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.

Aurantiochytrium是一种海洋破囊壶菌，是一种很有前景的生产二十二碳六烯酸 (DHA) 和角鲨烯的生物体。利用食品工业废物和副产品中的蛋白质等廉价物质进行种植是降低生产成本的一个不错的选择；然而， Aurantiochytrium spp的蛋白水解能力。与分类学上接近的Shizochytriumaggregatum相比较低。我们之前从培养物上清液中鉴定出S.aggregatum ATCC 28209中的胞外蛋白酶（胞外蛋白酶 1，EP1） ，并发现类似的蛋白酶基因（EP2）位于 EP1 基因的下游。在本研究中，我们创建了表达 SaEP1 和/或 SaEP2 的转化体，以增强Aurantiochytrium sp 的蛋白水解能力。18W-13a菌株并将其培养在含有酪蛋白作为测试蛋白底物的培养基中。通过SDS-PAGE分析，我们证实转化子比野生型更有效地降解上清液中的酪蛋白，这表明表达的蛋白酶得到了正确的表达和排泄。在酪蛋白培养基中培养4天后，同时表达SaEP1和SaEP2的转化体（命名为EP12菌株）在660 nm处的光密度值和培养物中的细胞数分别比其他菌株高1.48倍和1.38倍。分别为野生型。EP12菌株的DHA和角鲨烯产量分别为158.3和0.23 mg L ^-1，这些值分别比野生型高1.42和2.01倍，表明本研究中创建的EP12是有利的。用于使用含蛋白质培养基培养的菌株。