基于基因组学的新型地芽孢杆菌合成乙偶姻及其衍生物研究

We recently reported the finding of the first thermophile capable of acetoin biosynthesis in the nature-a novel Geobacillus strain, XT15. Large quantities of acetoin and 2,3-butanediol, two bio-based platform chemicals, can be produced by XT15 using a variety of renewable materials. Some other kinds of potentially valuable derivatives can also be synthesized, including a newly found natural compound. This project will sequence the genome of XT15 and study the metabolic pathway of acetoin and its derivatives in XT15 at the genomic level by comparative genomics. The pathway will be verified at the metabolite level by comparing the metabolites of XT15 with other sequenced homologous species. The key enzymes will be cloned, expressed, and purified. The pathway will also be verified at the enzymatic level by determining the basic properties of the key enzymes and mimicking the cellular enzymatic steps using the purified enzymes in vitro. The key enzymes will be mutated by site-directed mutagenesis and the changes of enzymatic activities, thermal stability and protein rigidity will be measured. Combining with the homology modeling analysis results of the structural changes and the comparison with mesophilic enzymes, the relationship between enzyme activity-thermal stability-rigidity-structure will be explored and the mechanism of thermophilic or heat-resistant properties will be elucidated. This project will open up new areas of research in the biosynthesis of acetoin and its derivatives in thermophilic bacteria. The fulfillment of the project will be helpful to find new functional genes and biosynthesis pathways in novel organisms. This project will also lay the foundation for the study of directed evolution of enzymes, the analysis of metabolic and regulation networks, the genetic manipulation and the final goal of biosythesis of highly valuable products using thermophilic bateria.

申请者最近报道了在自然界中首次发现可以合成乙偶姻的嗜热生物- - 一株新型地芽孢杆菌XT15。该菌株除了能够利用多种可再生原料大量合成乙偶姻和2,3-丁二醇等生物基平台化合物外，还产生多种其他有潜在价值的衍生物，包括一种新发现的天然化合物。本项目将对XT15基因组测序，与已测序近源菌株在基因组层面和代谢产物层面上对比研究XT15中的乙偶姻及其衍生物合成途径；克隆表达纯化关键酶，测定基本酶学性质，利用各个纯酶在体外组合,模拟体内途径的反应步骤；对关键酶定点突变，测定活性、热稳定性和刚性变化，结合同源建模对结构变化的分析和与常温酶的比较，探索关键酶活性-热稳定性-刚性-结构之间的关系和机理。本项目将开辟嗜热菌中乙偶姻及其衍生物合成研究的新领域，有助于挖掘新颖物种功能基因、认识新合成途径和酶的定向进化研究，并为将来代谢网络及调控分析、基因工程改造、最终实现高温生物合成高附加值产品等工作奠定基础。

乙偶姻天然存在于许多食物中，在食品、香料、化妆品、手性合成等领域有重要的用途，是一种新兴的生物基平台化合物。在本项目之前的研究为常温微生物合成，本项目主要以嗜热菌为研究对象，主要在基因组、代谢产物、酶三个层面开展了研究。在基因组层面，不仅完成了对目标菌株Geobacillus sp. XT15的测序，还对近源菌株Bacillus sp. H15-1、Bacillus sp. 1s-1、Bacillus sp. 50-1和远源菌株Myroides sp. ZB35完成了测序，进行了比较基因组学的部分研究，确定了部分功能基因和代谢途径；在代谢产物层面，采用嗜热菌首次实现了不灭菌发酵高产乙偶姻，并采用GC-EIMS技术，系统分析了以上多株菌的代谢产物，并发现和鉴定了两个新的乙偶姻类代谢产物；研究了乙偶姻最重要的衍生物——川芎嗪的绿色合成过程，通过发酵制备13C标记的乙偶姻，首次采用同位素示踪法证实了川芎嗪生成机制，证实了乙偶姻和氨是川芎嗪的前体；在酶学研究层面，本项目还研究了酶法合成乙偶姻脂肪酸酯，另外克隆表达并纯化了两个关键酶，并初步研究分析了其催化产生新型偶姻类代谢产物的机理。