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

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Abstract

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 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.

Conclusions
Our 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.
Original languageEnglish
JournalFungal Biology and Biotechnology
Volume6
Issue number24
Number of pages14
ISSN2054-3085
DOIs
Publication statusPublished - 2019

Keywords

  • Aspergillus
  • Bidirectional promoters
  • Histone
  • Motifs
  • Heterologous expression
  • Biosynthetic gene clusters
  • Malformin
  • Non-ribosomal peptide synthetase
  • Adenylation domain

Cite this

@article{4f826d7e2d114ae5b8450f9f1d3b7f38,
title = "Bidirectional histone‑gene promoters in Aspergillus: characterization and application for multi‑gene expression",
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.",
keywords = "Aspergillus, Bidirectional promoters, Histone, Motifs, Heterologous expression, Biosynthetic gene clusters, Malformin, Non-ribosomal peptide synthetase, Adenylation domain",
author = "Rendsvig, {Jakob Kr{\ae}mmer Haar} and Christopher Workman and Hoof, {Jakob Bl{\ae}sbjerg}",
year = "2019",
doi = "10.1186/s40694-019-0088-3",
language = "English",
volume = "6",
journal = "Fungal Biology and Biotechnology",
issn = "2054-3085",
publisher = "BioMed Central Ltd.",
number = "24",

}

TY - JOUR

T1 - Bidirectional histone‑gene promoters in Aspergillus: characterization and application for multi‑gene expression

AU - Rendsvig, Jakob Kræmmer Haar

AU - Workman, Christopher

AU - Hoof, Jakob Blæsbjerg

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Aspergillus

KW - Bidirectional promoters

KW - Histone

KW - Motifs

KW - Heterologous expression

KW - Biosynthetic gene clusters

KW - Malformin

KW - Non-ribosomal peptide synthetase

KW - Adenylation domain

U2 - 10.1186/s40694-019-0088-3

DO - 10.1186/s40694-019-0088-3

M3 - Journal article

VL - 6

JO - Fungal Biology and Biotechnology

JF - Fungal Biology and Biotechnology

SN - 2054-3085

IS - 24

ER -