Engineering a high-efficiency expression system in Tremella fuciformis using novel promoters and optimized...

The development of a high-efficiency expression system is crucial for advancing the molecular breeding and synthetic biology of Tremella fuciformis. Here, the system is reported through the identification of potent endogenous promoters and the systematic optimization of an Agrobacterium tumefaciens-mediated transformation protocol. Key parameters for transformation were established, including optimal antibiotic concentrations (12.5 µg/mL hygromycin, 250 µg/mL cefotaxime) and the critical yeast-like cells (YLCs) density (OD₆₀₀ 0.6-0.8) for co-culture. In addition, scanning electron microscope (SEM) observation revealed that surface damage on YLCs treated with brown aluminum oxide facilitated T-DNA transfer from A. tumefaciens and resulted in a 2.25-fold increase in the number of transformants. Central to this system, gene_sp10042040.1 (P4), gene_sp10011850.1 (P5) and gene_sp10036670.1 (P7) were validated in transformed YLCs, where they drove the expression of the eGFP to levels 67.8-fold, 11.8-fold, and 7-fold higher than the control wild-type strain, respectively. Strikingly, these novel promoters outperformed the constitutive gpd promoter, with an increase in expression efficiency of up to 1476.74%. This integrated expression system, comprising an optimized transformation workflow and a set of superior endogenous promoters, provides a powerful platform for genetic manipulation and metabolic engineering in T. fuciformis.