Background: Plastoquinone biosynthesis in cyanobacteria differs from that in plants.Results: Chorismate pyruvate-lyase and 4-hydroxy-3-solanesylbenzoate decarboxylase single-gene knock-out mutants are severely affected in plastoquinone synthesis, cell size/structure, and growth. Conclusion: The plastoquinone biosynthetic pathway likely evolved from a pre-existing ubiquinone pathway in cyanobacterial ancestors. Significance: Cyanobacterial mutants deficient in plastoquinone biosynthesis allow deeper understanding of processes related to energy transformation.