Biofilms can enhance industrial fermentation efficiency by increasing cell density, stability, and metabolic activity and have been successfully applied to the continuous production of many small-molecule chemicals. However, the continuous production of proteins by biofilms has been less studied. This study used secretory human epidermal growth factor (hEGF) as a representative product to evaluate and optimize biofilm-based continuous protein production. First, by deleting the protease and overexpressing eight key genes involved in protein secretion in Saccharomyces cerevisiae, the yield of hEGF was improved by 82.6% from 77.4 to 141.3 mg/L in shake flasks. Subsequently, the flocculation genes FLO11 and ALS3 were introduced to facilitate the establishment of a biofilm-based continuous immobilization fermentation model. The optimal strain SIC-ALS3-PDI1 produced 583.8 mg/L of hEGF, with a productivity of 4.9 mg/L/h during traditional free-cell fermentation, while it produced an average of 300.0 mg/L of hEGF in 10 continuous batches of biofilm-based fermentation, with a productivity of 6.3 mg/L/h. Although the hEGF production in biofilms was lower than that in free-cell fermentation, biofilm fermentation demonstrated greater productivity, with the advantage of not requiring seed culture for each batch of fermentation. This study provided a valuable reference for the biofilm-based production of other peptides.

Human epidermal growth factor (hEGF) holds significant importance in the fields of medicine and cosmetics. Therefore, it becomes imperative to develop a highly efficient fermentation system for hEGF production. In this study, a stable hEGF-secreting expression strain was created by integrating the hEGF gene into the genome of Escherichia coli (E. coli) BL21, and an immobilized fermentation system was developed based on biofilm to facilitate continuous hEGF production. After optimization of fermentation conditions and gene dosage, the production of hEGF was increased from 13.9 mg/L to 52.4 mg/L in free-cell fermentation. Moreover, genetic modifications targeting dgcC, csgD, bcsA, and bcsB proved to enhance biofilm formation. When the bcsB was overexpressed in BL21-hEGF-C5, the biofilm-forming ability was enhanced by 91.1% and the production of hEGF was increased by 28% in biofilm-immobilized continuous fermentation. In conclusion, this study successfully confirms the feasibility of continuous hEGF production through the biofilm system of E. coli, providing valuable insights for the development of other proteins in the field of continuous biomanufacturing.

Pleurotus ostreatus is a mushroom species renowned for its abundant nutritional and medicinal properties. Nevertheless, the yield of its fruiting bodies has long remained at a standstill, making it arduous to achieve substantial improvements. Because the traditional composting approach for enhancing the yield of Pleurotus ostreatus has drawbacks such as a long duration and a high susceptibility to mold contamination, incorporating nutritional supplements into the culture medium of P. ostreatus has emerged as a relatively straightforward yet effective approach to enhancing its yield. This study was predicated on the roles of nucleotides and nucleosides in cellular metabolism and signal transduction. These substances were applied during the cultivation process of P. ostreatus to investigate their impact on the growth and nutritional composition of this mushroom. The findings of this study demonstrate that the supplementation of nucleotides and nucleosides not only improved the yield and biological efficiency of P. ostreatus but also increased its dietary fiber content and amino acids. Furthermore, this research has disclosed that nucleotides and nucleosides exert a notable influence on the lignocellulolytic enzyme system. This investigation provides a scientific foundation for the development of novel yields—enhancing agents for P. ostreatus and offering new insights into cultivation techniques for the progress of P. ostreatus cultivation techniques in both academic and practical arenas.