蓝细菌生理与代谢工程新策略研究

QIBEBT-IR > 微生物代谢工程研究组

	蓝细菌生理与代谢工程新策略研究
	栾国栋
	2016-05-18
关键词	蓝细菌代谢工程生物乙醇糖原光合作用
学科分类	微生物代谢工程
报告类别	专题报告
中文摘要	Cyanobacteria were a group of important photoautotrophic microorganisms, converting CO2 and solar energy into organic compounds through photosynthesis and releasing O2. Photosynthetic production of diverse chemicals in cyanobacteria has been achieved by modification of natural metabolic pathways or introduction of artificial metabolic pathways. Ethanol was the most promising biofuel product, the first reported and the most representative cyanobacteria based biochemical. Development of novel strategies for physiological and metabolic engineering in cyanobacteria is of great significance. This project aimed to explore new strategy and designing principles for optimization of metabolic pathways and photosynthetic platforms. We adopted a combinatory strategy to understand and characterize the PDCzm-slr1192 pathway, essential for ethanol synthesis in cyanobacteria cell factories, by in vitro reconstitution and metabolic engineering. Based on the in vitro reconstitution system, we assessed the contributions of diverse components during the catalytic processes, and discovered that it was PDCzm rather than slr1192 holding control over the ethanol synthesis process, an optimal ratio of PDCzm-slr1192 was 4:6, and increasing the supply of NADPH and pyruvate should be the effective strategy for enhancing ethanol photosynthetic production. We performed metabolic engineering in cyanobacteria to confirm the hypothesis from in vitro reconstitution. The results provided a novel paradigm for characterizing metabolic pathways in cyanobacteria and inspired the direction for optimizing advanced ethanol photosynthetic production. For understanding and optimizing physiological and metabolic characteristics of cyanobacteria cells, we modified and analyzed the system of glycogen metabolism. Comparing with the traditional glycogen synthesis blocking strategy, we overexpresses glycogen phosphorylase to accelerate the glycogen digestion processes. We discovered that enhanced glycogen phosphorylase activities did not decrease but significantly increase glycogen accumulations. Further analysis revealed that enhanced glycogen phosphorylase caused an intracellular phosphate-deprivation status, allosterically activated GlgC, and finally enhanced glycogen synthesis and accumulations. In engineered cyanobacteria strain for ethanol synthesis, overexpression of glgP simultaneously enhanced carbon partitioning ratio to glycogen and ethanol; under nitrogen deprivation conditions, ethanol synthesis was improved by 1.5-fold comparing with the wildtype strain, indicating glycogen accumulation was not a simple carbon flow competitive pathway with metabolite synthesis. Our results demonstrated new strategies for physiological and metabolic engineering in cyanobacteria, and provided new understanding for function and regulation of metabolism and physiology, which would enable the development of more efficient photosynthetic platforms.
英文摘要	蓝细菌是重要的光合自养微生物，能够利用二氧化碳和太阳能合成有机物并释放氧气，也是最具潜力的生物燃料和生物基化学品光合平台，通过修饰蓝细菌天然代谢途径或向蓝细菌中引入人工构建的代谢途径已经实现了多种化合物的光合合成。乙醇是最具产业化潜力的生物燃料产品，也是最早报道、最具代表性的蓝细菌光合生物基化合物，以乙醇光合为模式进行蓝细菌生理和代谢工程策略的研究具有重要的示范意义。本研究从代谢途径和光合平台两个角度分别开展，探索开发新的优化策略和设计原则。针对蓝细菌光合细胞工厂中乙醇合成的核心代谢途径PDCzm-slr1192，我们系统运用了体外重构和代谢工程两种策略对该途径进行深入、全面的动态解析。在代谢途径体外重构的基础上，我们系统评价了催化过程各种组分对反应速率的影响发现PDCzm的含量和活性是乙醇合成速率的限制因素，PDCzm和slr1192的最优浓度比为4:6，提高NADPH和丙酮酸的供应也应该是优化乙醇合成速率的重要方向。对体外重构和定量分析的结果，我们通过对蓝细菌的代谢工程改造加以验证。该部分研究为蓝细菌乙醇光合细胞工厂的下一阶段改造明确了方向，也为代谢途径的解析与优化提供了新的研究模式。针对蓝细菌光合平台本身的生理和代谢特性与机制，我们主要进行糖原代谢系统进行改造和分析。与传统的阻断糖原合成的策略不同，我们采取过量表达糖原磷酸化酶来加速糖原降解的策略。结果我们发现糖原降解速率的加快，不但没有降低胞内糖原含量，反而使之大幅度提高，其原因是糖原磷酸化降解过程的加速降低了胞内磷酸的含量，别构激活了糖原合成的关键酶GlgC，从而使糖原合成得到加强，提高了糖原积累。在乙醇光合的蓝细菌工程藻株中，糖原磷酸化酶的过量表达也提高了糖原的积累，但同时乙醇产量和碳流分配比也得到提高；在限氮条件下，乙醇产量相对野生型藻株提高50%，证明糖原积累与代谢产物合成并非简单的碳流竞争关系。我们的结果为蓝细菌生理与代谢工程研究提供了新的策略，为蓝细菌生理和代谢功能与调控机制研究提供了新的认识，将有助于开发更高效的蓝细菌光合平台。
文献类型	研究报告
条目标识符	http://ir.qibebt.ac.cn/handle/337004/9802
专题	微生物代谢工程研究组
推荐引用方式 GB/T 7714	栾国栋. 蓝细菌生理与代谢工程新策略研究. 2016.

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