A toxicity study of organic acids to develop a marker-free DNA delivery system in Arthrospira

A cyanobacterium, Arthrospira, has a high potential for being used as a synthetic biology platform to produce bioactive compounds. However, antibiotic resistance genes in DNA transfer systems remain a significant biosafety concern. This study investigates the toxicity of three organic acids to Arthrospira platensis strain BP (Arthrospira BP) for their potential application in developing a marker-free selection system. These were acrylic acid, 3-hydroxypropionic acid, and propionic acid. The capable growth of mutant strains on organic acid containing media was used as a basis for marker-free selection due to the disruption of the acsA gene, which encodes acetyl-CoA ligase. The results revealed that acrylic acid was the most toxic to Arthrospira BP with the minimum inhibitory concentration at 0.1 millimolar. The findings were used to develop a DNA cassette containing the omega-3 fatty acid desaturase (desB) gene, designed to replace the acsA gene in the Arthrospira genome after transformation. This replacement facilitated the selection of a marker-free mutant strain capable of producing unsaturated fatty acids omega-3 on selective media containing acrylic acid. This system enhances the safety and efficiency of DNA transfer in Arthrospira, providing a marker-free approach for producing functional compounds without reliance on antibiotic resistance genes.