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Diosgenin is used widely to synthesize steroidal hormone drugs in the pharmaceutical industry. The conventional diosgenin production process, direct acid hydrolysis of the root of Dioscorea zingiberensis C. H. Wright (DZW), causes large amounts of wastewater and severe environmental pollution. To develop a clean and effective method, the endophytic fungus Fusarium sp. CPCC 400226 was screened for the first time for the microbial biotransformation of DZW in submerged fermentation (SmF). Statistical design and response surface methodology (RSM) were implemented to develop the diosgenin production process using the Fusarium strains. The environmental variables that significantly affected diosgenin yield were determined by the two-level Plackett-Burman design (PBD) with nine factors. PBD indicates that the fermentation period, culture temperature, and antifoam reagent addition are the most influential variables. These three variables were further optimized using the response surface design (RSD). A quadratic model was then built by the central composite design (CCD) to study the impact of interaction and quadratic effect on diosgenin yield. The values of the coefficient of determination for the PBD and CCD models were all over 0.95. P -values for both models were 0.0024 and &lt;0.001, with F -values of ∼414 and ∼2215, respectively. The predicted results showed that a maximum diosgenin yield of 2.22% could be obtained with a fermentation period of 11.89 days, a culture temperature of 30.17 °C, and an antifoam reagent addition of 0.20%. The experimental value was 2.24%, which was in great agreement with predicted value. As a result, over 80% of the steroidal saponins in DZW were converted into diosgenin, presenting a ∼3-fold increase in diosgenin yield. For the first time, we report the SmF of a Fusarium strain used to produce diosgenin through the microbial biotransformation of DZW. A practical diosgenin production process was established for the first time for Fusarium strains. This bioprocess is acid-free and wastewater-free, providing a promising environmentally friendly alternative to diosgenin production in industrial applications. The information provided in the current study may be applicable to produce diosgenin in SmF by other endophytic fungi and lays a solid foundation for endophytic fungi to produce natural products.

Development of a New Bioprocess for Clean Diosgenin Production through Submerged Fermentation of an Endophytic Fungus