Abstract: Arthrospirais a multicellular filamentous cyanobacterium with high economic value. However, genetic manipulation ofArthrospirahas been a major challenge, especially the lack of an easy-to-operate genetic transformation technology. To further develop the potential ofArthrospirafor genetic engineering applications, this study attempted to screen strains with natural transformation ability from 12 differentArthrospiraand evaluated the impact of homologous flanking sequence and insertion fragment lengths on natural transformation efficiency. The results showed that only strains S17 and S39 could be genetically modified through natural transformation. Both homologous flanking sequences and insertion DNA fragment lengths influenced transformation efficiency. It was found that increasing the homologous flanking sequences to over 1 500 bp could significantly increase the positive rate. Conversely, the length of the inserted DNA fragment was negatively correlated with the positive rate, so it was necessary to consider increasing the length of homologous flanking sequences when using longer inserted DNA fragments. In addition, this study also confirmed that the homogeneity ofArthrospirawas related to the duration of continuous screening, where after 120 days of screening, homogenous strains accounted for over 50% of the positive strains; these homogenous strains remained genetically stable for at least 6 months. It was expected that this work would provide technical support for the development ofArthrospiraas a bioreactor for producing high-value compounds.

Key words: Arthrospira, natural transformation, homologous recombinant, genetic engineering, vector