Nuclear genome shuffling significantly increases production of chloroplast-based recombinant protein in Chlamydomonas reinhardtii

• Published in: Algal research (Amsterdam) Vol. 41
• Main Authors: Fields, Francis J., Ostrand, Joseph T., Tran, Miller, Mayfield, Stephen P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier 15.05.2019
• Subjects: Algae; BASIC BIOLOGICAL SCIENCES; Biotechnology; FACS; Genome shuffling; Mutagenesis; Strain improvement
• ISSN: 2211-9264; 2211-9264

Algae biotechnology holds great promise for the production of sustainable and environmentally friendly products, however more robust strains of microalgae must be developed before algae can be used as a commercially relevant production platform. Here we demonstrate a timely and effective approach to significantly improve a desired phenotype in algae through a combination of mutagenesis and genome shuffling. In this study, we engineered a strain of Chlamydomonas reinhardtii to produce recombinant green fluorescent protein in the chloroplast and then subjected the nuclear genome to various rounds of UV-induced mutagenesis and genome shuffling via sexual recombination, followed by isolation of strains with enhanced recombinant protein expression using fluorescence-activated cell sorting (FACS). Here, the greatest success came from first UV-mutagenizing the host strain to create mutations in the nuclear genome, followed by FACS to isolate the best GFP-expressing mutant, then shuffling the nuclear genome of that mutant by mating it with selected wild-type strains, and then again isolating the best progeny via FACS. Using this strategy, a novel strain was evolved in <3 months to express GFP over 2% total soluble protein (TSP), a 15-fold increase from the initial recombinant strain which expressed GFP at 0.14% TSP. Furthermore, this significant increase in GFP expression from the chloroplast was accomplished without altering the GFP gene itself or reducing the growth rate of the strain, demonstrating the effectiveness of this strategy to enhance a specific phenotype and significantly improve product production within an algal species in a commercially relevant timeframe.