The price of fish oil has reached a historical peak due to a consistent downward production trend, and therefore, the search for sustainable alternative sources has received great attention. This research was conducted to evaluate dietary micro-algae, Schizochytrium sp. (SC) as fish oil (FO) replacer in rainbow trout, Oncorhynchus mykiss. In the first trial, apparent digestibility coefficient (ADC) was 92.4% for dry matter, 91.4% for crude protein, and 94.2% for crude lipid in rainbow trout. In the second trial, six diets were formulated to replace FO at 0% (CON), 20% (T20), 40% (T40), 60% (T60), 80% (T80), and 100% (T100) with SC in the rainbow trout (3.0 ± 0.4 g, mean ± SD) diet. After eight weeks’ feeding trial, weight gain (WG), specific growth rate (SGR), and feed efficiency (FE) of fish fed the T20 diet were significantly higher than those of fish fed other diets (p < 0.05). However, there were no significant differences in these parameters among those of fish fed CON, T40, T60, and T80 diets. Lysozyme activity of fish fed the T20 diet was significantly higher than those of fish fed other experimental diets (p < 0.05). After 10 days of disease challenge testing with pathogenic bacteria (Lactococcus garvieae 1 × 10⁸ CFU/mL), the cumulative survival rate of fish fed the T20 diet was significantly higher than those of fish fed the CON, T80, and T100 diets. Therefore, these results suggest dietary microalgae SC is well-digested and could replace up to 80% of fish oil in the diet of rainbow trout without negative effects on growth and immune responses.

Schizochytrium, a group of eukaryotic marine protists, is an oleaginous strain, making it a highly promising candidate for the production of lipid-derived products such as biofuels and omega-3 fatty acids. However, the insufficient advancement of genetic engineering tools has hindered further advancements. Therefore, the development and application of genetic engineering tools for lipid enhancement are crucial for industrial production. Transgene expression in Schizochytrium often encounters challenges such as instability due to positional effects. To overcome this, we developed a safe-harbor transgene expression system. Initially, the sfGFP gene was integrated randomly, and high-expressing transformants were identified using fluorescence-activated cell sorting. Notably, HRsite 2, located approximately 3.2 kb upstream of cytochrome c, demonstrated enhanced sfGFP expression and homologous recombination efficiency. We then introduced the 3-ketoacyl-ACP reductase (KR) gene at HRsite 2, resulting in improved lipid and docosahexaenoic acid (DHA) production. Transformants with KR at HRsite 2 exhibited stable growth, increased glucose utilization, and a higher lipid content compared to those with randomly integrated transgenes. Notably, these transformants showed a 25% increase in DHA content compared to the wild-type strain. This study successfully established a robust homologous recombination system in Schizochytrium sp. by identifying a reliable safe harbor site for gene integration. The targeted expression of the KR gene at this site not only enhanced DHA production but also maintained growth and glucose consumption rates, validating the efficacy of the safe-harbor approach. This advancement in synthetic biology and metabolic engineering paves the way for more efficient biotechnological applications in Schizochytrium sp.