High-throughput screening method and application of filamentous fungal promoter based on single-cell quantitative characterization

Recently, the Yin Wenbing research group of the Institute of Microbiology of the Chinese Academy of Sciences published a paper in Science China Life Sciences (Quantitative characterization of filamentous fungal promoters on single-cell resolution to discover cryptic natural products).
Using the filamentous fungus Aspergillus nipurus as the research model, a high-throughput screening method
for filamentous fungal promoters based on single-cell quantitative characterization was established.

In this study, a high-throughput protoplast preparation method for Aspergillus nidogen was established, and combined with the single-cell fluorescence detection method of flow cytometry, a high-throughput screening method for fungal promoters based on single-cell level characterization was created, which could quantitatively characterize promoter expression intensity
more efficiently, rapidly and accurately 。 The study systematically analyzed 454 transcription factors from Aspergillus nigae from Aspergillus nesting database (AspGD) database, screened 93 secondary metabolism-related transcription factor promoters as research objects, and accurately evaluated their expression intensity with the help of high-throughput screening methods, and obtained a natural promoter library
with a relative expression intensity of up to 37 times.
Among them, the expression intensity of promoters P_zipA and P_sltA is 2.
9 and 1.
5 times
that of the constitutive promoters P_gpdA.

Figure 1.
High-throughput screening and quantitative characterization of filamentous fungal promoters

To evaluate the accuracy of the new high-throughput screening method, promoters of different intensity levels were randomly selected and the expression of the SfGFP gene for which they were responsible was measured by qRT-PCR.
Fluorescence intensity is observed by confocal microscopy; Used to activate the silenced immunosuppressant neosartoricin biosynthetic gene cluster and calculate neosartoricin yield to reflect promoter expression levels
.
The results show that the promoter intensity levels characterized by the above three methods are consistent with the promoter library, which proves that the new high-throughput screening method for fungal promoters based on single-cell level characterization can reliably and accurately characterize promoter strength
.

In addition, the study also used the screened two strong promoters P_zipA and P_sltA to replace the original promoter of the non-ribosomal polypeptide synthase gene Afpes1 of Aspergillus fumigatus, successfully activated the silencing gene, and identified two novel cyclic tetrapeptide compounds by natural product isolation, named fumiganins A and B
.
Combined with the progress of previous research on the Afpes1 gene, this study speculates that its product fumiganins may be related to
the oxidative stress response mechanism and virulence of Aspergillus fumigastus.

Wei Penglin, doctoral student of the Institute of Microbiology, Chinese Academy of Sciences, Fan Jie, special research assistant, and Yu Jingwen, a graduate master's student, are the co-first authors of the paper, and researcher Yin Wenbing is the corresponding author
.
This work was supported
by the National Key Research and Development Program of China, the National Natural Science Foundation of China, the Biological Resources Derivatives Bank Project of the Strategic Biological Resources Service Network Program of the Chinese Academy of Sciences, the 0 to 1 Original Innovation Project of the Chinese Academy of Sciences and the Postdoctoral Science Foundation Project.