Improvement of fatty acid productivity of thraustochytrid, Aurantiochytrium sp. by genome editing.

Thraustochytrid strains belonging to the genus Aurantiochytrium accumulate significant amounts of lipids including polyunsaturated fatty acids and carotenoids and, therefore, are expected to be used for industrial production of various valuable materials. Although various efforts such as chemical mutagenesis and homologous gene recombination have been made to improve lipid productivity of Aurantiochytrium species, low specificity and efficiency in the conventional methods hinder the research progress. Here, we attempted to apply a genome editing technology, the CRISPR-Cas9 system as an alternative molecular breeding technique for Aurantiochytrium species to accelerate the metabolic engineering. The efficiency of specific gene knock-in by the homologous recombination increased more than 10-folds by combining the CRISPR-Cas9 system. As a result of disrupting the genes associated with ß-oxidation of fatty acids by the improved method, the genome edited strains with higher fatty acid productivity were isolated, demonstrating for the first time that the CRISPR-Cas9 system was effective for molecular breeding of the strains in the genus Aurantiochytrium to improve lipid productivity.

Metabolite Profile Analysis of Aurantiochytrium limacinum SR21 Grown on Acetate-based Medium for Lipid Fermentation.

Thraustochytrids, a group of marine protists, are continuously gaining attention due to their capability in producing lipids for various biotechnological applications towards foods, medicines, chemicals, and biofuels. Although various substrates, predominantly glucose, have been used as carbon source for this microalga, it is desirable to adopt cheaper and more diversified substrate to expand their application range. In this study, we aimed to examine the ability of acetate, which can be easily generated from various resources by acetogenic microorganisms, as a substrate of Aurantiochytrium limacinum SR21. As a result of flask-scale analysis, specific growth rates (µ) of the strain SR21 grown in 3% acetate- or glucose-based medium were 0.55 and 0.98 h-1, respectively. The maximum yield of total fatty acid in acetate medium was 4.8 g/L at 48 h while that in glucose medium was 6.8 g/L at 30 h, indicating that acetate has potential as substrate. Metabolome analysis was performed to comprehensively elucidate characteristic metabolic fluctuations caused by acetate assimilation and identify targets to improve the fatty acid productivity from acetate. It was found that the use of glyoxylate cycle, which bypasses release of energy molecules such as NADH and GTP, and the inhibition of utilization of compounds from TCA cycle for anabolic reactions, may cause the slow growth in acetate which has an effect also in lipid productivity. The activity of the pentose phosphate pathway was found to be weak in acetate cultivation, thus NADPH was mainly produced in malate-pyruvate cycle. Lastly, mevalonate pathway was found to be activated in acetate cultivation which additionally competes with acetyl-CoA as starting material of fatty acid synthesis.