Expression Of Xylanase Research Articles

Novel transcriptional regulation of the GAP promoter in Pichia pastoris towards high expression of heterologous proteins

BackgroundPichia pastoris (Komagataella phaffii) is a promising production host, but the usage of methanol limits its application in the medicine and food industries.ResultsTo improve the constitutive expression of heterologous proteins in P. pastoris, four new potential transcription regulators (Loc1p, Msn2p, Gsm1p, Hot1p) of the glyceraldehyde triphosphate dehydrogenase promoter (pGAP) were revealed in this study by using cellulase E4 as reporter gene. On this basis, a series of P. pastoris strains with knockout or overexpression of transcription factors were constructed and the deletion of transcription factor binding sites on pGAP was confirmed. The results showed that Loc1p and Msn2p can inhibit the activity of pGAP, while Gsm1p and Hot1p can enhance the activity of pGAP; Loc1p, Gsm1p and Hot1p can bind directly to pGAP, while Msn2p must be treated to expose the C-terminal domain to bind to pGAP. Moreover, manipulating a single transcription factor led to a 0.96-fold to 2.43-fold increase in xylanase expression. In another model protein, aflatoxin oxidase, knocking out Loc1 based on AFO-∆Msn2 strain resulted in a 0.63-fold to 1.4-fold increase in expression. It can be demonstrated that the combined use of transcription factors can further improve the expression of exogenous proteins in P. pastoris.ConclusionThese findings will contribute to the construction of pGAP-based P. pastoris systems towards high expression of heterologous proteins, hence improving the application potential of yeast.

Combined strategies for improving the heterologous expression of a novel xylanase from Fusarium oxysporum Fo47 in Pichia pastoris

Xylanase, an enzyme capable of hydrolyzing non-starch polysaccharides found in grain structures like wheat, has been found to improve the organizational structure of dough and thus increase its volume. In our past work, one promising xylanase FXYL derived from Fusarium oxysporum Fo47 and first expressed 779.64 U/mL activity in P. pastoris. It has shown significant potential in improving the quality of whole wheat bread, making it become a candidate for development as a new flour improver. After optimization of expression elements and gene dose, the xylanase activity of FXYL strain carrying three-copies reached 4240.92 U/mL in P. pastoris. In addition, 12 factors associated with the three stages of protein expression pathway were co-expressed individually in order in three-copies strain, and the translation factor Pab1 co-expression increased FXYL activity to 8893.53 U/mL. Nevertheless, combining the most effective or synergistic factors from three stages did not exhibit better results than co-expressing them alone. To further evaluate the industrial potential, the xylanase activity and protein concentration reached 81184.51 U/mL and 11.8 g/L in a 5 L fed-batch fermenter. These engineering strategies improved the expression of xylanase FXYL by more than 104-fold, providing valuable insights for the cost-effective industrial application of FXYL in the baking field.

INFLUENCE OF CULTIVATION CONDITIONS ON CELLULASE AND XYLANASE ACTIVITY OF RHIZOPUS ORYZAE F-1030

The paper examines the relationship between the activity of cellulase and xylanase enzymes and the lactic acid yield which was synthesized by the fungus Rhizopus oryzae F-1030 during its cultivation on a culture medium based on catalytically modified neutral sulfite liquors. It was shown that in cultivating by the deep method the fungus R. oryzae F-1030 synthesizes xylanase and cellulase enzymes that break down polysaccharides in a nutrient medium to simple sugars which are easily accessible for assimilation by the fungus. Accordingly, the fungus R. oryzae F-1030 can be used as a promising biotechnological object for the bioconversion of cellulose production secondary resources. It was found that the level of cellulase and xylanase expression in R. oryzae F-1030 depends on the type of substrate. The xylanase activity of the fungus is more pronounced than the cellulase activity since xylan oligomers predominate in birch wood. It was noted that the lactic acid yield during the cultivation of the fungus R. oryzae F-1030 on a catalytically modified neutral sulfite liquor depends on the action specifics of the hydrolyzing catalyst. As a nutrient medium for the microbiological synthesis of lactic acid the fungus R. oryzae F-1030 can use biocatalytically treated liquors of birch wood neutral sulfite pulping.

Optimization of the extracellular secretion of black goat rumen metagenome-derived KG42 xylanase by Bacillus subtilis

Xylanase (E.C. 3.2.1.8) is the enzyme that breaks down β-1,4 xylan by cleaving β-1,4 glycosidic linkages. Production of xylanases is important for various industrial applications. Here, we aimed to determine the optimal incubation conditions for expression and secretion of KG42 xylanase in Bacillus subtilis using response surface methodology based on Box-Behnken design in preparation for industrial applications. Among nine broth media tested in this study, Power Broth was chosen as a basal medium. In addition to the basal medium, the four other independent variables of extra carbon sources (glucose, lactose, mannose, fructose, and sucrose), extra nitrogen sources (beef extract, yeast extract, tryptone, urea, NaNO3, and (NH4)2SO4), isopropyl β-D-1-thiogalactopyranoside concentrations, and induction times were individually tested using one factor at a time in an optimization experiment. Next, a Box-Behnken design-based response surface methodology approach was used to identify and validate the optimized incubation conditions with the four variables in batch culture. The statistically optimized incubation conditions obtained from this study yielded a maximum of approximately 3- to 4-fold increases in the expression and secretion of KG42 xylanase by B. subtilis in comparison with unoptimized medium and incubation conditions.

Recent developments in heterologous production of cellulases and xylanases using the Pichia pastoris expression system

AbstractThe yeast expression system, Pichia pastoris, is one of the most robust and versatile expression systems in biotechnology and molecular biology, generally considered as a safe host for heterologous protein expression, especially for producing cellulases and xylanases at the industrial level. Despite the high recombinant protein expression rate, the potential of the P. pastoris expression system has still not been fully explored. Cultivation of P. pastoris under optimized conditions greatly relies on the strain and is associated with certain problems such as promoter strength, sensitivity to methanol, and oxygen demand. To address these issues, different genetic engineering strategies have been employed. Advancements in promoter engineering, optimization of gene dosage and codon usage, recombinant plasmid engineering using CRISPR/Cas9 system, and directed evolution strategies have proven beneficial to the yield of cellulase expression levels. This study will systemically review recent progress in various genetic engineering strategies to enhance cellulase and xylanase expression in the P. pastoris expression system. The utilization of alcohol oxidase 1 promoter (pAOX1), methanol‐free system, and recombinant plasmid engineering for improved production of these enzymes are highlighted. Additionally, we discuss the recent advancements in the P. pastoris expression system toolbox for improved cellulase and xylanase production, thus providing a deep insight into how P. pastoris is becoming the indispensable platform for heterologous protein production. © 2023 Society of Chemical Industry (SCI).

Multi-copy expression of a protease-resistant xylanase with high xylan degradation ability and its application in broilers fed wheat-based diets

The acidic thermostable xylanase (AT-xynA) has great potential in the feed industry, but its low activity is not conductive to large-scale production, and its application in poultry diets still needs to be further evaluated. In Experiment1, AT-xynA activity increased 3.10 times by constructing multi-copy strains, and the highest activity reached 10,018.29 ± 91.18 U/mL. AT-xynA showed protease resistance, high specificity for xylan substrates, xylobiose and xylotriose were the main hydrolysates. In Experiment2, 192 broilers were assigned into 3 treatments including a wheat-based diet, and the diets supplemented with AT-xynA during the entire period (XY-42) or exclusively during the early stage (XY-21). AT-xynA improved growth performance, while the performance of XY-21 and XY-42 was identical. To further clarify the mechanism underlying the particular effectiveness of AT-xynA during the early stage, 128 broilers were allotted into 2 treatments including a wheat-based diet and the diet supplemented with AT-xynA for 42 d in Experiment3. AT-xynA improved intestinal digestive function and microbiota composition, the benefits were stronger in younger broilers than older ones. Overall, the activity of AT-xynA exhibiting protease resistance and high xylan degradation ability increased by constructing multi-copy strains, and AT-xynA was particularly effective in improving broiler performance during the early stage.

Improved secretory expression and characterization of thermostable xylanase and β-xylosidase from Pseudothermotoga thermarum and their application in synergistic degradation of lignocellulose.

Xylanase and β-xylosidase are the key enzymes for hemicellulose hydrolysis. To further improve hydrolysis efficacy, high temperature hydrolysis with thermostable hemicellulases showed promise. In this study, thermostable xylanase (Xyn) and β-xylosidase (XynB) genes from Pseudothermotoga thermarum were cloned and secretory expressed in Bacillu subtilis. Compared with Escherichia coli expression host, B. subtilis resulted in a 1.5 time increase of enzymatic activity for both recombinant enzymes. The optimal temperature and pH were 95°C and 6.5 for Xyn, and 95°C and 6.0 for XynB. Thermostability of both recombinant enzymes was observed between the temperature range of 75-85°C. Molecular docking analysis through AutoDock showed the involvement of Glu525, Asn526, Trp774 and Arg784 in Xyn-ligand interaction, and Val237, Lys238, Val761 and Asn76 in XynB-ligand interaction, respectively. The recombinant Xyn and XynB exhibited synergistic hydrolysis of beechwood xylan and pretreated lignocellulose, where Xyn and XynB pre-hydrolysis achieved a better improvement of pretreated lignocellulose hydrolysis by commercial cellulase. The observed stability of the enzymes at high temperature and the synergistic effect on lignocellulosic substrates suggested possible application of these enzymes in the field of saccharification process.

Extracellular Expression of Feruloyl Esterase and Xylanase in Escherichia coli for Ferulic Acid Production from Agricultural Residues.

There is still a large amount of ferulic acid (FA), an outstanding antioxidant, present in agricultural residues. Enzymatic hydrolysis has been regarded as the most effective way to release FA. This present study therefore selected feruloyl esterase (FAE) and xylanase (XYN) from the metagenomes of a cow rumen and a camel rumen, respectively, for their recombinant expression in Escherichia coli BL21 and further application in releasing FA. After screening the candidate signal peptides, the optimal one for each enzyme, which were selected as SP1 and SP4, respectively, was integrated into the vectors pET22b(+) and pETDuet-1. Among the generated E. coli strains SP1-F, SP4-X, and SP1-F-SP4-X that could express extracellular enzymes either separately or simultaneously, the latter one performed the best in relation to degrading the biomass and releasing FA. Under the optimized culture and induction conditions, the strain SP1-F-SP4-X released 90% of FA from 10% of de-starched wheat bran and produced 314.1 mg/L FA, which was deemed to be the highest obtained value to the best of our knowledge. This result could pave a way for the re-utilization of agricultural residues and enhancing their add-value.

Review of Recent Advances in the Physiology of the Regulation of Cellulase and Xylanase Production by Basidiomycetes

The potential of wood-rotting and litter-deconstructing basidiomycetes to convert lignocellulose into a wide variety of products has been extensively studied. In particular, wood-rotting basidiomycete secretomes are attracting much attention from researchers and biotechnology companies due to their ability to produce extracellular hydrolytic and oxidative enzymes that effectively degrade cellulose, hemicellulose, and lignin of plant biomass. An analysis of the available literature data shows that Basidiomycota fungi, which are most adapted to the depolymerization of plant polysaccharides, are promising but so far unexploited sources of new hydrolytic enzymes. The review summarizes the latest data on the great variety, common features, and unique properties of individual fungi and the production of cellulases and xylanases by various physiological and ecological groups of basidiomycetes. The most important microbial cellulase-producing strains for submerged and solid-phase fermentation, as well as the main substrates, including the use of agro-industrial waste, are considered. It highlights ways to increase both cellulase and xylanase expression levels and the cost-effectiveness of producing these enzymes for various biotechnological applications. It is anticipated that this review will be particularly useful to novice scientists working in the lignocellulose biorefinery, as it describes current knowledge and issues related to the production and regulation of polysaccharide hydrolyzing enzyme synthesis.

The Ras small GTPase RSR1 regulates cellulase production in Trichoderma reesei

BackgroundLignocellulose is the most abundant renewable resource in the world and has attracted widespread attention. It can be hydrolyzed into sugars with the help of cellulases and hemicellulases that are secreted by filamentous fungi. Several studies have revealed that the Ras small GTPase superfamily regulates important cellular physiological processes, including synthesis of metabolites, sporulation, and cell growth and differentiation. However, it remains unknown how and to what extent Ras small GTPases participate in cellulase production.ResultsIn this study, we found that the putative Ras small GTPase RSR1 negatively regulated the expression of cellulases and xylanases. Deletion of rsr1 (∆rsr1) significantly increased cellulase production and decreased the expression levels of ACY1-cAMP-protein kinase A (PKA) signaling pathway genes and the concentration of intracellular cyclic adenosine monophosphate (cAMP). Loss of acy1 based on ∆rsr1 (∆rsr1∆acy1) could further increase cellulase production and the expression levels of cellulase genes, while overexpression of acy1 based on ∆rsr1 (∆rsr1-OEacy1) significantly reduced cellulase production and transcriptional levels of cellulase genes. In addition, our results revealed that RSR1 negatively controlled cellulase production via the ACY1-cAMP-PKA pathway. Transcriptome analysis revealed significantly increased expression of three G-protein coupled receptors (GPCRs; tre62462, tre58767, and tre53238) and approximately two-fold higher expression of ACE3 and XYR1, which transcriptionally activated cellulases with the loss of rsr1. ∆rsr1∆ tre62462 exhibited a decrease in cellulase activity compared to ∆rsr1, while that of ∆rsr1∆tre58767 and ∆rsr1∆tre53238 showed a remarkable improvement compared to ∆rsr1. These findings revealed that GPCRs on the membrane may sense extracellular signals and transmit them to rsr1 and then to ACY1-cAMP-PKA, thereby negatively controlling the expression of the cellulase activators ACE3 and XYR1. These data indicate the crucial role of Ras small GTPases in regulating cellulase gene expression.ConclusionsHere, we demonstrate that some GPCRs and Ras small GTPases play key roles in the regulation of cellulase genes in Trichoderma reesei. Understanding the roles of these components in the regulation of cellulase gene transcription and the signaling processes in T. reesei can lay the groundwork for understanding and transforming other filamentous fungi.

An inducible Komagataella phaffii system for protein expression using sorbitol dehydrogenase promoter

The aim of the present work was todevelop a methanol-independent Komagataella phaffii (K. phaffii) strain using a non-methanol promoter. In this study, the food grade enzyme xylanase from Aspergillus niger ATCC 1015 was used as the reporter protein, a recombinant K. phaffii containing a cascade gene circus was designed and constructed using sorbitol as inducer. Sorbitol induced PSDH leading to MIT1 expression firstly, and heterologous protein xylanase expression finally. This system showed 1.7 fold of xylanase activity at the condition of single copy number of extra MIT1, and 2.1 fold of xylanase activity at condition of multi-copy extra MIT1 gene. This sorbitol-induced expression system of K. phaffii avoided toxic and explosive methanol. It was a novel cascade gene expression and a food safety system.

Characterization and High Expression of a Novel Alkalinegh11 Xylanase in Pichia pastoris

Characterization and High Expression of a Novel Alkalinegh11 Xylanase in Pichia pastoris

Enhanced expression of xylanase in Aspergillus niger enabling a two-step enzymatic pathway for extracting β-glucan from oat bran

The high cost and process complexity limit the enzymatic extraction of β-glucan. In this study, β-glucan was extracted from oat bran in a two-step enzymatic pathway using a recombinant strain of Aspergillus niger AG11 overexpressing the endogenous xylanase (xynA) and amylolytic enzyme. First, co-optimization of promoter and signal peptide and a fusion of glucoamylase (glaA) fragment were integrated into the β-glucosidase (bgl) locus to improve xynA expression. Then, the optimized expression cassette was simultaneously integrated into bgl, α-amylase amyA, and acid α-amylase ammA loci, yielding the Rbya with 3,650-fold and 31.2% increase in xynA and amylolytic enzyme activity than the wild-type strain, respectively. Finally, Rbya’s supernatants at 72 h (rich in xynA and amylolytic enzyme) and 10 d (rich in proteases) were used to decompose xylan/starch and proteins in oat bran, respectively, to obtain 85.1% pure β-glucan. Rbya could be a robust candidate for the cost-effective extraction of β-glucan.

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

Enhancing xylanase expression by Komagataella phaffii by formate as carbon source and inducer

Komagataella phaffii (syn. Pichia pastoris), a methylotrophic yeast, has many advantages as a protein expression system, but has the disadvantage of hazardous methanol as an inducer and carbon source. To enable substitution of formate for methanol, a formate assimilation pathway was constructed by the co-expression of acetyl-CoA synthase, acetaldehyde dehydrogenase, and transcription factor Mit1, resulting in a 103.5 ± 12.5% increase in xylanase production. Recombinant K. phaffii was able to use formate as a carbon source, indicating successful substitution of formate for methanol. Xylanase production, using the safe and sustainable formate as an inducer and carbon source, is a major advance in the field of industrial enzyme production. KEY POINTS: • Change to formate assimilation by recombinant K. phaffii instead of methanol • K. phaffii expressed xylanase by formate induction instead of methanol induction • Increased xylanase expression by transcription factor co-expression.

Transcriptomic analysis of lignocellulose degradation by Streptomyces coelicolor A3(2) and elicitation of secondary metabolites production.

Streptomyces coelicolor A3(2) is considered as the model strain among the Streptomyces and has the capacity to produce several natural molecules. Our hypothesis was that cultivation of the strain onto a complex carbon source such as wheat bran (WB) would induce the production of various secondary metabolites due to the presence of complex polysaccharides. A multiapproach has been performed in order to investigate: (1) whether that strain could degrade lignocellulose; (2) which enzymatic and metabolic pathways secondary were over-expressed when grown on WB. The transcriptomic approach showed the expression of several CAZymes significantly expressed when grown on WB such as endoglucanases (encoding for GH74, GH5_8, and GH12) and xylanases (GH11 and CE4 encoding for respectively endo-1,4-beta-xylanase and an acetyl-xylan esterase). Enzymatic activities showed an expression of xylanase (115.3±32.2 mUI/ml) and laccase-peroxidase (101.5±10.9 mUI/ml) during WB degradation by S. coelicolor A3(2). Metabolomics showed that the production of secondary metabolites differed between growth on either glucose or WB as carbon source, which may be correlated to the complexity of carbon compounds within WB, which are similar to the ones encountered in soils and should represent more the in situ carbon conditions which Streptomyces might face off. This opens opportunities for the bioproduction of molecules of interest from WB.

Heterologous expression of family GH11 Aspergillus niger xylanase B (AnXylB11) in Pichia pastoris and competitive inhibition by riceXIP: An experimental and simulation study

The family GH11 Aspergillus niger JL15 xylanase B (AnXylB11) was heterologously expressed in Pichia pastoris X33. The recombinant AnXylB11 (reAnXylB11) was secreted into the culture medium with a molecular weight of approximately 33.0 kDa. The optimal temperature and pH of reAnXylB11 were 40 ℃ and 5.0, respectively. reAnXylB11 released xylobiose (X2)-xylohexaose (X6) from beechwood xylan, with xylotriose (X3) as the primary product. The hydrolysates showed significant antioxidant activity. reAnXylB11 was also competitively inhibited by recombinant rice xylanase inhibitory protein (rePriceXIP), with an inhibition constant (Ki) of 106.9 nM. Molecular dynamics (MD) simulations, non-covalent interactions (NCI), and binding free energy calculation and decomposition were conducted to decipher the interactional features between riceXIP and AnXyB11. Representative conformation of riceXIP-AnXylB11 complex showed that a U-shaped long loop between α4 and β5 (K143-L152) of riceXIP was protruded into the catalytic groove and formed tight interaction with many key residues of AnXylB11. The binding free energy of riceXIP-AnXylB11 was calculated to be − 46.1 ± 10.5 kcal/mol, with Coulomb and van der Waals forces contributing the most. NCI analysis showed that the hydrogen bonding networks such as R148riceXIP-E98AnXyl11B, K143riceXIP-D138AnXyl11B and R148riceXIP-E189AnXyl11B provided terrific contributions to the interface interaction. The Laplacian of electron density values of atom pairs R148riceXIP@ 2HH1-E89AnXylB11@OE2 and N142riceXIP@ 1HD2-D138AnXylB11@OD1 were 0.12190 and 0.16009 a.u., respectively. Exploring the interactional features between xylanase and inhibitor protein may aid in constructing mutant xylanase that is insensitive to xylanase inhibitory proteins (XIs).

Expression of an extremophilic xylanase in Nicotiana benthamiana and its use for the production of prebiotic xylooligosaccharides

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported.

PCold-assisted expression of a thermostable xylanase from Bacillus amyloliquefaciens: cloning, expression and characterization.

The online version contains supplementary material available at 10.1007/s13205-022-03315-y.

Enhancing xylanase expression of Komagataella phaffii induced by formate through Mit1 co-expression.

Komagataella phaffii (K. phaffii) is a famous microbial cell of heterologous protein and value-added chemicals production because of its strict and strong promoter (alcohol oxidase 1 promoter, PAOX1). Formate is an attractive substitute of traditional inducer methanol because methanol is toxic and explosive. To obtain high level of Aspergillus niger ATCC1015 xylanase as a model of heterologous protein by K. phaffii at formate induction, insertion of three-copy cis-acting element W3A into PAOX1 additionally, and co-expression of transcription factor Mit1 under another PAOX1 were carried out separately and simultaneously. The yield of xylanase increased by 41% at formate induction when Mit1 was co-expressed. Furtherly, the yield of xylanase increased by 42% using sorbitol as supplemental carbon source with the result of 408.3 × 103 U‧L-1 xylanase. Therefore, a non-methanol needed and inducible heterologous protein expression system of Komagataella phaffii was developed successfully.