Bidirectional histone-gene promoters in : characterization and application for multi-gene expression.

BACKGROUND

Filamentous fungi are important producers of enzymes and bioactive secondary metabolites and are exploited for industrial purposes. Expression and characterization of biosynthetic pathways requires stable expression of multiple genes in the production host. Fungal promoters are indispensable for the accomplishment of this task, and libraries of promoters that show functionality across diverse fungal species facilitate synthetic biology approaches, pathway expression, and cell-factory construction.

RESULTS

In this study, we characterized the intergenic region between the genes encoding histones H4.1 and H3, from five phylogenetically diverse species of , as bidirectional promoters (P). By expression of the genes encoding fluorescent proteins mRFP1 and mCitrine, we show at the translational and transcriptional level that this region from diverse species is applicable as strong and constitutive bidirectional promoters in . Bioinformatic analysis showed that the divergent gene orientation of and appears maintained among fungi, and that the P display conserved DNA motifs among the investigated 85 Aspergilli. Two of the heterologous Ps were utilized for single-locus expression of four genes from the putative malformin producing pathway from in . Strikingly, heterologous expression of encoding the non-ribosomal peptide synthetase is sufficient for biosynthesis of malformins in , which indicates an iterative use of one adenylation domain in the enzyme. However, this resulted in highly stressed colonies, which was reverted to a healthy phenotype by co-expressing the residual four genes from the putative biosynthetic gene cluster.

CONCLUSIONS

Our study has documented that P is a strong constitutive bidirectional promoter and a valuable new addition to the genetic toolbox of at least the genus .