蓝细菌能量-还原力代谢重平衡新策略及其生理影响研究

Cyanobacteria are ideal model organisms for exploring photosynthesis mechanisms and developing photosynthetic platforms for biochemicals and biofuels. Photosynthetic carbon fixation is the functional basis for physiological and metabolic activities in cyanobacteria. In the photosynthesis process, the light reactions provide ATP and NADPH as energy and reducing force respectively for carbon fixation in the dark reactions. However, the generation-consumption ratio between ATP and NADPH during photosynthesis is not in balance, with insufficient ATP supply and relatively excessive NADPH for carbon fixation and cellular metabolism. Such and unbalanced energy/reducing-force metabolism status reduces the total efficiency of photosynthesis and restricts the cellular physiological functionalities in cyanobacteria. In this project, we proposed a novel strategy for rebalancing the metabolism of energy and reducing force by introducing and regulating metabolic pathways coupling ATP generation and NADPH consumption. The novel strategy aimed to promote the dynamic adaptation and matching between light reactions and dark reactions in photosynthesis and optimize the cellular physiological functionalities. Taking photosynthetic synthesis of ethanol as an example, we redesigned the synthesis route and would regulate the expression strengths of different metabolic modules to achieve serial disturbance strengths for ATP/NADPH metabolism. Through systematic analysis on photosynthesis, physiology and metabolism in engineered cyanobacteria, we would explore the impacts of energy/reducing-force metabolism rebalance on photosynthetic carbon fixation, physiological functionality and metabolites production. This project aimed to provide more comprehensive understanding for photosynthesis mechanisms in cyanobacteria, simultaneously with novel designing principles for metabolic engineering.

蓝细菌是研究光合作用机制、开发化学品光合平台的理想模式生物。光合作用固碳是蓝细菌生理与代谢活动的功能基础。光合作用中光反应为暗反应固碳提供能量ATP和还原力NADPH，但是ATP和NADPH的合成-消耗比率并不平衡，ATP供应不足而NADPH相对过剩。这种失衡状态限制了光合作用的整体效率，也全局性的影响了细胞生理功能。本项目提出一种新的能量-还原力代谢重平衡策略，即引入偶联ATP合成与NADPH消耗的化学品合成途径来平衡ATP和NADPH的周转，促进光反应-暗反应动态适配并优化细胞整体生理功能。本研究以蓝细菌乙醇光合为模式，重新设计了合成路线，通过代谢模块的表达强度调控来实现不同程度的ATP-NADPH代谢扰动；进而通过系统的光合、生理和代谢分析，探索能量-还原力代谢比率重平衡对蓝细菌光合固碳、生理功能和代谢产物合成的影响；在加深对光合作用机制理解的同时，开发新的代谢工程设计策略。

蓝细菌是研究光合作用机制、开发化学品光合平台的理想模式生物。光合作用固碳是蓝细菌生理与代谢活动的功能基础。光合作用中光反应为暗反应固碳提供能量ATP和还原力NADPH，但是ATP和NADPH的合成-消耗比率并不平衡，ATP供应不足限制了光合作用的整体效率，也全局性的影响了细胞生理功能。本研究以蓝细菌能量-还原力代谢重平衡策略开发和生理功能研究为导向，开展了蓝细菌能量-还原力代谢参数分析方法的建立和优化、蓝细菌能量-还原力代谢重平衡策略的建立、蓝细菌能量-还原力重平衡的细胞生理功能影响和机制解析三个层次的研究。首先建立了可靠的生理和代谢功能参数分析方法，获得了对不同模式藻株能量-还原力代谢状态的基础性认识，发现集胞藻PCC6803、聚球藻PCC7002以及聚球藻UTEX2973中能量物质ATP和还原力NADPH的丰度差异巨大，而导入乙醇合成途径会显著的降低胞内能荷以及能量-还原力比率；第二阶段，在原先设计的通过新代谢途径的引入来扰动能量-还原力代谢平衡的策略无法如期起作用的背景下，根据对速生聚球藻UTEX2973的比较基因组和生理分析，设计了基于ATP合成酶扰动的能量-还原力重平衡策略，通过向聚球藻ATP合成酶alpha亚基导入C252Y位点突变即可大幅提升藻株的光合效率以及在高温高光胁迫条件下的稳定性；第三阶段，通过后续的生理和代谢分析发现，该策略有效的提升了ATP合成酶alpha亚基的活性和稳定性，提高了胞内ATP合成水平和ATP丰度，有利于高光利用过程中各种生理损伤的修复。在本项目资助下，我们还利用高光效蓝细菌底盘细胞构建了乙醇合成藻株，并基于相关研究成果和研究经验针对蓝细菌生理和代谢调控、蓝细菌合成生物学、蓝细菌光合生物制造技术的现状和发展方向进行了总结，提出了新的观点和认识，在生物技术领域权威期刊上发表了多篇高水平综述论文。