动态启动子调控高产前体alpha-酮戊二酸解脂亚洛酵母异源合成1,4-丁二醇

1,4-Butanediol (1,4-BDO) is a high-valued compound. As a precursor, alpha-ketoglutarate (a-KG) was used for 1,4-BDO synthesis. To eliminate the obstacle that low intracellular a-KG was accumulated in Escherichia coli, presently, the heterogeneous 1,4-BDO synthesis route will be introduced and established in Yarrowia lipolytica, an a-KG overproducer. According to module optimization, the expression level of each heterogenous gene in 1,4-BDO synthesis route will be regulated by applying different promotors to enhance 1,4-BDO synthesis. Also bottle neck reaction for 1,4-BDO synthesis will be identified. A global transcriptomic analysis will be applied to exploit and develop putative promotors which can dynamically response to intracellular 1,4-BDO content. To increase and achieve efficient 1,4-BDO accumulation, a dynamic metabolic strategy both for central pathway and heterogeneous 1,4-BDO synthesis pathway will be established using dynamic promotors. Applying such strategy, a 1,4-BDO accumulation pattern in which metabolic flux will be balance between cellular growth and 1,4-BDO accumulation can be achieved. The results obtained will provide essential theoretical and technical references for metabolic engineering central pathway for heterogenous synthesis of compounds of high-valued.

1,4-丁二醇(1,4-butanediol, 1,4-BDO)是具有重要价值化学品，alpha-酮戊二酸(alpha-ketoglutarate, a-KG)作为前体物质被用于异源合成1,4-BDO。为解决大肠杆菌细胞内a-KG含量低的困扰，本项目拟以富含1,4-BDO合成前体a-KG的解脂亚洛酵母为宿主细胞，异源引入1,4-BDO合成途径。为强化1,4-BDO合成，基于模块化优化理论，优化合成途径中异源基因表达量，并寻找1,4-BDO合成的瓶颈步骤。通过转录组分析开发并建立能动态响应1,4-BDO含量变化的启动子，构建动态调控1,4-BDO合成策略，平衡细胞生长与1,4-BDO合成竞争关系，并配合动态调控中心代谢途径强化前体供应，实现1,4-BDO高效合成。本项目将为代谢改造中心代谢途径异源合成其他重要经济价值化合物提供理论与技术支持。

在Y.lipolytica细胞中构建了从头合成1,4-BDO途径，在Y.lipolytica SS细胞外发酵上清液中检测到85.9 mg/L的1,4-BDO，证明由Pg4Hbd、PgCat2、CsBld和CsBld组成的外源代谢途径能利用内源谷氨酸-琥珀酸旁路中的碳代谢中间产物合成1,4-BDO。并利用模块化改造策略在细胞体内调控4个异源蛋白的表达量，1,4-BDO积累量从85.9 mg/L上升至330.6 mg/L。从平衡外源基因表达量的角度解析，上游基因表达量对异源产物合成起至关重要的作用，单独强化下游基因表达对强化产物合成产生副反应；综合分析菌株最大干重、最大比生长速率和底物最大比消耗速率、alpha-KG积累量和NADPH/NADP+等参数推断，异源引入合成1,4-BDO途径,引起辅因子供应不平衡;.通过强化甘油途径中关键酶甘油脱氢酶表达，发现强化以NADP+为辅因子的YlGDH的表达是强化1,4-BDO积累关键因素，与出发菌株Y. lipolytica MM相比，YlGDH的表达促使1,4-BDO积累量提高102.9%至670.8 mg/L，从辅因子层面证明NADP+是限制1,4-BDO积累关键因素之一；而过量表达以NAD+为辅因子的EcgldA是强化甘油利用的关键因素，而对1,4-BDO积累影响不显著；.通过过量表达CgSucA和CgSucA361突变体，相对于出发菌株Y.lipolytica MM，在1,4-BDO积累量最高点60 h时，重组菌株Y.lipolytica MMK1和Y.lipolytica MMK2细胞内酮戊二酸脱羧酶比酶活力分别提高71.4%和21.4%。证明CgSucA的N端1-361氨基酸残基的截短不影响该酶分子的酮戊二酸脱羧酶催化活力。同时，1,4-BDO最大积累量分别为：619.1 mg/L和417.1 mg/L，相对于Y.lipolytica MM菌株，1,4-BDO积累量分别提高87.3%与26.3%。综合UGA2基因敲除实验结果UGA2敲除的重组菌株Y.lipolytica MMU2L的发酵液中1,4-BDO最大积累量为：2776.1 mg/L从碳代谢流层面证明4HB供应是限制1,4-BDO积累关键因素之一。