Engineering the <i>ptxD</i> Gene into Chlorella sorokiniana via <i>Agrobacterium</i> -Mediated Transformation Enables Selective Metabolism of the Reduced Phosphorous Compound Phosphite

The microalga Chlorella sorokiniana is a promising platform for sustainable biomanufacturing due to its rapid growth rate and scalability. This study established an Agrobacterium -mediated genetic transformation system to introduce the phosphite dehydrogenase (PtxD) gene into C. sorokiniana, with a genus-specific efficiency of 278 ± 5 transformants per 10 6 cells. The transformants were verified via PCR, RT-qPCR, and Western blot analysis, and the genetic stability of the transformant was confirmed over a ten-week period, suggesting that the transgene was integrated into the nuclear genome. Under phosphite (Phi) cultivation conditions, the transformant grew normally, whereas the wild-type (WT) lines did not grow, indicating that the transformant has the ability to metabolize Phi into phosphate (Pi). When cultured under open conditions, the transformant demonstrated enhanced resistance against contamination compared with the WT, indicating that Phi suppresses certain contaminant microorganisms and enables a selective cultivation method for C. sorokiniana . RNA-seq analysis revealed 524 differentially expressed genes (DEGs) between the WT and transformant strains cultured on TAP media, whereas 5364 DEGs were identified for the transformant strains cultured on TAP and TAP-Phi media, suggesting dramatic differences during the use of Phi. A genome-wide analysis revealed 38 phosphate transporter (PHT) genes, among which three PHT genes were upregulated over 100-fold in TAP-Phi medium, implying that these genes play important roles in transporting phosphite into C. sorokiniana cells.