Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC2 in and secretion of the heterologous enzyme by Hypocrea jecorina (original) (raw)
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Catalysis Today, 2011
Hypocrea 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.
D-Xylose as a Repressor or Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)
Applied and Environmental Microbiology, 2010
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.
Biotechnology for Biofuels, 2015
Background: One of the primary industrial-scale cellulase producers is the ascomycete fungus, Hypocrea jecorina, which produces and secretes large quantities of diverse cellulolytic enzymes. Perhaps the single most important biomass degrading enzyme is cellobiohydrolase I (cbh1or Cel7A) due to its enzymatic proficiency in cellulose depolymerization. However, production of Cel7A with native-like properties from heterologous expression systems has proven difficult. In this study, we develop a protein expression system in H. jecorina (Trichoderma reesei) useful for production and secretion of heterologous cellobiohydrolases from glycosyl hydrolase family 7. Building upon previous work in heterologous protein expression in filamentous fungi, we have integrated a native constitutive enolase promoter with the native cbh1 signal sequence. Results: The constitutive eno promoter driving the expression of Cel7A allows growth on glucose and results in repression of the native cellulase system, severely reducing background endo-and other cellulase activity and greatly simplifying purification of the recombinant protein.
Expression of xylanase enzymes from thermophilic microorganisms in fungal hosts
Extremophiles, 2002
Aims: To express a gene encoding a heterologous fungal xylanase in Trichoderma reesei. Methods and Results: Humicola grisea xylanase 2 (xyn2) cDNA was expressed in Trichoderma reesei under the main cellobiohydrolase I (cbh1) promoter (i) as a fusion to the cellobiohydrolase I (CBHI) secretion signal and (ii) the mature CBHI core-linker. The recombinant xylanase (HXYN2) was secreted into the cultivation medium and processed in a similar fashion to the endogenous T. reesei xylanases, resulting in an active enzyme. Conclusions, Signi®cance and Impact of the Study: HXYN2 was successfully processed in T. reesei. Composition of the culture medium affected the HXYN2 yields, favouring Avicellactose as a carbon source. Best yields (about 0á5 g l )1 ) in shake¯ask cultivations were obtained from a transformant where xyn2 was fused directly to the CBHI secretion signal.
Molecular characterization of a glycosyl hydrolase family 10 xylanase from Aspergillus niger
Protein Expression and Purification, 2013
A gene coding for an endo-b-1,4-xylanase (XlnA) (glycosyl hydrolase family 10) from Aspergillus niger DSM 1957 was cloned and sequenced. The cDNA sequence (984 bp) and its putative endoxylanase (327 aa protein with a predicted molecular mass of 35.5 kDa and pI 6.23) showed 91.3-99.5% and 96.3-99.1% identities with cDNA sequences and their corresponding endoxylanases from A. niger strains from GenBank, respectively. The cDNA was expressed in Pichia pastoris GS115 under the control of AOX1 promoter at a level of 46.4 U/ml culture supernatant, after 144 h of growth at 30°C in YP medium induced with 0.5% (v/v) of methanol. The molecular mass of the purified XlnA determined by SDS-PAGE was 35.5 kDa with a specific activity of 808.5 U/mg towards 1% (w/v) of birch wood xylan. Temperature and pH optimum were observed at 50°C and pH 7.0, respectively. The enzyme was stable over a temperature range of 25-40°C and at pH range of 4.5-8.5 and resistant to Tween 80 and acetone. The K m and V max value obtained for the purified xylanase were 25.5 mg/ml and 5000 lmol/min/mg protein with birch wood xylan as substrate, respectively. The xylanase was free of cellulase and mannanase activity but highly active towards birch wood xylan. The major products of the birch wood xylan hydrolysis were predicted as xylotriose, xylotetraose, and xylopentose. The biochemical characteristics suggested that the recombinant xylanase has a potential application, including use as a feed enzyme.
Screening and Identification of Thermophilic Glycosyl Hydrolases Producing Microorganisms
Journal of Biotechnology & Bioresearch
This study focuses on screening and identification of bacteria, which can produce alkaline xylanase at alkaline pH and high temperature. Bacterial isolates from corncob decaying soil, capable of hydrolyzing xylan were screened. Selected and purified 108 bacterial colonies grown on xylan-nutrient agar slants were activated and transferred into the fermentation medium. Six highest xylanase producing isolates were selected for further studies. Isolates CS 1 [132.0(±0.09)], CS 27 [121.3(±0.11)]& CS 88 [124.8(±0.44) UmL-1 ] showed highest xylanase production at pH 8.5 while isolates CS 52 [124.4(±0.01) UmL-1 ], CS 93 [113.0(±0.48) UmL-1 ] and CS 104 [110.1(±0.54) UmL-1 ] showed at pH 8.0 and 45 o C. Therefore isolates CS 1 , CS 27 & CS 88 were selected and the xylanase produced by them were screened for the kinetic properties. The crude enzymes of the isolates CS 1 , CS 27 & CS 88 showed zero order kinetics up to 4 min. The optimum temperature for the activity of the xylanase from isolates CS 1 and CS 27 was 55 o C, while that of isolate CS 88 was 60 o C. The optimum pH value for the xylanase from isolate CS 1 and CS 88 was 8.4 and that of isolate CS 27 was 8.0. Based on the kinetic properties of xylanase, isolates CS 1 and CS 88 were selected and characterized and found to be belonging to genus Bacillus. As Bacillus CS 1 produced highest xylanase activities, 16S rDNA was analyzed and identified as Bacillus pumilus and selected for further studies to produce xylanase at 45 o C and pH 8.5.
FEMS Yeast Research, 2005
A thermostable glycoside hydrolase family-10 xylanase originating from Rhodothermus marinus was cloned and expressed in the methylotrophic yeast Pichia pastoris (SMD1168H). The DNA sequence from Rmxyn10A encoding the xylanase catalytic module was PCR-amplified and cloned in frame with the Saccharomyces cerevisiae a-factor secretion signal under the control of the alcohol oxidase (AOX1) promotor. Optimisation of enzyme production in batch fermentors, with methanol as a sole carbon source, enabled secretion yields up to 3 g l À1 xylanase with a maximum activity of 3130 U l À1 to be achieved. N-terminal sequence analysis of the heterologous xylanase indicated that the secretion signal was correctly processed in P. pastoris and the molecular weight of 37 kDa was in agreement with the theoretically calculated molecular mass. Introduction of a heat-pretreatment step was however necessary in order to fold the heterologous xylanase to an active state, and at the conditions used this step yielded a 200-fold increase in xylanase activity. Thermostability of the produced xylanase was monitored by differential-scanning calorimetry, and the transition temperature (T m) was 78°C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.
African Journal of Biotechnology, 2011
The xyn5 gene, which encodes an endo-β-1,4-xylanase (Xyn5), in Aspergillus niger GS1 was cloned into an expression cassette under the control of constitutive glyceraldhehyde-3-phosphate dehydrogenase gene promoter. The expression system was designed to produce the recombinant enzyme containing a six-histidine peptide fused to the carboxyl end of the protein. The efficiency of Xyn5 production under submerged (SmF) and solid-state (SSF) fermentation was investigated using the homologous cotransformed A. niger AB4.1. A productivity of 17.1 U/(l·h) was estimated for SSF and 3.2 U/(l·h) for SmF calculated at peak value of enzyme titers. Recombinant Xyn5 obtained by SSF on polyurethane fiber, was purified 5.1-fold by anion exchange and immobilized metal affinity chromatography, with 35.7% recovery. The purified recombinant enzyme showed an apparent molecular weight of 30 kDa and optimal activity (522 U/mg protein) at pH 5.5 and 50°C.
L-Arabitol Is the Actual Inducer of Xylanase Expression in Hypocrea jecorina (Trichoderma reesei)
Applied and Environmental Microbiology, 2011
The saprophytic fungus Hypocrea jecorina (anamorph, Trichoderma reesei) is an important native producer of hydrolytic enzymes, including xylanases. Regarding principles of sustainability, cheap and renewable raw materials, such as D-xylose (the backbone monomer of xylan), have been receiving increasing attention from industries. Recently, it was demonstrated that small (0.5 to 1 mM) amounts of D-xylose induce the highest expression of xylanase in H. jecorina. However, it was also reported that active metabolism of D-xylose is necessary for induction. In this report, we demonstrate that xylitol, the next intermediate in the pentose pathway after D-xylose, does not trigger transcription of xylanase-encoding genes in H. jecorina QM9414. The highest level of transcription of xylanolytic enzyme-encoding genes occurred in an xdh1 (encoding a xylitol dehydrogenase) deletion strain cultured in the presence of 0.5 mM D-xylose, suggesting that a metabolite upstream of xylitol is the inducer. The expression levels of xylanases in an xdh1-lad1 double-deletion strain were lower than that of an xdh1 deletion strain. This observation suggested that L-xylulose is not an inducer and led to the hypothesis that L-arabitol is the actual inducer of xylanase expression. A direct comparison of transcript levels following the incubation of the H. jecorina parental strain with various metabolites of the pentose pathway confirmed this hypothesis. In addition, we demonstrate that xyr1, the activator gene, is not induced in the presence of pentose sugars and polyols, regardless of the concentration used; instead, we observed low constitutive expression of xyr1.
Journal of Biotechnology, 1994
The medium components for the production of extracellular xylanase by Thermoascus aurantiacus was optimized in shake-flask culture using the Box-Wilson method and a central composite design. The traditional one-factor-at-atime method was employed for selecting the effective factors and the initial test range of each ingredient. The optimized medium composition was found to be 3.24% wheat straw (steam pretreated, particle size approx. 0.25 mm), 1.32% pharmamedia and 0.49% KHEPO 4. Other compounds such as inorganic nitrogen, MgSO4, CaCI2, trace elements and vitamins showed no marked positive effect on the enzyme yield by the fungus, while Tween-80 exhibited slight enhancing effect. The optimized culture medium and conditions gave 5347.4 nkat ml-1 of xylanase, 3.0 nkat ml-1 of/3-xylosidase, 1.0 nkat ml-1 of acetyl esterase, 89.7 nkat ml-1 of acetyl xylan esterase and 3.5 nkat ml-~ of t~-arabinosidase activities. In addition to xylanase activities, the culture filtrates exhibited cellulases (filter paper cellulase, carboxymethyl cellulase and /3-glucosidase), mannanases (endo-mannanase, fl-mannosidase and a-galactosidase) and other polysaccharases (pectinase, chitinase and a-amylase) activities. In a bioreactor culture, the production of xylanase and /3-xylosidase was markedly influenced by the pH and inoculum type. The fungus produced maximum enzymes when the pH was not controlled and repressed mycelial inoculum was used. Under these conditions the yield of xylanase was about 30% lower and that of/3-xylosisdase was at least 2-fold higher in comparison to those achieved in shake-flask cultures. The pH optimum of xylanase and /3-xylosidase was 5.0. The optimum temperature for xylanase was 80°C, while /3-xylosidase functioned optimally at 75°C. The enzymes exhibited remarkable stabilities at high temperatures (50-70°C) for prolonged period. At 70°C, the half-lives of xylanase and/3-xylosidase were approx. 204 h and 113 h, respectively.