Construction of a Cosmid-Based Ultraefficient Genomic Library System for Filamentous Fungi of the Genus Aspergillus

Abstract

Filamentous fungi of the genus Aspergillus include producers of industrially important organic acids, enzymes, and secondary metabolites, as well as pathogens of many plants and animals. Novel genes in the Aspergillus genome are potentially crucial for the fermentation and drug industries (e.g., agrochemicals and antifungal drugs). A research approach based on classical genetics is effective for identifying functionally unknown genes. During analyses based on classical genetics, mutations must be identified easily and quickly. Herein, we report the development of a cosmid-based plasmid pTOCK1 and the use of a genomic library of Aspergillus nidulans constructed using pTOCK1. The cosmid-based genomic library was used for convenient auxotrophic mutants (pyroA and pabaB), as well as mutants with abnormal colony morphology (gfsA) and yellow conidia (yA), to obtain library clones complementary to these phenotypes. The complementary strain could be obtained through a single transformation, and the cosmid could be rescued. Thus, our cosmid library system can be used to identify the causative gene in a mutant strain.

Keywords: Aspergillus; classical genetics; cosmid vector; genomic library.

Figure 1

Cosmid vector map of pTOCK1. The DDBJ/ENA/GenBank accession number is LC795782. The addgene catalog number is #215566.

Figure 2

Plates coated with AKU89PPAP transformed with pTOCK1-gAKU89; MM supplemented with biotin, arginine, pyridoxine, and 4-aminobenzoic acid (PABA) (left panel) as a positive control; MM supplemented with biotin, arginine, and PABA to select pyroA complementation strains (middle panel); MM supplemented with biotin, arginine, and pyridoxine to select pabaB complementation strains (right panel); all plates were incubated at 37 °C for 3 days.

Figure 3

Cosmid rescue technique using in vitro packaging; (A) colonies of Escherichia coli ED8767 propagated after in vitro packaging with 0.36 µg of the genome of the AKU89PPAP + pyroA candidate strain; (B) Ten colonies were randomly selected for colony PCR for pyroA.

Figure 4

Complementation of the gfsA mutant by pTOCK1-gAKU89; ΔgfsA ΔpyrG was transformed with pTOCK1-gAKU89 and incubated on MM supplemented with biotin at 37 °C for 3 days. Transformants were diluted such that 100 colonies appeared on one plate and were applied to several plates. Red arrows indicate transformants complemented with gfsA.

Figure 5

Complementation of the yA mutant by pTOCK1-gAKU89; AKU89PY was transformed with pTOCK1-gAKU89 and incubated on MM supplemented with arginine and biotin at 37 °C for 3 days. Transformants were diluted such that 100 colonies appeared on one plate and applied to several plates. Red arrows indicate that the transformants complement yA.

Figure 6

Reamplification of pTOCK1-gAKU89 using in vitro packaging; reconstituted lambda phage was constructed using 0.06 µg of pTOCK1-gAKU89 and propagated through Escherichia coli ED8767.

Figure 7

Overall view of the E. coli-filamentous fungi shuttle cosmid-based library system.