Chlamydomonas mutant strain and its uses

Non-GMO microalgae with increased zeaxanthin and lipid content via simultaneous CRISPR-mediated knockout of ZEP and AGP

This technology involves a mutant strain of the microalga Chlamydomonas reinhardtii, created by introducing specific insertions, deletions, or substitutions into the ZEP and AGP genes. By using CRISPR to knock out both genes simultaneously without inserting foreign DNA, the strain achieves higher lipid, zeaxanthin, and lutein production compared to the wild type.

While fish oil is rich in omega-3s, it is prone to rancidity and raises concerns regarding heavy metal/dioxin contamination in large fish and the depletion of marine resources. Vegetable oils, such as soybean oil, are high in polyunsaturated fatty acids and oxidize easily during production and storage. Hydrogenated oils, developed to address this, create trans fats, and microalgae oil—a popular alternative—also faces limitations due to its high unsaturated fatty acid content, making it susceptible to oxidation.

This technology uses ZEP mutations to accumulate zeaxanthin and AGP mutations to boost lipid production, enabling the intracellular synthesis of vegetable oil containing antioxidant xanthophyll pigments. The mutant strain grows rapidly, improving culture efficiency. The resulting lipids can be extracted as oil, or the biomass can be used directly as food or feed. Applicable to edible microalgae oils, omega oils, antioxidant-rich cosmetic ingredients, and aquaculture/livestock feed, it provides a stable, long-lasting oil without the need for synthetic antioxidants, free from GMO regulatory burdens.

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Key Features:

• ZEP gene mutation characterized by the insertion of adenine or a specific nucleotide sequence between the 816th and 817th nucleotides of the ZEP gene sequence of SEQ ID NO: 1
• AGP gene mutation characterized by the insertion of adenine between the 116th and 117th nucleotides, or the deletion of the 115th nucleotide and substitution of the 116th nucleotide with thymine in the AGP gene sequence of SEQ ID NO: 2
• Chlamydomonas reinhardtii mutant strain with improved lipid, zeaxanthin, and lutein production compared to the wild-type strain
• Process of culturing the mutant strain and extracting lipids from the strain or culture to produce vegetable oil

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This invention was developed with support from the Ministry of Science and ICT for the development of high-efficiency CO2-fixing microalgae through molecular biological improvement.

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