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Development of an activity-directed selection system enabled significant improvement of the carboxylation efficiency of Rubisco |
Zhen Cai1,Guoxia Liu1,Junli Zhang2,Yin Li1,*( ) |
1. CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2. Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China |
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Abstract 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-bisphosphate 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 photosynthetic 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 efficient selection system towards its activity. To address the unsuccessful molecular engineering of Rubisco, we developed an Escherichia coli-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 substitutions 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 carboxylation 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.
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| Keywords
carboxylation efficiency
CO2 fixation
directed evolution
Rubisco
Synechococcus sp. PCC7002
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Corresponding Author(s):
Yin Li
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Issue Date: 31 July 2014
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