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

Area of Science:

• * Biotechnology and Synthetic Biology
• * Microalgal Molecular Biology

Background:

• * Chlorella vulgaris is a promising microalga for various applications due to its rapid growth and heterotrophic capabilities.
• * Efficient nuclear transformation systems are hindered by the alga's rigid cell wall and low homologous recombination (HR) activity.

Purpose of the Study:

• * To establish an antibiotic-free nuclear transformation strategy for Chlorella vulgaris.
• * To enhance homologous recombination efficiency for genome engineering.

Main Methods:

• * Developed donor constructs with NR homology arms for targeted HR at the nitrate reductase (NR) locus.
• * Utilized codon-optimized GFP expression cassette and electroporation for DNA delivery.
• * Quantified transformation efficiency using flow cytometry and confirmed integration via junction PCR.

Main Results:

• * Achieved GFP-positive cell frequencies of 5.1-6.3% using the optimized HR strategy.
• * Demonstrated reproducible integration of donor DNA at the targeted NR locus.
• * Observed significantly higher transformation efficiencies compared to previous reports.

Conclusions:

• * The developed strategy enables efficient, antibiotic-free nuclear genome engineering in Chlorella vulgaris.
• * This method facilitates rapid screening via GFP fluorescence and selection via nitrate metabolism.
• * Provides a foundation for advanced recombinant protein expression and genetic manipulation in Chlorella vulgaris.