Safety evaluation and optimization of high-efficiency expression of 5hsCT in Saccharomyces cerevisiae

To evaluate the oral safety of the industrial strain Saccharomyces cerevisiae YS-5hsCT (S. cerevisiae YS-5hsCT) expressing human/salmon chimeric calcitonin (5hsCT) and optimize its fermentation conditions, providing scientific basis for industrial production of oral recombinant calcitonin. The strain demonstrated an LD₅₀ > 10 g kg⁻¹, with all toxicological tests yielding negative results and normal hematological/biochemical parameters (P > 0.05). mRNA analysis revealed optimal gene expression at 96 h, 30 °C and 225 rpm. Single-factor experiments established optimal growth conditions: 8% initial inoculum size, 72 h culture, 30 °C, 200-250 rpm agitation, and 7-8.5 l min⁻¹ aeration. Under optimized conditions (10% inoculum size, 72 h, 25 °C, 225 rpm, 7 l min⁻¹ aeration), the biomass reached 7.175 g l⁻¹ d⁻¹ and the 5hsCT protein yield was 149.24 mg l⁻¹ d⁻¹. These findings confirm the oral safety of YS-5hsCT and providetechnical support for industrial calcitonin production. Safety evaluations demonstrated transgenic S. cerevisiae YS-5hsCT exhibits no toxicity or mutagenicity, with optimized cultivation conditions enhancing biomass yield for potential calcitonin production applications.