解脂耶氏酵母合成β-紫罗兰酮的代谢调控研究

Terpenoid flavor and fragrance compounds (TFFCs) show extensive application in nutraceutical, pharmaceutical and food industries that have a rapid grow market demand. With the development of synthetic biology technology, many TFFCs have been demonstrated could be biosynthesized in microorganism via the pathway reconstruction and regulation. Moreover, the use of microorganisms to convert natural raw materials into aroma compounds can be described as natural, which suggest engineering of microbes for production of TFFCs has great potential and will be one of the most promising strategies. However, fermentive TFFCs produced by engineered microbes mostly only obtain intermediates or low yields of end-product currently. The choice of a suitable host strain (chassis) determines the success of developing an efficient TFFCs biosynthesis process. This project proposes a non-conditional yeast Yarrowia lipolytica as a chassis for TFFCs production, in which β-ionone that have an annual market volume of 10 thousand tons is chose as target product. By employing synthetic biology technology and "pull-push" strategy, this proposal aims to develop Y. lipolytica as an alternative platform for production of TFFCs. Firstly, we modify the strain and the technology for rapid assemble and integrate the pathway into the genome of Y. lipolytica. And then, optimization and regulation of the precursor synthesis module for “push” and the β-ionone synthesis module for “pull” to enhancing the β-ionone production. This proposal will provide an alternative platform and metabolic engineering strategy for biosynthesis of TFFCs.

萜类香料在食品、医疗等领域上有着重要的作用，市场需求量快速增长。随着合成生物学的发展，在微生物中重构合成途径可实现萜类香料的生物合成，能极大的缓解市场需求压力。特别是，微生物法生产的萜类香料已被认定为天然产物，更显示出其巨大的潜力。然而，当前缺乏高效的底盘细胞，大部分萜类香料通过微生物异源合成产量低或只是简单的中间体。因此，挖掘新一代底盘细胞，建立相应的代谢调控研究策略具有重要意义。本项目以市场年需求量近万吨的重要萜类香料β-紫罗兰酮的高效生物合成作为切入点，挖掘解脂耶氏酵母成为新型萜类香料的合成底盘。通过采用合成生物学的快速组装技术、基因编辑技术和模块化理念并结合“pull-push”策略，进行前体合成模块和β-紫罗兰酮合成模块的优化，构建基于解脂耶氏酵母的萜类香料合成平台。本项目的研究将为解决目前萜类香料产量低的问题提供新的平台与研究思路。

萜类香料在食品、医疗等领域上有着重要的作用，市场需求量快速增长。随着合成生物学的发展，在微生物中重构合成途径可实现萜类香料的生物合成，能极大的缓解市场需求压力。特别是，微生物法生产的萜类香料已被认定为天然产物，更显示出其巨大的潜力。然而，当前缺乏高效的底盘细胞，大部分萜类香料通过微生物异源合成产量低或只是简单的中间体。因此，挖掘新一代底盘细胞，建立相应的代谢调控研究策略具有重要意义。本项目利用市场年需求量近万吨的重要萜料香料β-紫罗兰酮的高效生物合成作为切入点，以解脂耶氏酵母为出发菌株，采用合成生物学的快速组装技术、基因编辑技术和模块化理念并结合“pull-push”策略，构建了高产β-紫罗兰酮的解脂耶氏酵母菌株。通过发酵优化，在3-L发酵水平上采用YPD培养基可获得1 g/L的β-紫罗兰酮，在摇瓶水平采用厨余水解液可获得4g/L的β-紫罗兰酮。项目研究发现：1）通过引入磷酸转酮酶途径来提高胞浆中乙酰辅酶A供应是提高 β-紫罗兰酮产量的有效策略；2）通过CCD1的亲膜突变或融合亲膜标签可有效提高β-紫罗兰酮的产量；3）培养基中的溶氧水平对β-紫罗兰酮的合成有重要的影响；4）工程菌可有效利用厨余水解液高产β-紫罗兰酮（4g/L）。本项目将β-紫罗兰酮产量提高1000倍以上（从3.5mg/L到4g/L），为目前微生物法的最高生产水平，具有良好的工业应用前景，为挖掘解脂耶氏酵母成为新型萜类香料合成底盘、解决目前萜类香料产量低的问题提供新的平台菌株与研究思路。