Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco

作     者：Zhen Cai Guoxia Liu Junli Zhang Yin Li 

作者机构：CAS Key Laboratory of Microbial Physiological and Metabolic Engineering Institute of Microbiology Chinese Academy of Sciences Beijing 100101 China Tianjin Institute of Industrial Biotechnology Chinese Academy of Sciences Tianjin 300308 China 

出 版 物：《Protein & Cell》 (蛋白质与细胞（英文版）)

年 卷 期：2014年第5卷第7期

页      面：552-562页

核心收录：

学科分类：0710[理学-生物学] 0831[工学-生物医学工程（可授工学、理学、医学学位）] 1007[医学-药学(可授医学、理学学位)] 1002[医学-临床医学] 09[农学] 0903[农学-农业资源与环境] 0703[理学-化学] 0901[农学-作物学] 090302[农学-植物营养学] 0836[工学-生物工程] 

基　　金：国家自然科学基金 国家973计划 中国科学院知识创新工程项目 

主　　题：carboxylation efficiency CO2 fixation directed evolution Rubisco Synechococcus sp. PCC7002 

摘      要：Photosynthetic CO2 fixation is the ultimate source of organic carbon on earth and thus is essential for crop production and carbon sequestration, Ribulose-1,5-bis- phosphate carboxylase/oxygenase （Rubisco） catalyzes the first step of photosynthetic CO2 fixation. However, the extreme low carboxylation efficiency of Rubisco makes it the most attractive target for improving pho- tosynthetic efficiency. Extensive studies have focused on re-engineering a more efficient enzyme, but the effort has been impeded by the limited understanding of its structure-function relationships and the lack of an effi- cient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coil-based activity-directed selection system which links the growth of host cell solely to the Rubisco activity therein. A Synechococcus sp. PCC7002 Rubisco mutant with E49V and D82G sub- stitutions in the small subunit was selected from a total of 15,000 mutants by one round of evolution. This mutant showed an 85% increase in specific carboxyla- tion activity and a 45% improvement in catalytic efficiency towards CO2. The small-subunit E49V mutation was speculated to influence holoenzyme catalysis through interaction with the large-subunit Q225. This interaction is conserved among various Rubisco from higher plants and Chlamydomonas reinhardtii. Knowledge of these might provide clues for engineering Rubisco from higher plants, with the potential of increasing the crop yield.