The Promoter Toolbox for Recombinant Gene Expression in Trichoderma reesei

Abstract

The ascomycete Trichoderma reesei is one of the main fungal producers of cellulases and xylanases based on its high production capacity. Its enzymes are applied in food, feed, and textile industry or in lignocellulose hydrolysis in biofuel and biorefinery industry. Over the last years, the demand to expand the molecular toolbox for T. reesei to facilitate genetic engineering and improve the production of heterologous proteins grew. An important instrument to modify the expression of key genes are promoters to initiate and control their transcription. To date, the most commonly used promoter for T. reesei is the strong inducible promoter of the main cellobiohydrolase cel7a. Beside this one, there is a number of alternative inducible promoters derived from other cellulase- and xylanase encoding genes and a few constitutive promoters. With the advances in genomics and transcriptomics the identification of new constitutive and tunable promoters with different expression strength was simplified. In this review, we will discuss new developments in the field of promoters and compare their advantages and disadvantages. Synthetic expression systems constitute a new option to control gene expression and build up complex gene circuits. Therefore, we will address common structural features of promoters and describe options for promoter engineering and synthetic design of promoters. The availability of well-characterized gene expression control tools is essential for the analysis of gene function, detection of bottlenecks in gene networks and yield increase for biotechnology applications.

Assessment of the ptxD gene as a growth and selective marker in Trichoderma atroviride using Pccg6, a novel constitutive promoter

“…Trichoderma species have gained reputation as biotechnology workhorses for the expression of heterologous and homologous genes for industrial applications and also as a model to study functional genomics in fungi. For recombinant protein production, both constitutive and inducible promoters are essential tools [11]. However, constitutive promoters are often preferable because they are simple to use and do not require external stimulus or agents to activate expression.…”

“…EG2; xyn1, xylanase XYN1) derived from xylanase or cellulase genes are available for the expression of homologous and heterologous genes in different Trichoderma species [10,11,12]. The inducible promoter from the cel7a gene is one of the most often used for industrial purposes [11,13].…”

“…EG2; xyn1, xylanase XYN1) derived from xylanase or cellulase genes are available for the expression of homologous and heterologous genes in different Trichoderma species [10,11,12]. The inducible promoter from the cel7a gene is one of the most often used for industrial purposes [11,13]. However, some drawbacks from using this promoter have been detected because it requires high concentrations of an inducer (cellulose, sophorose, and lactose) which can also activate the expression of other genes leading to the production of contaminant proteins.…”

Genetically Modified Micro-Organisms for Industrial Food Enzyme Production: An Overview

“…Constitutive promoters are expressed independently of the environmentally induced transcription factors and regulate the expression of basal genes. In contrast, tunable promoters react to the presence or absence of biotic or abiotic factors that can induce or repress the promoter [36]. In Bacillus species, the amyL (B. licheniformis α-amylase) and amyM (B. stearothermophilus maltogenic amylase) genes are often used as promoters [22].…”

Promoter tools for further development of Aspergillus oryzae as a platform for fungal secondary metabolite production

“…A variety of basidiomycete terpenes have been successfully produced in A. oryzae by heterologous expression of their respective biosynthetic genes using the genome-editing system [21]. Although the number of promoters that can be used for heterologous expression in filamentous fungi is limited in comparison with that in the yeast Saccharomyces cerevisiae, where a well-established set of promoters covers virtually all patterns of expression [22], promoter tools have been developed for filamentous fungi including Trichoderma reesei [23], A. niger [24], Penicillium chrysogenum [25] and Ustilago maydis [26]. In A. oryzae, the maltose-inducible promoter of the Taka-amylase A gene (amyB) [27][28][29] is often used for heterologous expression [16,20,30], as are the thiamine-inducible promoter of the thiamine thiazole synthase gene (thiA) [31] and the constitutive promoter of the translation-elongation factor 1α (tef1) [32].…”