Engineering the membrane rotor ring of ATP synthases involved in photosynthesis

Abstract

The F-type ATP synthase is a macromolecular machine capable of coupling the proton-motive force with the synthesis of adenosine triphosphate, ATP, the universal energy currency in biology. A key component of the ATP synthase machinery is the cn-ring with a stoichiometry that is constant within a given species but varies across different species, presumably due to the different bioenergetic demands placed on an organism in various environments. The cn-ring stoichiometries of F-type ATP synthases involved in the light dependent reactions of photosynthesis are among the largest known, ranging from c13 to c15-rings. The recent increase in availability of genome-editing tools paves the way for investigating the importance of large cn-rings for photosynthesis. In this study, CRISPR-Cas12a genome editing is used to alter the stoichiometry of the cn-ring of the F-type ATP synthase found in the model cyanobacterium, Synechococcus sp. PCC 7002, by replacing the endogenous c-subunit gene, atpE, with the atpE gene from Spirulina platensis which natively possess a c15-ring. The native stoichiometry of the Synechococcus sp. PCC 7002 cn-ring was unknown but suspected to be in the range of 13 to 15 subunits. Electron cryo-microscopy was used to identify a native c14-ring stoichiometry in the wild-type organism and later confirm the formation of a chimeric F-type ATP synthase possessing all the native subunits of the Synechococcus sp. PCC 7002 enzyme with the exception of a c15-ring from Spirulina platensis. Functional comparisons between prepared spheroplasts of the wild-type and mutant strains showed a decreased ATP synthesis rate under high light for the mutant. Furthermore, the mutant strain showed improved growth in alkaline media. These findings highlight the potential for targeting cn-ring stoichiometries to improve photosynthesis.