Abstract
Filamentous fungi are extensively used as an important expression host for the production of a variety of essential industrial proteins. They have significant promise as an expression system for protein synthesis due to their inherent superior secretory capabilities. The purpose of this study was to develop a novel expression system by utilizing a Penicillium oxalicum strain that possesses a high capacity for protein secretion. The expression of glycoside hydrolases in P. oxalicum was evaluated in a cleaner extracellular background where the formation of two major amylases was inhibited. Four glycoside hydrolases (CBHI, Amy15B, BGL1, and Cel12A) were expressed under the highly constitutive promoter PubiD. It was found that the proteins exhibited high purity in the culture supernatant after cultivation with starch. Two inducible promoters, Pamy15A and PempA, under the activation of the transcription factor AmyR were used as elements in the construction of versatile vectors. When using the cellobiohydrolase CBHI as the extracellular quantitative reporter, the empA promoter screened from the AmyR-overexpressing strain was shown to be superior to the amy15A promoter based on RNA-sequencing data. Therefore, we designed an expression system consisting of a cleaner background host strain and an adjustable promoter. This system enables rapid and high-throughput evaluation of glycoside hydrolases from filamentous fungi.
Key points
• A new protein expression system derived from Penicillium oxalicum has been developed.
• The expression platform is capable of secreting recombinant proteins with high purity.
• The adjustable promoter may allow for further optimization of recombinant protein synthesis.
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All data generated or analyzed during this study are included in this published article and its supplementary files.
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Acknowledgements
We thank Prof. Yinbo Qu of the State Key Laboratory of Microbial Technology, for providing Penicillium oxalicum strains and pyrG marker recycling plasmid.
Funding
This study was funded by National Natural Science Foundation of China (No. 31700019), the Research Foundation of Education Bureau of Hunan Province (No. 17K058), Hunan province college students research learning and innovative experiment project (No. S202110542106), and the Natural Science Foundation of Hunan Province (No. 2017JJ3203).
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ZZ carried out the experiments and drafted the manuscript. XBY, ZSF, and YL participated in the design of the study. CY and HSB supervised the experiments and helped to draft the manuscript. HYB conceived the study and reviewed the final manuscript. All authors read and approved the final manuscript.
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Zhang, Z., **ang, B., Zhao, S. et al. Construction of a novel filamentous fungal protein expression system based on redesigning of regulatory elements. Appl Microbiol Biotechnol 106, 647–661 (2022). https://doi.org/10.1007/s00253-022-11761-0
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DOI: https://doi.org/10.1007/s00253-022-11761-0