Development of marker-less counter selection system for production of thermostable phycocyanin in Spirulina

Phycocyanin is a blue protein pigment found in cyanobacteria. It is widely utilized in various industries, including food, cosmetics, and medicine. However, commercially extracted phycocyanin from Spirulina has limitations in stability, particularly its sensitivity to high temperatures. The development of a mutant Spirulina strain capable of producing thermostable phycocyanin without selectable marker genes is a promising alternative. To achieve this goal, this study employed a counter-selection system to develop a Spirulina strain deficient in phycocyanin production, which serves as a host cell. A DNA cassette was designed and constructed to eliminate the phycocyanin genes from the genome of Spirulina strain BP (Spirulina BP). This cassette contains the hygromycin resistance and the lethal mazF genes from bacteria, which are controlled by the promoter and the regulatory genes of Spirulina BP that respond to nickel compounds. Additionally, genetic modifications based on codon preference in the genome of Spirulina BP were implemented in the selection genes and the DNA sequence of the cassette to prevent recognition by restriction enzymes within Spirulina. After DNA transfer and selection of transformants on hygromycin selective media, the Spirulina mutants have been confirmed to be free of phycocyanin genes in their genomes. The phycocyanin-defective mutant will serve as a host strain for developing Spirulina mutants capable of producing thermostable phycocyanin in future studies.