Cnb proteins affect the intracellular 2-MIB retention in heterologous gene expression systems

2-methylisoborneol (2-MIB) is one of the main cyanobacterial metabolites responsible for unpleasant taste and odor in drinking water. 2-MIB biosynthesis involves a two-step enzymatic reaction catalyzed by enzymes encoded by the mtf and mtc genes. Two homologous genes, cnbA and cnbB, which encode cyclic nucleotide-binding proteins of unknown function are located upstream and downstream of the mtf and mtc genes, respectively. This study investigated the involvement of cnb genes of 2-MIB-producing cyanobacterium Pseudanabaena foetida var. intermedia NIES 512 in 2-MIB synthesis using heterologous gene expression systems. Transgenic Escherichia coli and Synechococcus elongatus PCC 7942 strains expressing the cnbA-mtf-mtc-cnbB gene cluster exhibited a notable intracellular 2-MIB retention which was not shown by the transgenic strains expressing only the mtf-mtc gene unit. Immunoelectron microscopy detected that the Cnb proteins are associated with unknown intracellular compartments in transgenic S. elongatus PCC 7942 cells. This study suggests that 2-MIB-related Cnb proteins are involved in the formation of intracellular compartments, which are likely associated with intracellular 2-MIB retention.