Modification of essential factors mediating post-translational processing for high-quality protein expression in Penicillium

Abstract

The formation of mature proteins requires complex post-translational modification and processing. Efficient post-translational processing machinery is beneficial for the high-quality expression of proteins. To comprehensively evaluate the role of post-translational mediating factors (PTMFs) in protein synthesis, two reporter strains expressing a homologous protein, Amy15A, and a heterologous protein, TaEG, were constructed in Penicillium oxalicum. Three PTMFs including a conserved basic leucine zipper transcription factor, HacA; an endoplasmic reticulum chaperone-binding protein, BipA; and a protein disulfide isomerase, PdiA, were individually overexpressed in the both reporter strains. The findings showed that overexpression of these PTMFs enhanced the enzymatic activity of both homologous and heterologous proteins. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that, upon overexpression of the PTMFs, heterologous protein secretion remained stable or slightly increased, whereas that of homologous proteins remained unchanged or decreased. Neither the vegetative growth rate nor reporter transcription levels accounted for these variations in protein production or enzymatic activity. Conclusively, this study suggests that PTMFs play a positive role in protein expression and can be leveraged to optimize filamentous fungal chassis cells in the future.

Keywords

Penicillium oxalicum / Post-translational processing / Protein synthesis / HacA / BipA / PdiA

Data Availability Statement

All data generated during this study are included in this published article and its supplementary information files. The original data and resources are available from the corresponding author on reasonable request.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Yibo Hu reports financial support was provided by National Natural Science Foundation of China. Yibo Hu reports financial support was provided by Natural Science Foundation of Hunan Province. Yibo Hu reports financial support was provided by Natural Science Foundation of Changsha. Reports a relationship with that includes:. Has patent pending to. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

CRediT authorship contribution statement

Demin Guo: Investigation, Formal analysis. Shengfang Zhao: Investigation, Formal analysis. Jie Chen: Validation. Shuhui Han: Validation. Yangtao Li: Formal analysis. Yu Chen: Validation, Supervision. Shengbiao Hu: Writing - review & editing, Supervision, Resources. Yibo Hu: Writing - review & editing, Writing - original draft, Validation, Supervision, Funding acquisition.

Acknowledgments

This study was funded by the National Natural Science Foundation of China (31700019), the Natural Science Foundation of Hunan Province (2023JJ30395), and the Natural Science Foundation of Changsha (kq2208169).

Supplementary Materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.engmic.2025.100194.

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