, cloning, in vitro and in vivo expression and, functional identification in this project. Based on bioinformatics analysis, the key amino acid sites of SaSS that catalyze substrate FPP will be mutated. The relationship between amino acid sequence and function, as well as spatial structure and function of SaSS will be explored. The consequences will help us to understand the biosynthetic mechanism of santalene, and to expand our knowledge about the function of sesquiterpene synthase genes, as well as their relationship of systematic evolution. An expression cassette module combined the high-activity SaSS mutant gene with Sa-CYP76F39v1 gene will be constructed by synthetic biology technique. Applying the CRISPR-Cas9 gene editing system, the copy number of expression cassette module will be increased by cutting the multi-copy δ loci of S. cerevisiae. Meanwhile, the MVA metabolic pathway of S. cerevisiae chassis cells will be optimized. The yield of santalene/santalenol will be improved by these approaches. The results of this project will provide the engineered S. cerevisiae with high yield of santalene/santalenol for spice industry to production of santalenol. It will be helpful to gradually reduce the dependence of sandalwood oil on sandalwood and to promote the development of synthetic biology and related emerging industries.

檀香油是世界上最珍贵的精油之一。檀香资源匮乏，化学合成难以替代，无法满足檀香油市场的强劲需求。本项目选择印度白檀的檀香烯合酶（SaSS）基因和细胞色素P450单加氧酶（Sa-CYP76F39v1）基因，进行密码子优化与合成、克隆、体内外表达和功能鉴定；基于生物信息学分析，定点突变SaSS催化FPP的关键氨基酸位点，探索SaSS氨基酸序列与功能、空间结构与功能之间的关系，理解其在檀香烯生物合成中的作用机制，拓展倍半萜合酶基因功能及系统进化的知识。构建高酶活SaSS突变基因与Sa-CYP76F39v1基因的表达盒子模块；运用CRISPR-Cas9基因编辑技术对酿酒酵母的多拷贝δ位点进行切割，增加表达盒子模块的拷贝数；优化酿酒酵母底盘细胞的MVA代谢途径。本项目将为香料工业生产檀香醇提供优产檀香烯/檀香醇的酿酒酵母工程菌，逐步减少檀香油对檀香木的依赖，促进合成生物学及相关新兴产业的发展。

本项目依据合成生物学基本思路，采用“推-拉-抑制-减毒”策略，以S. cerevisiae为宿主，利用多物种来源与人工合成的30多条基因和元件序列，分别构建了葡萄糖→檀香烯→檀香醇→檀香醇糖苷的代谢途径及其工程菌，最终在S. cerevisiae细胞工厂内优化重建Santalenes和Santalols合成通路，成功合成稀缺化合物檀香烯和檀香醇，并首次将檀香醇转化得到檀香醇糖苷，为高效生物合成S. album的呈香和潜香物质奠定了基础。此外还对关键酶进行生信分析。主要结果包括：.1. 在S. Cerevisiae的δ位点分别整合了3条合成檀香烯的途径，摇瓶发酵7天获得α-santalene、α-bergamotene、epi-β-santalene和β-santalene的产量分别达到28.49 mg/L、13.97 mg/L、4.08 mg/L和20.54 mg/L。.2. 系统验证了2个P450与2个CPR的融合基因对檀香烯氧化能力的差异，证实Sa-CYP736A167和2个CPR（SaCPR2，t46ATR1）融合酶能在S. cerevisiae体内完成檀香烯的氧化，合成的檀香醇达到29.56 mg/L。.3. 通过构建一个可兼容糖基转移酶表达和ERG9下调功能的表达质粒，发现PHXT1和PERG1下调ERG9后，工程菌檀香烯和檀香醇产量最高分别达到164.71 mg/L和68.81 mg/L，分别比出发菌株增加74.0%和132.8%，同时角鲨烯分别下降54.3%和51.3%。选择A. thaliana的3个糖基转移酶 UGT73C1、UGT73C5、UGT75D1进行S. cerevisiae体内的活性测试，首次证明UGT73C1能在S. cerevisiae体内将Santalols糖基化得到糖苷化合物。.4. 对檀香烯合酶SaSS进行蛋白三维结构同源建模，推测SaSS的293Trp、417 Ile、422 Leu、545Phe位点可能是定向突变的潜在位点；通过生信分析2个P450均具有N端信号肽序列，但CYP76F39v1的亚细胞定位模糊，而CYP736A167亚细胞定位于内质网；4个CPR均具备明确的N-端跨膜信号肽，实验证实截短信号肽的46tATR1具备更好的酶活性。