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.

Systematic Analysis of Functionally Related Gene Clusters in the Opportunistic Pathogen, Candida albicans

“…The mechanism that links transcription within clusters has yet to be decisively elucidated, however there is evidence supporting the role of chromatin remodelers mediating changes at the local level to modulated transcription within a genomic neighborhood [ 35 ]. While genes that are found in a divergent orientation oftentimes have bidirectional promoters, as seen in the histone protein coding genes, this does not appear to be a major factor in the regulation of clusters in C. albicans [ 52 , 53 ]. The fact that there is a disproportionate incidence of gene clusters found in a tandem orientation is surprising—there are incidences of tandem oriented pairs exhibiting mutually exclusive transcription patterns, as in SER3 transcription and heat shock protein gene family [ 49 , 54 , 55 ].…”

Engineered Fungus Thermothelomyces thermophilus Producing Plant Storage Proteins

“…Only exchanging the terminators TtrpC for Ttef1 for a bidirectional histone-gene promoters system in Aspergillus resulted in a significant increase in the relative multigene expression by a factor of 1.8, while they were interchangeable and equally efficient for many other expression constructs [48]. Moreover, the use of the similar promoters in a simultaneously operating dual-promoter vector system was thought to interfere with their effectiveness, because the promoter interference has the potential to occur between vectorborne promoters and endogenous promoters following vector integration [40].…”

“…Evidently, the promoter interference cannot always be anticipated and empirical validation of functionality should be a prerequisite, as vector containing more than one target gene and promoters and promoter design features can significantly impact performance [40,44]. Only exchanging the terminators TtrpC for Ttef1 for a bidirectional histone-ge promoters system in Aspergillus resulted in a significant increase in the relative multige expression by a factor of 1.8, while they were interchangeable and equally efficient f many other expression constructs [48]. Moreover, the use of the similar promoters in simultaneously operating dual-promoter vector system was thought to interfere wi their effectiveness, because the promoter interference has the potential to occur betwe vector-borne promoters and endogenous promoters following vector integration [4 However, the 5′-end of OpIE2 insect viral promoter was found to be blocking the activi of the CMV promoter, constructed in a tandem arrangement, when transforming mam malian cells [49].…”

Glycoengineering of Aspergillus nidulans to produce precursors for humanized N-glycan structures

“…The vector pAC986 was constructed by USER-fusion of six PCR fragments amplified by primer pairs. Three of the fragments formed the vector backbone and were amplified from pAC5 using primer pairs DC88/C435, C436/DC103 and DC102/C434, whereas gDNA from A. nidulans was used for amplifying the dual promoter Ph4h3 (Rendsvig et al, 2019) and the heterodimeric αglucosidase II subunits (GII), encoded by AN10702 and AN11054, by the primer pairs DC305/DC306, DC387/DC388, and DC385/DC389, respectively. All plasmids were linearized by digestion with SwaI prior to transformation.…”