Thaumatin (Tha), a natural sweetener derived from the fruit of Thaumatococcus daniellii, is 1 600 times sweeter than sucrose.It is characterized by its minimal caloric content and its complete degradation into amino acids essential for human nutrition, aligning with the modern food industry’s demand for safe, healthy, and high-potency sweeteners.To achieve its industrial-scale production in the yeast Pichia pastoris, a systematic strategy was implemented using the GS115 strain.This strategy involved promoter screening, signal peptide engineering, gene dosage optimization, and the coordinated expression of multiple chaperone pathways.Initial screening of three promoters, including PAOX1, PGAP, and PGCW14, revealed that PAOX1 yielded the highest extracellular Tha titre, reaching 33.37 mg/L.Subsequently, fourteen hybrid signal peptides were constructed.Among these, KexEA-α enhanced secretion to 47.89 mg/L.Using an isoschizomer-based assembly method, expression vectors carrying 1 to 6 copies of the Tha gene were built.The optimal gene dosage was identified as 4 copies, achieving a titre of 60.12 mg/L.Further increases in copy number led to a decline in production, likely due to excessive cellular burden.Through single-factor screening, seven beneficial chaperone proteins were identified:Ero1, Pdi1, Pdi2, Sec53, Hrd1, Ubc1, and Sso2.Co-expression of Ero1 alone increased Tha production to 72.02 mg/L.A synergistic five-factor combination was then constructed by co-expressing chaperones from the oxidative folding module (Pdi1-Pdi2-Ero1) with those from the quality control module (Hrd1-Ubc1).This strategy boosted the titre to 154.29 mg/L, which is 6.29 times higher than that of the original strain.Scale-up was performed using a 3-L bioreactor for high-cell-density fed-batch fermentation.After 156 hours of induction, the Tha concentration reached 1.09 g/L, representing a 7.06-fold increase over the shake-flask yield.To our knowledge, this study reports the first achievement of g/L secretion of Tha in P.pastoris, establishing a critical technical foundation for its low-cost, industrial-scale production.
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