An expression system for regulated protein production in Synechocystis sp. PCC 6803 and its application for construction of a conditional knockout of the ferrochelatase enzyme

Synechocystis sp. PCC 6803 is a model organism for the study of photosynthetic processes. Methods to genetically manipulate this bacterium are essential to investigate these processes and to evaluate potential biotechnological applications. We developed a vector for controllable expression of proteins using a platform for stable integration of the expression cassette into the genome. The respective gene is translationally fused to the promoter of the petJ gene encoding cytochrome c553 that is repressed by copper. Maximal expression from this promoter is achieved under copper depletion, whereas normal copper concentrations in standard medium lead to low expression rates. We show here the application of this system for construction of a conditional knockout mutant for the ferrochelatase, which is an essential enzyme in heme biosynthesis. Using different amounts of copper in the medium we were able to control the amount of ferrochelatase in the cell resulting in a varying expression of the phenotype.

► We describe an expression vector for synthesis of proteins in Synechocystis 6803.
► The promoter activity can be tightly regulated by copper availability.
► We use this system for creation of a conditional knockout mutant for ferrochelatase.
► Different concentrations of copper in the medium lead to different phenotypic effects of varying ferrochelatase deficiency.