Schizochytrium microalgae expressing recombinant bovine lactoferrin as a route to improve intestinal health in calves

Enteric diseases are one of the leading causes of neonatal calf mortalities and result in prolonged reduced performance for surviving animals, causing an estimated €10 million in losses to the global dairy industry annually. With increasing restrictions on antibiotic use due to antimicrobial resistance, alternative strategies are required to enhance intestinal health and immunity in calves. Nutritional interventions, particularly those supplementing bioactive compounds, offer a promising solution. This thesis investigates the genetic engineering of the microalga Schizochytrium sp. (SZ) to co-produce docosahexaenoic acid (DHA) and recombinant bovine lactoferrin (rbLF) and evaluates its potential role in improving intestinal health in calves. In Chapter 2, an rbLF expression cassette was constructed and integrated into SZ via 18S rRNA gene recombination. The recombinant strain (rSZ) exhibited comparable growth performance to wild-type SZ while expressing rbLF at 4.63% of total protein yield and producing 8.77 g/L DHA. The expression and structure of rbLF were confirmed through Western blot, ELISA, and mass spectrometry, achieving 66.81% amino acid coverage and correct antigenicity. Chapter 3 assessed the biological activity of rbLF on calf intestinal epithelial cells (CIECs). rbLF demonstrated no cytotoxicity and promoted cell growth at an optimal concentration of 175 μg/mL. Treatment enhanced tight junction protein expression, reduced oxidative stress, and decreased inflammatory markers, with a sustained reduction in TNF-α observed 24 h post-incubation. In Chapter 4, the immunomodulatory capacity of rbLF was examined under oxidative and inflammatory challenges. rbLF provided protection against H2O2 - induced cellular stress when applied either as pretreatment or post-challenge, though preventive application sometimes aggravated damage. Nevertheless, both treatment modalities increased antioxidant activity, strengthened tight junction protein expression, and attenuated LPS - and H2O2 - induced inflammation. Given the potential degradation of bioactive proteins during gastrointestinal passage, Chapter 5 evaluated digestion dynamics of SZ and rSZ using the INFOGEST in vitro digestion system. Maximum digestion occurred at substrate-to-fluid ratios of 1:3 - 1:4, with stability maintained at higher ratios. Ball-milling improved digestion efficiency without compromising rbLF stability. Digestion products promoted CIEC growth at a concentration of 800 μg/mL. Chapter 6 investigated the influence of ball - milled and non - ball - milled SZ and rSZ on intestinal barrier integrity using a transwell model. CIEC monolayers were robustly established within 15 days of inoculation with rSZ. Both SZ and rSZ enhanced barrier function under E.coli K99 challenge, though rSZ exerted superior effects, evidenced by significantly higher ZO-1 expression (2.30 to 2.28 - fold) and lower IL-1β expression (0.83 - fold). Collectively, this thesis demonstrates that DHA-enriched SZ can be engineered as a production and delivery platform for functional rbLF. Recombinant SZ-derived rbLF survives gastrointestinal digestion, retains bioactivity, and contributes to enhanced epithelial barrier integrity and resilience against infection. These findings support the application of recombinant SZ as a nutritional strategy to address calf enteric disease, warranting further in vivo validation.