微生物光合转化CO2合成高值天然产物香兰素的研究

Vanillin, a fine example of valuable natural products, is one of the most widely used flavoring agents in the world. However, plants tend to accumulate only low quantities of phenylpropanoids, and extraction or purification is often problematic. Recent advances in synthetic biology and metabolic engineering have enabled the tailored production of vanillin via microbial fermentation of carbohydrates. Nevertheless, these systems have some limitations, such as inadequate supply of NADPH, problematic expression of cytochrome P450s, require the addition of expensive precursor chemicals and carbohydrate feedstocks. As the existence of these production bottlenecks, the research offers a subtle blend of photosynthetic microorganism with the production of vanillin. By mimicking the natural pathway of vanillin biosynthesis, a new metabolic route containing two microbial genes and three plant genes was developed in cyanobacterium Synechococcus elongatus PCC7942 to directly convert the greenhouse gas CO2 into vanillin. The bottleneck of the biosynthetic pathway was identified by dynamic flux profiling, then the selection of enzymes and replacement of promoters were used to relieve the bottlenecks. Meanwhile, various strategies including introducing a feedback-inhibition-resistant enzyme, creating functional fusion proteins, and screening the mutant would be systematically investigated to increase the production of vanillin. The novel synthetic strategy makes the environmentally friendly, low-cost and sustainable production of vanillin a reality. It also provides new ideas for the production of other valuable natural products and strengthen the core competitiveness of industrial biotechnology.

植物来源的香兰素是典型的高值天然产物，市场需求量大；目前天然香兰素主要从植物香荚兰豆中提取，生产成本很高。植物源酶适配性、还原力供应和对昂贵底物的依赖限制了微生物合成香兰素技术的开发；合成生物学和代谢工程的发展使微生物法从头合成香兰素成为可能。针对上述技术瓶颈，本研究拟利用植物近源光合微生物Synechococcus elongatus PCC7942作为底盘，引入两个微生物源基因和三个植物源基因，模拟重构香兰素的天然合成途径，直接将温室气体CO2转化为香兰素；利用代谢流动态分析等方法鉴定合成途径的限速步骤；利用酶筛选和启动子替换等手段来解除合成代谢的瓶颈；通过芳香化合物合成主途径的反馈抑制解除、融合酶和突变株筛选等策略进行系统优化。这种合成策略将使得环境友好、低成本和可持续合成香兰素成为可能，并可为其它高值天然产物的生产提供新的思路。

香兰素是典型的高值天然产物，有“香料之王”的美誉，需求量巨大。合成生物学和代谢工程的发展使得微生物法合成香兰素成为现实，然而，植物源酶适配性、还原力供应和对昂贵底物的依赖等限制了生物法的应用。针对这些生产瓶颈，本项目将光合微生物与香兰素合成相融合，以Synechococcus elongatus PCC7942作为底盘，模拟和重构了阿魏酸的合成途径，重组菌株S111直接将温室气体CO2转化为23.1 mg/L的阿魏酸。同时，构建了可以将阿魏酸转化为香兰素的催化体系LV，含有酚酸脱羧酶和芳香双加氧酶。将S111分别与LV连用，可以直接将CO2转化为最高16.64mg/L的香兰素。随后，将两个微生物源基因和三个植物源基因直接引入Synechococcus elongatus PCC7942，模拟和重构香兰素的天然合成途径，重组菌株可以直接将CO2转化为香兰素。我们通过在培养基中添加苯丙氨酸的类似物3-氟苯丙氨酸筛选了S. elongatus PCC7942的抗类似物自发突变株，并将人工的香兰素合成途径转入其中，重组菌可以产生5.2 mg/l的香兰素。这是首次在原核系统中利用CO2和太阳能来直接合成香兰素，这种新颖的合成策略使得环境友好、低成本和可持续合成香兰素成为现实。同时，我们还用这种方法生产了苯乙醇，基于香兰素和苯乙醇较高的相对经济价值，本研究可能会推动商业可行的光合反应器的出现，进而推动相关的放大设备和生产方法的发展，随后，在蓝藻中光合生产生物燃料和大宗化学品将可能成为现实。相关研究成果发表论文3篇，均标注该项目资助，项目负责人为相关论文的第一（含共同一作）或通讯作者，申请中国发明专利1项，培养研究生3名，完成项目目标。