大肠杆菌光控诱导表达系统中光敏蛋白YF1的定向进化

Optogenetics is a non-destructive, reversible, and non-invasive biotechnology. It has the ability to selectively turn on or off certain physiological activities in cells at a specific time and space using light. In many developments of optogenetics, in order to minimize the damage to subjects, researchers expect to set the light source in vitro. In this case, the light signal needs to pass through a variety of living cells or tissues, thus resulting in an unavoidable attenuation. Unfortunately, there is little systematic study on the reduction of light response threshold in the report of photoreceptors at present. In our previous study, we established a light-induced expression system for heterologous β-glucosidase producing in Eshcerichia coli, which showed an excellent light regulation effect. Therefore, this project intends to use directed evolution and semi-rational design methods to construct photoreceptor YF1’s derived mutant library. by high-throughput screening, it is desirable to obtain some YF1 mutants that possesses a lower optical response threshold. Our results will contribute to provide the new evidence to gain some insight into the response mechanisms of photoreceptor to light, and provide some guidance for the expression of heterogeneous proteins in engineering strains.

光遗传学技术可以选择性地在特定时间、空间范围内利用光信号开启或关闭细胞内某些既定的生理活动，是一种无损、可逆和侵入性低的研究方法。在一些研究中，光信号需要穿过各种活体组织或细胞，因而发生不可避免的衰减，这就需要降低光敏蛋白的光响应阈值来提高诱导效率。然而目前对光敏蛋白的报道中，尚未有针对其光响应阈值降低的系统性研究。前期，我们基于YF1/FixJ光调控体系成功搭建了异源蛋白β-葡萄糖苷酶的大肠杆菌光诱导表达系统，并首次在大肠杆菌高密度发酵中获得了较好的光调控诱导效果。在此基础上，本项目拟采用定向进化和半理性设计等方法对光敏蛋白YF1进行分子改造，通过高通量筛选获得光响应阈值降低的YF1突变体，系统研究其在细胞中的光响应性能，初步阐明其光响应阈值降低的相关机制，研究结果能为深入了解光敏蛋白信号响应和传递机制提供一定的理论基础，也可以为工程菌改造提供一定的借鉴。

光诱导表达系统可以通过控制光信号的开启或关闭，在特定的时间和空间范围内对目标蛋白的表达进行调节，是一种快速响应、无损和侵入性低的蛋白诱导表达方案。本项目基于大肠杆菌搭建了以绿色荧光蛋白为目标产物和筛选标记的光诱导表达基因工程菌株，进一步通过易错PCR手段构建了光敏蛋白YF1的突变体文库。筛选获得了光信号响应调控能力发生改变的光敏蛋白YF1突变体并进行了相关分析。其中两个氨基酸突变处于光敏蛋白YF1的N端靠近其活性二聚体结构的中轴位置，推测这些改变影响了所在区域对光感受结构域光信号的传导能力。另一个氨基酸突变位于该蛋白光感受结构域C端的α螺旋结构中，光敏蛋白YF1的光感受结构域通过此α螺旋结构连接至激酶活性结构域，其氨基酸残基的改变也严重影响了光信号的正确传递。此外，本项目还考察了光诱导表达系统对深海海单胞菌来源漆酶的光照诱导表达能力，并初步完成了相关蛋白的性质分析。本项目研究结果为大肠杆菌光诱导表达系统的进一步研究提供了一定的理论基础。