Quantitative Evaluation of NR Locus-Targeted Nuclear Transformation in Chlorella vulgaris Using eGFP and Flow...

Chlorella vulgaris is a haploid green microalga with cost-effective cultivation, high biomass productivity, and heterotrophic growth capability, making it an attractive platform for food, pharmaceutical, and recombinant protein production. However, development of efficient nuclear transformation systems has been limited by its rigid cell wall and intrinsically low homologous recombination (HR) activity. Here, we established an antibiotic-free nuclear transformation strategy targeting the endogenous nitrate reductase (NR) locus in C. vulgaris PKVL7422 using donor constructs designed to promote homologous recombination. Transformation outcomes were quantitatively evaluated by flow cytometry based on GFP-positive frequency. A codon-optimized GFP expression cassette driven by the heterologous CaMV 35S promoter and terminated by the RBCS2 3' untranslated region was flanked by NR homology arms and introduced as linear donor DNA via electroporation. GFP-positive frequency was quantified by flow cytometry. GFP-positive cells were detected at frequencies of 5.1-6.3%. Optimization of homology arm length, donor DNA amount, and electroporation voltage enabled reproducible enrichment of targeted GFP integration events. Junction PCR confirmed precise insertion at the NR locus. Compared with previous reports, the GFP-positive frequencies observed in this study were substantially higher under our experimental conditions. Disruption of the endogenous NR gene enabled antibiotic-free selection based on nitrate metabolism, while GFP fluorescence allowed rapid quantitative screening. These results provide a basis for HR-mediated nuclear genome engineering and recombinant protein expression in C. vulgaris.