Background: Due to its ability to perform fast and high-density fermentation, Pichia pastoris is not only used as an excellent host for heterologous protein expression but also exhibits good potential for efficient biosynthesis of small-molecule compounds. However, basic research on P. pastoris lags far behind Saccharomyces cerevisiae, resulting in a lack of available biological elements. Especially, fewer strong endogenous promoter elements available for foreign protein expression or construction of biosynthetic pathways were carefully evaluated in P. pastoris. Thus, it will be necessary to identify more available endogenous promoters from P. pastoris. Results: Based on RNA-seq and LacZ reporter system, eight strong endogenous promoters contributing to higher transcriptional expression levels and β-galactosidase activities in three frequently-used media were screened out. Among them, the transcriptional expression level contributed by P₀₀₁₉, P₀₁₀₇, P₀₂₃₀, P₀₃₉₂, or P₀₇₈₅ was basically unchanged during the logarithmic phase and stationary phase of growth. And the transcriptional level contributed by P₀₂₀₈ or P₀₆₂₇ exhibited a growth-dependent characteristic (a lower expression level during the logarithmic phase and a higher expression level during the stationary phase). After 60 h growth, the β-galactosidase activity contributed by P₀₂₀₈, P₀₆₂₇, P₀₀₁₉, P₀₄₀₇, P₀₃₉₂, P₀₂₃₀, P₀₇₈₅, or P₀₁₀₇ was relatively lower than P_GAP but higher than P_ACT1. To evaluate the availability of these promoters, several of them were randomly applied to a heterogenous β-carotene biosynthetic pathway in P. pastoris, and the highest yield of β-carotene from these mutants was up to 1.07 mg/g. In addition, simultaneously using the same promoter multiple times could result in a notable competitive effect, which might significantly lower the transcriptional expression level of the target gene. Conclusions: The novel strong endogenous promoter identified in this study adds to the number of promoter elements available in P. pastoris. And the competitive effect observed here suggests that a careful pre-evaluation is needed when simultaneously and multiply using the same promoter in one yeast strain. This work also provides an effective strategy to identify more novel biological elements for engineering applications in P. pastoris.

Adeno-associated virus (AAV) vectors face a critical translational challenge in ocular gene therapy due to pre-existing neutralizing antibodies (NAbs) whose seroprevalence limits patient eligibility. Standard NAb detection using non-ocular cell models (Human Embryonic Kidney 293T) may inadequately predict retinal transduction inhibition due to cell type-related variations in receptor usage and immunogenicity. This study established parallel NAb detection platforms utilizing human retinal pigment epithelial (ARPE-19) cells and standard 293T cells to systematically evaluate clinical serum samples against ophthalmologically relevant AAV serotypes (2, 5, 8, 9) via luciferase reporter-based transduction inhibition assays. Comparative analysis demonstrated ARPE-19 exhibited 42–48% higher NAb titers against AAV5/9 compared to 293T cells, with distinct serotype-biased neutralization hierarchies observed between cellular models. Furthermore, female-derived sera exhibited significantly elevated NAbs against particular serotypes in the ARPE-19 system. Critically, inter-serotype cross-neutralization correlation patterns differed substantially between cellular platforms. These findings demonstrate that physiologically relevant retinal cellular models provide essential immunological profiling data, revealing NAb characteristics obscured in standard assays. Consequently, employing retinal cell-based platforms is crucial for optimizing AAV serotype selection, patient stratification, and predicting clinical outcomes in ocular gene therapy.