Macular pigment-enriched oil production from genome-edited microalgae

Area of Science:

• Biotechnology
• Microalgal cultivation
• Genetic engineering

Background:

• Chlamydomonas reinhardtii, a GRAS microorganism, can overproduce carotenoid pigments and fatty acids.
• Zeaxanthin epoxidase (ZEP) and ADP-glucose pyrophosphorylase (AGP) are key regulatory genes in xanthophyll and starch pathways.
• Editing these genes is crucial for enhancing pigment and lipid production.

Purpose of the Study:

• To create a double knock-out mutant (dZA) in C. reinhardtii by simultaneously deleting ZEP and AGP genes.
• To enhance the production of macular pigments and lipids for food applications.
• To develop a one-step cultivation process for efficient microalgal oil production.

Main Methods:

• Utilized CRISPR-Cas9 RNP-mediated gene editing for sequential knock-out of ZEP and AGP genes.
• Cultivated the generated double knock-out mutants (dZAs) under nitrogen-deprivation conditions.
• Optimized culture medium and process for one-step pigment and oil production.
• Developed a hexane/isopropanol extraction method for edible microalgal oil.

Main Results:

• Generated dZA mutants with high accumulation of lutein, zeaxanthin, and lipids.
• Achieved 81% higher oil productivity in a one-step cultivation process compared to two-step methods.
• Produced edible microalgal oil rich in lutein and zeaxanthin (196 mg/g DCW oil).

Conclusions:

• Simultaneous induction of lipids and pigments was observed in the dZA strain.
• The dZA mutant is advantageous for food applications as it lacks antibiotic resistance genes.
• Enriched macular pigment oil from dZAs is suitable for various food products and nutraceuticals.