張衛(wèi)文 陳磊 周杰 馬鋼 曹旭鵬

摘 要：成功開發(fā)了高效、穩(wěn)定的藍(lán)細(xì)菌遺傳改造工具。使用該工具可以實(shí)現(xiàn)對(duì)不同基因進(jìn)行高表達(dá)或低表達(dá)；通過理性設(shè)計(jì)基因組插入位點(diǎn)，實(shí)現(xiàn)對(duì)藍(lán)細(xì)菌內(nèi)容碳流進(jìn)行重新分配。該工具正在藍(lán)細(xì)菌創(chuàng)建生物合成途徑中測(cè)試。成功創(chuàng)建了從CO2到異丙醇的生物合成途徑，實(shí)現(xiàn)利用CO2生物合成異丙醇。突變株生理特性測(cè)試及優(yōu)化正在進(jìn)行中。構(gòu)建了針對(duì)雙組件相應(yīng)調(diào)控蛋白，轉(zhuǎn)錄調(diào)控因子，以及細(xì)胞小RNA等的藍(lán)細(xì)菌刪除株共273株，以及18株含有不同功能基因的藍(lán)細(xì)菌過表達(dá)株。使用基于96孔板的底盤檢測(cè)技術(shù)，對(duì)構(gòu)建獲得的藍(lán)細(xì)菌底盤進(jìn)行了生物產(chǎn)品耐受性（針對(duì)乙醇、丁醇、3羥基丙酸3HP等）以及環(huán)境脅迫因子耐受（氮源脅迫、金屬離子脅迫、高鹽脅迫等）進(jìn)行了系統(tǒng)的分析，獲得多株對(duì)于各種脅迫因素以及生物產(chǎn)品發(fā)生抗性改變的底盤細(xì)胞。確定了一個(gè)與集胞藻脂肪酸代謝相關(guān)的轉(zhuǎn)錄因子，其在脂肪酸代謝網(wǎng)絡(luò)中的位置及功能的確定還在進(jìn)一步分析中。完成了兩個(gè)重要蛋白的晶體結(jié)構(gòu)解析。

關(guān)鍵詞：人工合成細(xì)胞工廠 光合藍(lán)細(xì)菌 底盤細(xì)胞

Abstract：A highly efficient and stable genetic transformation tool for cyanobacteria was successfully developped of. High or low expression of different genes can be achieved by use of this tool. A biosynthetic pathway from CO2 to isopropyl alcohol was successfully created. 273 cyanobacteria gene knockout mutants were constructed for the corresponding two- component regulatory proteins， transcription factors， or small RNAs. 18 cyanobacteria overexpression strains containing different functional genes were also constructed. Phenotype analysis was performed and a series of mutants that are tolerant to ethanol， butanol ， 3HP and some environmental stress factors such as nitrogen stress， metal ion stress， high salt stress， were identified. A fatty acid metabolism related transcription factor in Synechocystis was identified. Crystal structure analysis of two important proteins was completed.

Key Words：Synthetic cell factories；Photosynthetic cyanobacteria；Chassis cell

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