Functional characterization of an inducible bidirectional promoter from Fusarium oxysporum f. sp. cubense

Abstract

Bidirectional promoters (BDPs) are regulatory DNA sequences (~1000 bp long) intervening two genes arranged on opposite strands with their 5′ ends in close proximity. these genes are mostly co-expressed; but, instances of anti-correlation and independent transcription have been observed. In fungal systems, BDPs have shown to provide an improved genetic circuit by assembling and regulating transcription of different genes of a common metabolic pathway. We have identified an intergenic region (1063 bp) from the genome of Fusarium oxysporum f. sp. cubense (Foc), a banana root pathogen. this intergenic region regulates the expression of a gene pair required for the breakdown of hemicellulose. For characterization, it was cloned into pCSN44 vector backbone between two reporter genes, namely β-glucuronidase (GUS) and enhanced green fluorescent protein (EGFP). The newly formed vector was transformed into Foc and tested for its bidirectional expression activity. Using histochemical staining and fluorescence microscopy, the kinetics for both, GUS and EGFP expression were tested under different growth conditions respectively. The activity was differentially regulated by inducers such as xylan, arabinogalactan and pectin. This is the first report on the isolation of the intergenic region with inducible bidirectional promoter activity from Fusarium. characterization of such BDPs will find applications in genetic engineering, metabolic engineering and synthetic biology using fungal systems.

Identification of a Bidirectional Promoter from Trichoderma reesei and Its Application in Dual Gene Expression

“…Compared with several other reported BDPs from filamentous fungi with a length ranging from ~800 bp to 1200 bp [ 8 , 9 , 10 , 12 , 13 ], T. reesei P sor has a shorter length of 767 bp. Nonetheless, it is currently unclear whether the flanking regions of P sor (the coding sequences within sor1 or sor2 ) are involved in the activation of sor genes, although the 767 bp-intergenic region is competent to drive the expression of two reporter genes and two cellulase genes.…”

“…In contrast, the application of bidirectional promoter (BDPs) helps to facilitate genetic engineering and expand expression flexibility. Natural BDPs have been found in almost all kingdoms, ranging from bacteria to mammals [ 7 , 8 ]. It has been shown that BDPs are always intergenic regions regulating the flanking two genes that encode proteins relevant to the same biological process.…”

“…Up to now, only a few BDPs have been identified and characterized in filamentous fungi, most of which are present in Aspergillus and Penicillium . These BDPs were discovered in the intergenic regions between two divergently oriented genes involved in penicillin production [ 9 , 10 ], nitrogen metabolism [ 10 , 11 , 12 ], histone proteins [ 13 ], or hemicellulases synthesis [ 8 ], and some of them have been successfully used for dual gene co-expression [ 13 ]. However, no BDPs have been characterized in T. reesei , which limits the development of the multiple gene expression system in such an important eukaryotic chassis for the efficient synthesis of recombinant proteins or secondary metabolites.…”

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum

“…Previously identified bidirectional promoters mostly initiated genes involved in co‐regulatory metabolic pathways. [ 29 ] However, several strong bidirectional promoters identified in this study were found to regulate structural proteins rather than functional proteins. It is widely recognized that ribosomal protein genes are characterized by strong promoters, [ 41 ] and this finding was further validated during the exploration of bidirectional promoters which has significant implications for the development of effective genetic engineering strategies for A. chrysogenum .…”

“…The construction of a bidirectional promoter library will expand the possibilities, diversity, and flexibility of gene bidirectional expression. [ 29 ] Compared to unidirectional expression cassettes in tandem, bidirectional cloning of target genes offers a simpler and faster approach to achieve multigene co‐expression. [ 30 ] Furthermore, the use of shorter expression frames can also improve the efficiency of homologous recombination.…”