BackgroundTrichoderma reesei is an organism involved in degradation of (hemi)cellulosic biomass. Consequently, the corresponding enzymes are commonly used in different types of industries, and recently gained considerable importance for production of second-generation biofuel. Many industrial T. reesei strains currently in use are derived from strain Rut-C30, in which cellulase and hemicellulase expression is released from carbon catabolite repression. Nevertheless, inducing substances are still necessary for a satisfactory amount of protein formation.ResultsHere, we report on a T. reesei strain, which exhibits a very high level of xylanase expression regardless if inducing substances (e.g. D-xylose, xylobiose) are used. We found that a single point mutation in the gene encoding the Xylanase regulator 1 (Xyr1) is responsible for this strong deregulation of endo-xylanase expression and, moreover, a highly elevated basal level of cellulase expression. This point mutation is localized in a domain that is common in binuclear zinc cluster transcription factors. Only the use of sophorose as inducer still leads to a slight induction of cellulase expression. Under all tested conditions, the formation of cbh1 and cbh2 transcript level strictly follows the transcript levels of xyr1. The correlation of xyr1 transcript levels and cbh1/cbh2 transcript levels and also their inducibility via sophorose is not restricted to this strain, but occurs in all ancestor strains up to the wild-type QM6a.ConclusionsEngineering a key transcription factor of a target regulon seems to be a promising strategy in order to increase enzymes yields independent of the used substrate or inducer. The regulatory domain where the described mutation is located is certainly an interesting research target for all organisms that also depend so far on certain inducing conditions.
The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum. In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1.IMPORTANCE Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum. The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.
Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2′,3′-dihydrosorbicillin to sorbicillinol and 2′,3′-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2′,3′-dihydrosorbicillin and 2′,3′-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.
The state-of-the-art procedure for gene insertions into Trichoderma reesei is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes asl1 (encoding an enzyme of the l-arginine biosynthesis pathway), the hah1 (encoding an enzyme of the l-lysine biosynthesis pathway), and the pyr4 (encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in T. reesei. By employing goxA of Aspergillus niger as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.
BackgroundGlobal climate change and fossil fuels limitations have boosted the demand for robust and efficient microbial factories for the manufacturing of bio-based products from renewable feedstocks. In this regard, efforts have been done to enhance the enzyme-secreting ability of lignocellulose-degrading fungi, aiming to improve protein yields while taking advantage of their ability to use lignocellulosic feedstocks. Access to sugars in complex polysaccharides depends not only on their release by specific hydrolytic enzymes, but also on the presence of transporters capable of effectively transporting the constituent sugars into the cell. This study aims to identify and characterize xylose transporters from Aspergillus niger and Trichoderma reesei, two fungi that have been industrially exploited for decades for the production of lignocellulose-degrading hydrolytic enzymes.ResultsA hidden Markov model for the identification of xylose transporters was developed and used to analyze the A. niger and T. reesei in silico proteomes, yielding a list of candidate xylose transporters. From this list, three A. niger (XltA, XltB and XltC) and three T. reesei (Str1, Str2 and Str3) transporters were selected, functionally validated and biochemically characterized through their expression in a Saccharomyces cerevisiae hexose transport null mutant, engineered to be able to metabolize xylose but unable to transport this sugar. All six transporters were able to support growth of the engineered yeast on xylose but varied in affinities and efficiencies in the uptake of the pentose. Amino acid sequence analysis of the selected transporters showed the presence of specific residues and motifs recently associated to xylose transporters. Transcriptional analysis of A. niger and T. reesei showed that XltA and Str1 were specifically induced by xylose and dependent on the XlnR/Xyr1 regulators, signifying a biological role for these transporters in xylose utilization.ConclusionsThis study revealed the existence of a variety of xylose transporters in the cell factories A. niger and T. reesei. The particular substrate specificity and biochemical properties displayed by A. niger XltA and XltB suggested a possible biological role for these transporters in xylose uptake. New insights were also gained into the molecular mechanisms regulating the pentose utilization, at inducer uptake level, in these fungi. Analysis of the A. niger and T. reesei predicted transportome with the newly developed hidden Markov model showed to be an efficient approach for the identification of new xylose transporting proteins.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0564-4) contains supplementary material, which is available to authorized users.
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