Genetic Modification of Aurantiochytrium sp. 18W-13a for Enhancement of Proteolytic Activity by Heterologous Expression of Extracellular Proteases

Abstract

A marine thraustochytrid, Aurantiochytrium, is a promising organism to produce docosahexaenoic acid (DHA) and squalene. Utilization of inexpensive substances such as proteins in wastes and by-products from the food industry for cultivation is a considerable option to reduce production cost; however, the proteolytic ability of Aurantiochytrium spp. is low compared to taxonomically close Shizochytrium aggregatum. We previously identified extracellular protease (extracellular protease 1, EP1) in S. aggregatum ATCC 28209 from the supernatant of the culture and found that a similar protease gene (EP2) was located downstream of the EP1 gene. In the present study, we created the transformants expressing SaEP1 and/or SaEP2 to enhance the proteolytic ability of Aurantiochytrium sp. 18W-13a strain and cultivated them in the medium containing casein as a test protein substrate. Through SDS-PAGE analysis, we confirmed that casein in the supernatant was more efficiently degraded by the transformants than the wild type, suggesting that the expressed protease(s) were properly expressed and excreted. After 4-day cultivation in the casein medium, the value of optical density at 660 nm and the cell number in the culture of the transformant that expressed both SaEP1 and SaEP2 (designated as EP12 strain) showed 1.48- and 1.38-fold higher than those of wild type, respectively. The DHA and squalene yield of the EP12 strain were respectively 158.3 and 0.23 mg L⁻¹, and these values were 1.42- and 2.01-fold higher than those of wild type, respectively, suggesting that the EP12 created in the present study is a favorable strain for the cultivation using protein-containing medium.