Construction of an expression platform for fungal secondary metabolite biosynthesis in Penicillium crustosum

Abstract Filamentous fungi are prolific producers of bioactive natural products and play a vital role in drug discovery. Yet, their potential cannot be fully exploited since many biosynthetic genes are silent or cryptic under laboratory culture conditions. Several strategies have been applied to activate these genes, with heterologous expression as one of the most promising approaches. However, successful expression and identification of new products are often hindered by host-dependent factors, such as low gene targeting efficiencies, a high metabolite background, or a lack of selection markers. To overcome these challenges, we have constructed a Penicillium crustosum expression host in a pyrG deficient strain by combining the split-marker strategy and CRISPR-Cas9 technology. Deletion of ligD and pcribo improved gene targeting efficiencies and enabled the use of an additional selection marker in P. crustosum. Furthermore, we reduced the secondary metabolite background by inactivation of two highly expressed gene clusters and abolished the formation of the reactive ortho-quinone methide. Finally, we replaced the P. crustosum pigment gene pcr4401 with the commonly used Aspergillus nidulans wA expression site for convenient use of constructs originally designed for A. nidulans in our P. crustosum host strain. As proof of concept, we successfully expressed a single polyketide synthase gene and an entire gene cluster at the P. crustosum wA locus. Resulting transformants were easily detected by their albino phenotype. With this study, we provide a highly efficient platform for heterologous expression of fungal genes. Key points Construction of a highly efficient Penicillium crustosum heterologous expression host Reduction of secondary metabolite background by genetic dereplication strategy Integration of wA site to provide an alternative host besides Aspergillus nidulans Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00253-024-13259-3.


Fig. S1
Fig. S1 Schematic illustration of the Cas9 and gRNA expression plasmid pJZ66 (gRNA: guide RNA, PAM: protospacer adjacent motif, TEF promoter: Aureobasidium pullulans translation elongation factor promoter, NLS: nuclear localization signal, gla terminator: Aspergillus awamori glucoamylase terminator, lac: lactose operon, ori: origin of replication, AmpR: ampicillin resistance) Fig. S9 Schematic illustration of wA expression in P. crustosum JZ35 and PCR verification of JZ37 (wA::pyrG) and JZ38 (wAΔpyrG) by amplification of different partial fragments (P1-P5) from genomic DNA.Transformants were confirmed by amplification of the upstream and downstream sequence with primers binding outside of the deletion cassette and in the wA gene for P1 and P3, and by amplification of parts of the wA gene with or without the pyrG sequence for P2 and P4.The presence of pyrG was verified with primers binding in the pyrG sequence for P5.Primer sequences and their corresponding Primer-IDs are given in Supplemental TableS3.(5F: upstream flanking region, 3F: downstream flanking region)

Fig. S11
Fig. S11 Schematic illustration of pcribo (pcr11223) deletion in P. crustosum JZ38 and PCR verification of JZ51 (Δpcribo::pyrG) and JZ52 (ΔpcriboΔpyrG) by amplification of different partial fragments (P1-P5) from genomic DNA.The presence of pcribo was verified with primers binding in the pcribo sequence for P1.Transformants were confirmed using primers binding both outside of the deletion construct and in the pyrG sequence for P2 and P3, outside the deletion construct for P4, and in the pyrG sequence for P5.Primer sequences and their corresponding Primer-IDs are given in Supplemental TableS3.(5F: upstream flanking region, 3F: downstream flanking region)

Table S1
Aspergillus nidulans strains used in this study

Table S3
Primers used in this study

Table S4
Spacer sequences targeting the claF, pcr4401 and pcribo genes