Genomic Locus of a Penicillium crustosum Pigment as an Integration Site for Secondary Metabolite Gene Expression

Abstract

Heterologous expression of secondary metabolite genes and gene clusters has been proven to be a successful strategy for identification of new natural products of cryptic or silent genes hidden in the genome sequences. It is also a useful tool to produce designed compounds by synthetic biology approaches. In this study, we demonstrate the potential usage of the gene locus pcr4401 in the fast-growing filamentous fungus Penicillium crustosum as an integration site for heterologous gene expression. The deduced polyketide synthase (PKS) Pcr4401 is involved in the dihydroxynaphthalene (DHN)-melanin pigment formation, and its deletion in P. crustosum PRB-2 led to an albino phenotype. Heterologous expression of pcr4401 in Aspergillus nidulans proved its function as the melanin precursor YWA1 synthase. To ensure gene expression after genomic integration and to easily identify the potential transformants by visualization, the gene locus of pcr4401 was chosen as an integration site. For heterologous expression in P. crustosum, the expression constructs were created by ligation-independent homologous recombination in Escherichia coli or Saccharomyces cerevisiae. A pyrG deficient strain was also created, so that both the pyrG and hph resistance gene can be used as selection markers. Successful expression in P. crustosum was demonstrated by using one uncharacterized PKS gene from Aspergillus and two from Penicillium strains. All three genes were successfully introduced, heterologously expressed, and their biosynthetic products elucidated. The results presented in this study demonstrated that P. crustosum can be used as a suitable host for heterologous expression of secondary metabolite genes.