l-Methionine repressible promoters for tuneable gene expression in Trichoderma reesei

Abstract

BackgroundTrichoderma reesei is the main producer of lignocellulolytic enzymes that are required for plant biomass hydrolysis in the biorefinery industry. Although the molecular toolbox for T. reesei is already well developed, repressible promoters for strain engineering and functional genomics studies are still lacking. One such promoter that is widely employed for yeasts is that of the l-methionine repressible MET3 gene, encoding ATP sulphurylase.ResultsWe show that the MET3 system can only be applied for T. reesei when the cellulase inducing carbon source lactose is used but not when wheat straw, a relevant lignocellulosic substrate for enzyme production, is employed. We therefore performed a transcriptomic screen for genes that are l-methionine repressible in a wheat straw culture. This analysis retrieved 50 differentially regulated genes of which 33 were downregulated. Among these, genes encoding transport proteins as well as iron containing DszA like monooxygenases and TauD like dioxygenases were strongly overrepresented. We show that the promoter region of one of these dioxygenases can be used for the strongly repressible expression of the Aspergillus niger sucA encoded extracellular invertase in T. reesei wheat straw cultures. This system is also portable to other carbon sources including d-glucose and glycerol as demonstrated by the repressible expression of the Escherichia coli lacZ encoded ß-galactosidase in T. reesei.ConclusionWe describe a novel, versatile set of promoters for T. reesei that can be used to drive recombinant gene expression in wheat straw cultures at different expression strengths and in an l-methionine repressible manner. The dioxygenase promoter that we studied in detail is furthermore compatible with different carbon sources and therefore applicable for manipulating protein production as well as functional genomics with T. reesei.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0308-3) contains supplementary material, which is available to authorized users.

A complete annotation of the chromosomes of the cellulase producer Trichoderma reesei provides insights in gene clusters, their expression and reveals genes required for fitness

“…This finding could have potential biotechnological prospects for strain engineering in T. reesei : our data suggest that placing a promoter into any of the CEC areas may lead to a tight shut-off of expression under non-induced conditions. Such expression systems are strongly looked for in industry, but only one has recently been published for T. reesei [ 66 ].…”

The Post-genomic Era of Trichoderma reesei : What's Next?

“…The use of inducible systems has mainly focused on naturally strong promoters such as Pcbh1 and Pcbh2 [61][62][63][64][65]. Additionally, genomic and transcriptomic approaches have been used to identify novel inducible systems, such as copper and L-methionine inducible promoters with strong activity in T. reesei [66,67]; both systems display high performance for constructing stable phenotypes. On the other hand, several studies have tried to identify and characterize strong constitutive expression systems for T. reesei, using, for example, promoters from ribosomal proteins, such as Prp2 [68], the pyruvate decarboxylase promoter Ppdc [69], and the enolase promoter Peno [70].…”

The Promoter Toolbox for Recombinant Gene Expression in Trichoderma reesei

“…The efficient inactivation of the most commonly used promoter of the l -methionine repressible ATP sulphurylase MET3 was demonstrated for different yeasts including Saccharomyces cerevisiae (Cherest et al, 1985 ; Mao et al, 2002 ), P. pastoris (Delic et al, 2013 ), Candida albicans (Care et al, 1999 ), and Ashbya gossypii (Dünkler and Wendland, 2007 ). Addition of l -methionine to the medium does not interfere with T. reesei biomass formation or native cellulase production making this amino acid a suitable substance to repress gene expression (Gremel et al, 2008 ; Bischof et al, 2015 ). The use of different orthologues of these met genes as source for repressible promoters was therefore also tested for T. reesei .…”

“…A strong repression of invertase expression was found in wheat straw cultures, even hours after addition of l -methionine. This repressible promoter is not restricted to wheat straw cultures and was also functional during growth on other carbon sources including d -glucose and glycerol (Bischof et al, 2015 ).…”