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

Abstract

The cellulolytic filamentous fungus Trichoderma reesei has a strong capability in protein synthesis and secretion and is increasingly used as a fungal chassis for the production of heterologous proteins or secondary metabolites. However, bidirectional promoters that would significantly facilitate multiple genes’ expression have not been characterized in T. reesei. Herein, we show that a 767-bp intergenic region between two polyketide synthase encoding genes that were involved in the biosynthesis of the typical yellow pigment served as a bidirectional promoter in T. reesei. This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. Quantitative RT-PCR analysis demonstrated that the driving strength of this bidirectional promoter from each direction reached about half of that of the commonly used promoter PgpdA. Moreover, the co-expression of two cellulase genes driven by this bidirectional promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the carbon source. Our work identified the first bidirectional promoter in T. reesei, which would facilitate gene co-expression and find applications in synthetic biology using fungal systems.

Transcript CD81‐215 may be a long noncoding RNA of stromal origin with tumor‐promoting role in colon cancer

“… 11 The CpG islands were examined using two different approaches. The first approach involves the use of EMBOSS Cpgplot (https://www.ebi.ac.uk/Tools/seqstats/emboss_cpgplot/, Softberry CpG Finder (http://www.softberry.com/berry.phtml?topic=cpgfinder%26group=programs%26subgroup=promoter), and MethPrimer (https://www.urogene.org/methprimer/), with search parameters set according to the Gardiner−Garden and Frommer Algorithm (minimal length of CpG island 200 bp, minimal percentage of G + C 50%, minimal average observed to expected ratio of C + G 0.60) and Takai and Jones Algorithm (minimal length of CpG island 500 bp, minimal percentage of G + C 55%, minimal average observed to expected ratio of C + G 0.65) 12–16 . The second approach entails the use of CLC Genomics Workbench 22.0.2 (http://www.qiagenbioinformatics.com; QIAGEN).…”

Strategies for the Development of Industrial Fungal Producing Strains

“…Recently, natural BDPs have been found in filamentous fungi. It has been shown that BDPs are always intergenic regions regulating the flanking two genes that encode proteins relevant to the same biological process [ 270 ]. The histone H4.1 and H3 promoters can act as natural BDPs, allowing simultaneous expression of enzyme encoding genes in the case of metabolic engineering of Aspergillus sp.…”

“…This region was shown to be able to drive the simultaneous expression of two fluorescence reporter genes when fused to each end. This promoter enabled T. reesei to produce cellulases on glucose and improved the total cellulase activities with cellulose Avicel as the sole carbon source [ 270 ].…”

Promoter screening and identification for metabolic regulation in Acremonium chrysogenum

“…They typically regulate the transcription of gene pairs that have the same function or participate in the same metabolic process. [ 26 ] Fungi naturally possess bidirectional promoters, such as the pcbAB ‐ pcbC promoters located on the CPC synthesis gene cluster in A. chrysogenum , [ 27 ] two polyketide synthase promoters essential for yellow pigment production in T. reesei , [ 28 ] and two promoters of arabinoxylan degradation enzyme in Fusarium oxysporum . [ 29 ] However, it is notable that current studies are primarily focused on identifying the functions and activities of individual bidirectional promoters, and studies on the establishment of a bidirectional promoter library for filamentous fungi have not been reported yet.…”