A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina)

Abstract

BackgroundThe industrially applied filamentous fungus Trichoderma reesei has received substantial interest due to its highly efficient synthesis apparatus of cellulytic enzymes. However, the production of heterologous enzymes in T. reesei still remains low mainly due to lack of tools for genetic engineering.ResultsIn this study we present new genetic tools for T. reesei to further expand its use in industrial production. We have developed an expression platform where genes are inserted into a versatile expression vector via highly efficient uracil-excision cloning and subsequently inserted into a defined position in the T. reesei genome ensuring that enzyme production from different transformants can be directly compared. The ade2 locus was selected as integration site since ade2 mutants develop red pigment that facilitates easy and rapid detection of correctly targeted transformants. In addition, our system includes a tku70 disruption to increase gene targeting efficiency and a new bidirectional marker, pyr2, for iterative gene targeting. The dual selection system, color and prototrophism, ensures that correct transformants containing the desired gene inserted into the defined expression site can be selected with an efficiency approaching 100%.ConclusionsThe new genetic tools we have developed are suitable for high-throughput integration of genes into the genome of T. reesei and can easily be combined with techniques for generation of defined mutants. Moreover, the usability of the novel expression system with ade2 as integration site was confirmed by expression of a Thermomyces lanuginosus lipase.

Several steps of lateral gene transfer followed by events of ‘birth-and-death’ evolution shaped a fungal sorbicillinoid biosynthetic gene cluster

“…1). To indicate that these two genes are part of the sorbicillin biosynthetic cluster, we will – in agreement with [26] - further name them sor1 (= pks11 ) and sor2 (= pks10 ) throughout the manuscript.

Fig. 1Phylogenetic analysis of SOR1/SorA ( T. reesei PKS11) and SOR2/SorB ( T. reesei PKS10) proteins by PhyML.

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“…Baker et al [24] have recently compared the polyketide synthase (PKS) inventory of three Trichoderma species ( T. reesei , T. virens and T. atroviride ) and showed that two polyketide synthase encoding genes - pks10 , pks11 - were unique to T. reesei. Pks10 and pks11 are located head-to-head in the center of chromosome 5 [25] and were shown to be responsible for the synthesis of sorbicillinoids [26]. These are complex cyclic polyketides, some of which have been shown to exhibit cytostatic and neuroprotective effects [27].…”

“…Verticillium , Acremonium , Paecilomyces; for review see [27]) and the Eurotiomycete Penicillium notatum [30]. In support of this, a putative sorbicillinoid synthesizing cluster similar to the T. reesei cluster, is present in P. rubens [26, 31]. Moreover, the P. rubens orthologue of T. reesei pks11 ( pks13 ) was shown to be essential for sorbicillinoid biosynthesis [32].…”

Heterologous Expression of an Alkali and Thermotolerant Lipase from Talaromyces thermophilus in Trichoderma reesei

“…The decisive advantage of this strain for its extensive application is the salient secretory capacity, not only for the three cellulolytic enzymes (cellobiohydrolase, endo-β-1,4-glucanase, β-glucosidase) [5], but also for various heterologous proteins [6]. Additionally, excellent post-translational modifications such as glycosylation and phosphorylation together with simplified downstream purification make these versatile model fungi more suitable for heterologous expression [7].…”

“…Whereas, low productivity of this fungus held back its further applications. Among the solutions, heterologous expression is always counted upon for its high efficiency and practicability [6]. There are various host candidates for removing this bottleneck [10], such as the prevalent prokaryotic Escherichia coli, eukaryotic yeast, and T. reesei.…”