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

Jakob K. H. Rendsvig,Jakob B. Hoof,Christopher T. Workman

doi:10.1186/s40694-019-0088-3

Jakob K. H. Rendsvig, Jakob B. Hoof + Show 1 more

Open Access

https://doi.org/10.1186/s40694-019-0088-3

Copy DOI

Abstract

BackgroundFilamentous 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.ResultsIn this study, we characterized the intergenic region between the genes encoding histones H4.1 and H3, from five phylogenetically diverse species of Aspergillus, as bidirectional promoters (Ph4h3). 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 Aspergillus nidulans. Bioinformatic analysis showed that the divergent gene orientation of h4.1 and h3 appears maintained among fungi, and that the Ph4h3 display conserved DNA motifs among the investigated 85 Aspergilli. Two of the heterologous Ph4h3s were utilized for single-locus expression of four genes from the putative malformin producing pathway from Aspergillus brasiliensis in A. nidulans. Strikingly, heterologous expression of mlfA encoding the non-ribosomal peptide synthetase is sufficient for biosynthesis of malformins in A. nidulans, 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.ConclusionsOur study has documented that Ph4h3 is a strong constitutive bidirectional promoter and a valuable new addition to the genetic toolbox of at least the genus Aspergillus.

Highlights

Filamentous fungi are important producers of enzymes and bioactive secondary metabolites and are exploited for industrial purposes
Certain species of filamentous fungi of genus Aspergillus are widely applied for production of primary metabolites, industrial enzymes, pharmaceutical proteins, and bioactive secondary metabolites (SMs)
The h4.1 and h3 genes of each species were inferred from protein sequence homology to hhfA (h4.1, in this study h4 for simplicity) and hhtA (h3) of A. nidulans using BLASTp [22]

Summary

Introduction

Filamentous fungi are important producers of enzymes and bioactive secondary metabolites and are exploited for industrial purposes. Heterologous expression of SM pathways is important for elucidation of biosynthetic pathways originating from non-tractable hosts or for successful activation of a silent pathway [6]. It requires the expression of all pathway-contributing genes, which are often organized as a physically coherent biosynthetic gene cluster (BGC). An alternative approach to ensure activation of the BGC is by the functional expression of each gene This requires multiple promoters reacting to the same stimuli, since consecutive use of the same promoter would be prone to genetic instability and genome defense mechanisms like repeatinduced point mutations [11]

Methods

Results

Conclusion