A modified expression of the major hydrolase activator in Hypocrea jecorina (Trichoderma reesei) changes enzymatic catalysis of biopolymer degradation

AbstractHypocrea jecorina (anamorph Trichoderma reesei) is a saprophytic fungus that produces hydrolases, which are applied in different types of industries and used for the production of biofuel. A recombinant Hypocrea strain, which constantly expresses the main transcription activator of hydrolases (Xylanase regulator 1), was found to grow faster on xylan and its monomeric backbone molecule d-xylose. This strain also showed improved ability of clearing xylan medium on plates. Furthermore, this strain has a changed transcription profile concerning genes encoding for hydrolases and enzymes associated with degradation of (hemi)celluloses. We demonstrated that enzymes of this strain from a xylan cultivation favoured break down of hemicelluloses to the monomer d-xylose compared to the parental strain, while the enzymes of the latter one formed more xylobiose. Applying supernatants from cultivation on carboxymethylcellulose in enzymatic conversion of hemicelluloses, the enzymes of the recombinant strain were clearly producing more of both, d-xylose and xylobiose, compared to the parental strain. Altogether, these results point to a changed hydrolase expression profile, an enhanced capability to form the xylan-monomer d-xylose and the assumption that there is a disordered induction pattern if the Xylanase regulator 1 is de-regulated in Hypocrea

Quantitative modeling and analytic assessment of the transcription dynamics of the XlnR regulon in Aspergillus niger

Background: Transcription of genes coding for xylanolytic and cellulolytic enzymes in Aspergillus niger is controlled by the transactivator XlnR. In this work we analyse and model the transcription dynamics in the XlnR regulon from time-course data of the messenger RNA levels for some XlnR target genes, obtained by reverse transcription quantitative PCR (RT-qPCR). Induction of transcription was achieved using low (1 mM) and high (50 mM) concentrations of D-xylose (Xyl). We investigated the wild type strain (Wt) and a mutant strain with partial loss-of-function of the carbon catabolite repressor CreA (Mt). Results: An improved kinetic differential equation model based on two antagonistic Hill functions was proposed, and fitted to the time-course RT-qPCR data from the Wt and the Mt by numerical optimization of the parameters. We show that perturbing the XlnR regulon with Xyl in low and high concentrations results in different expression levels and transcription dynamics of the target genes. At least four distinct transcription profiles were observed, particularly for the usage of 50 mM Xyl. Higher transcript levels were observed for some genes after induction with 1 mM rather than 50 mM Xyl, especially in the Mt. Grouping the expression profiles of the investigated genes has improved our understanding of induction by Xyl and the according regulatory role of CreA. Conclusions: The model explains for the higher expression levels at 1 mM versus 50 mM in both Wt and Mt. It does not yet fully encapsulate the effect of partial loss-of-function of CreA in the Mt. The model describes the dynamics in most of the data and elucidates the time-dynamics of the two major regulatory mechanisms: i) the activation by XlnR, and ii) the carbon catabolite repression by CreA.</p

Identification and functional characterization of novel xylose transporters from the cell factories Aspergillus niger and Trichoderma reesei

Background: Global 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. Results: A 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. Conclusions: This 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.</p

In Vivo Study of the Sorbicillinoid Gene Cluster in Trichoderma reesei

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

d-Xylose as a Repressor or Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)▿

For Hypocrea jecorina (anamorph Trichoderma reesei), a filamentous fungus used for hydrolase production in different industries, it has been a long-term practice to use d-xylose as an inducing substance. We demonstrate in this study that the degree of xylanase-encoding gene induction strictly depends on the concentration of d-xylose, which was found to be optimal from 0.5 to 1 mM for 3 h of cultivation. At higher concentrations of d-xylose, a reduced level of xylanase gene expression was observed. In the present study, we also provide evidence that the d-xylose concentration-dependent induction is antagonized by carbon catabolite repressor 1. This repressor mediates its influence on d-xylose indirectly, by reducing the expression of xylanase regulator 1, the main activator of most hydrolase-encoding genes. Additionally, a direct influence of the repressor on xylanase 1 expression in the presence of d-xylose was found. Furthermore, we show that d-xylose reductase 1 is needed to metabolize d-xylose to achieve full induction of xylanase expression. Finally, a strain which expresses xylanase regulator 1 at a constant level was used to partially overcome the negative influence exerted by carbon catabolite repressor 1 on d-xylose