Heterologous Xylanase Research Articles

Improvement of homologous GH10 xylanase production by deletion of genes with predicted function in the Aspergillus nidulans secretion pathway.

SummaryFilamentous fungi are important cell factories for large‐scale enzyme production. However, production levels are often low, and this limitation has stimulated research focusing on the manipulation of genes with predicted function in the protein secretory pathway. This pathway is the major route for the delivery of proteins to the cell exterior, and a positive relationship between the production of recombinant enzymes and the unfolded protein response (UPR) pathway has been observed. In this study, Aspergillus nidulans was exposed to UPR‐inducing chemicals and differentially expressed genes were identified by RNA‐seq. Twelve target genes were deleted in A. nidulans recombinant strains producing homologous and heterologous GH10 xylanases. The knockout of pbnA (glycosyltransferase), ydjA (Hsp40 co‐chaperone), trxA (thioredoxin) and cypA (cyclophilin) improved the production of the homologous xylanase by 78, 171, 105 and 125% respectively. Interestingly, these deletions decreased the overall protein secretion, suggesting that the production of the homologous xylanase was specifically altered. However, the production of the heterologous xylanase and the secretion of total proteins were not altered by deleting the same genes. Considering the results, this approach demonstrated the possibility of rationally increase the production of a homologous enzyme, indicating that trxA, cypA, ydjA and pbnA are involved in protein production by A. nidulans.

Enhancement of xylanase expression by Komagataella phaffii through pexophagy inhibition

Xylanase is widely used in agriculture and food industry. To increase heterologous xylanase expression by Komagataella phaffii under the condition of mixed carbon source feeding, the atg30 gene was knocked out from this yeast to inhibit pexophagy for increasing sorbitol consumption. Komagataella phaffii atg30Δ was constructed through homologous recombination that resulted in the inhibition of pexophagy. When compared with the control, the yield of secretory xylanase increased by 4.1 ± 0.2%, 5.7 ± 0.5%, 11.8 ± 0.4% and 11.4 ± 0.7% under the conditions of 1.5% methanol and 0.5, 1, 1.5 and 2% sorbitol addition, respectively. The residual sorbitol analysis showed that sorbitol consumed by Komagataella phaffii atg30Δ increased by 121.3 ± 9.3%, 66.8 ± 15.2% and 31.2 ± 3.2% following 1, 1.5 and 2 sorbitol addition, respectively. Thus, non-methanol carbon source, co-feeding with methanol, provided more energy without pexophagy by atg30 gene knockout. Pexophagy inhibition increased the heterologous protein expression by Komagataella phaffii, which provided basis for increased yield of xylanase through pexophagy monitoring.

Using an Inducible Promoter of the Glucoamylase Gene to Construct New Multienzyme Complexes from Penicillium verruculosum

New recombinant strains of Penicillium verruculosum are created using a new plasmid construct based on an inducible promoter of glucoamylase gene (gla1) that secretes heterologous xylanase E (XylE) from P. canescens. New biocatalysts are produced that contain cellulolytic enzyme preparations (EPs) enriched with XylE. The amount of XylE in the recombinant EPs varies in the optimum range of 11–24% of the total protein while generally preserving the P. verruculosum cellulose complex. The hydrolytic activity of the new EPs with respect to polymeric plant-derived substrates exceeds that of EPs produced using other expression systems and commercial preparations. The new EP glaX-17 surpasses in particular a control EP based on the recipient strain by 13% in the efficiency of hydrolyzing aspen wood and is 20% more efficient than the commercial EP Accelerase Duet. The new EP glaX-17 displays 25% greater efficiency (35–43%) during the hydrolysis of wheat bran than the commercial EP Accelease Duet. The effectiveness is demonstrated of using the new gla1 promoter for the production of EPs (biocatalysts) while preserving the balanced cellulose complex of the strain and optimum yield of heterologous XylE required for the deep hydrolysis of xylan-containing plant biomass.

Preparation and properties of new biocatalysts for the degradation of nonstarch plant polysaccharides

Recombinant strains of Penicillium verruculosum are developed that produce the homologous endoglucanase 2 (Eg2) and the P. canescens heterologous xylanase E (XylE). The recombinant strains are used to obtain new biocatalysts, i.e., enzyme preparations (EPs) that are substantially enriched with Eg2 and XylE. These preparations are highly active with respect to nonstarch plant polysaccharides (NPSes): cellulose, β-glucan, and xylan. The qualitative and quantitative compositions of the new EPs are studied by protein chromatography. It was shown that the EPs contained (in terms of total protein content) ~16–17% Eg2, 48–63% XylE, and 17–30% cellobiohydrolases, while the EP obtained using the recipient strain contained 1.4% Eg2, ~60% cellobiohydrolase and no XylE. The optimum pH values for cellulase (with respect to carboxymethylcellulose, CMC) and the xylanase activity of the EPs are 4.0 and 5.5, respectively. The EPs exhibit the abovementioned activities within a wide range of pH (3 to 7). The EPs exhibit CMC-ase and xylanase activities in the temperature range of 20–80°С with maxima at 60 and 70°C, respectively. The xylanase activity of the new EPs is virtually uninhibited by protein inhibitors of rye.

Preparation and Properties of New Biocatalysts for Destruction of Plant non-Starch Polysaccharides

Recombinant strains Penicillium verruculosum were created which produce homologous endoglucanase 2 (Er2) and heterologous xylanase E (XylE) P. canescens. The recombinant strains were used for preparing new biocatalysts which are enzyme preparations (EP) enriched considerably with Er2 and XylE; the biocatalysts are highly active to plant non-starch polysaccharides (NPS) such as cellulose, β-glucan, xylan. Proteic chromatography was used for determining the qualitative and quantitative composition of the new EP to show that they contain (expressed as proportions of the total protein content) ca. 16–17 % Er2, 48–63 % XylE, 17–30 % cellobiohydrolase, while EP prepared with the recipient strain contained 1.4 % Er2, ca. 60 % cellobiohydrolase and no XylE. pH equal to 4.0 and 5.5 were optimal for the activity to cellulose (with respect to carboxymethylcellulose, CMC) and to xylanase, respectively, EP being active over a wide range of pH from 3 to 7. EP were active to CMC and xylanase at the temperature range from 20 to 80 °C with maxima at 60 and 70 °C. The activity of new EP to xylanase was practically not inhibited by protein inhibitors of rye.

Characterization of Two GAS1 Genes and Their Effects on Expression and Secretion of Heterologous Protein Xylanase B in Kluyveromyces lactis.

β-1,3-glucanosyltransferases play essential roles in cell wall biosynthesis in yeast. Kluyveromyces lactis has six putative β-1,3-glucanosyltransferase genes. KlGAS1-1 and KlGAS1-2 are homologs of Saccharomyces cerevisiae gene GAS1. RT-qPCR indicated the transcription level of KlGAS1-1 was significantly reduced while heterologous protein (thermostable xylanase B) secretion was enhanced during medium optimization. To evaluate if these two events were related, and to improve xylanase B secretion in K. lactis, we constructed KlGAS1-1 and KlGAS1-2 single deletion strains and double deletion strain, respectively. KlGAS1-1 gene deletion resulted in the highest xylanase B activity among the three mutants. Only the double deletion strain showed morphology similar to that of the GAS1 deletion mutant in S. cerevisiae. The two single deletion strains differed in terms of cell wall thickness and xylanase B secretion. Transcription levels of β-1,3-glucanosyltransferase genes and genes related to protein secretion and transport were assayed. The β-1,3-glucanosyltransferase genes displayed transcription complementation in the cell wall synthesis process. KlGAS1-1 and KlGAS1-2 affected transcription levels of secretion- and transport-related genes. Differences in protein secretion ratio among the three deletion strains were associated with changes of transcription levels of secretion- and transport-related genes. Our findings indicate that KlGAS1-1 deletion is an effective tool for enhancing industrial-scale heterologous protein secretion in K. lactis.

A novel pathway construction in Candida tropicalis for direct xylitol conversion from corncob xylan

In this study, an integrated xylitol production pathway, directly using xylan as the substrate, was constructed in Candida tropicalis BIT-Xol-1 which could efficiently convert xylose into xylitol. In order to consolidate this bioprocessing, a β-1,4-xylanase gene (atn) and a β-xylosidase gene (atl) were cloned from Aspergillus terreus, and were constructed onto episomal plasmid pAUR123. Additionally, combination of the individual atn and atl expression cassette was also cloned onto pAUR123. After transforming, the positive C. tropicalis transformants co-expressing xylanase and xylosidase produced larger hydrolysis zones than those expressing xylanase alone, when incubated on xylan-congo red plates. The engineered C. tropicalis/pAUR-atn-atl-3 (C. tropicalis PNL3) secrete heterologous xylanase and xylosidase simultaneously, with the activities of 48.17 and 11.56U/mL, respectively. The xylitol yields by C. tropicalis PNL3 utilizing xylan and corncob were 77.1% and 66.9%, respectively. The integrated pathway of xylitol production was feasible and efficient in utilization of xylan-rich renewable biomass via combining saccharification and transformation of xylan in engineered C. tropicalis.

Production of biocatalysts on the basis of recombinant heterologous xylanase producer strains in the Penicillium verruculosum fungus: Their application in the hydrolysis of timber and wood processing industry wastes

The aim of this work was to create biocatalysts with an increased heterologous expression of endo-β-1,4-xylanase of P. canescens using recombinant P. verruculosum strains, to analyze the properties of new enzyme preparations, and to study the saccharifying activity of these preparations in the hydrolysis of plant raw materials, such as hogged aspen and detarred pine wood wastes of the timber and wood processing industries. The xylanase activity of the existing enzymatic preparations is insufficiently high to hydrolyze a xylan-rich biomass. The creation of increasingly xylanolytically active P. verruculosum-based recombinant strains containing homologous or heterologous genes of xylanase and mannanase is therefore a problem of great interest.

Purification and characterisation of a xylanase from Thermomyces lanuginosus and its functional expression by Pichia pastoris

A xylanase produced by Thermomyces lanuginosus 195 by solid state fermentation (SSF) was purified 9.3-fold from a crude koji extract, with a 7.6% final yield. The purified xylanase (with an estimated mass of 22 kDa by SDS-PAGE) retained 18% relative activity when treated for 10 min at 100 °C and approximately 90% relative activity when incubated at pH values ranging from 6 to 10. Xylanase activity in the purified preparation was significantly enhanced following treatment with manganese and potassium chlorides ( p < 0.05) but significantly reduced by calcium, cobalt and iron ( p < 0.05). The purified enzyme was also shown to be exclusively xylanolytic. The gene encoding xylanase activity from T. lanuginosus 195 was functionally expressed by Pichia pastoris. MALDI-ToF mass spectrometry and zymography were employed to confirm functional recombinant expression. Maximum xylanase titres were achieved following 120 h induction of the recombinant culture, yielding 26.8 U/mL. Achieving functional protein expression facilitates future efforts to optimise the cultivation conditions for heterologous xylanase production.

The methylotrophic yeast as a host for the expression and production of thermostable xylanase from the bacterium

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 alpha-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 3gl(-1) xylanase with a maximum activity of 3130Ul(-1) 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 37kDa 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 degrees C. R. marinus xylanase is the first reported thermostable gram-negative bacterial xylanase efficiently secreted by P. pastoris.

Amino acid supplementation, controlled oxygen limitation and sequential double induction improves heterologous xylanase production by

Heterologous endo-beta-1,4-xylanase was produced by Pichia stipitis under control of the hypoxia-inducible PsADH2-promoter in a high-cell-density culture. After promoter induction by a shift to oxygen limitation, different aeration rates (oxygen transfer rates) were applied while maintaining oxygen-limitation. Initially, enzyme production was higher in oxygen-limited cultures with high rates of oxygen transfer, although the maximum xylanase activity was not significantly influenced. Amino acid supplementation increased the production of the heterologous endo-beta-1,4-xylanase significantly in highly aerated oxygen-limited cultures, until glucose was depleted. A slight second induction of the promoter was observed in all cultures after the glucose had been consumed. The second induction was most obvious in amino acid-supplemented cultures with higher oxygen transfer rates during oxygen limitation. When such oxygen-limited cultures were shifted back to fully aerobic conditions, a significant re-induction of heterologous endo-beta-1,4-xylanase production was observed. Re-induction was accompanied by ethanol consumption. A similar protein production pattern was observed when cultures were first grown on ethanol as sole carbon source and subsequently glucose and oxygen limitation were applied. Thus, we present the first expression system in yeast with a sequential double-inducible promoter.

Amino acid supplementation improves heterologous protein production by Saccharomyces cerevisiae in defined medium

Supplementation of a chemically defined medium with amino acids or succinate to improve heterologous xylanase production by a prototrophic Saccharomyces cerevisiae transformant was investigated. The corresponding xylanase production during growth on ethanol in batch culture and in glucose-limited chemostat culture were quantified, as the native ADH2 promoter regulating xylanase expression was derepressed under these conditions. The addition of a balanced mixture of the preferred amino acids, Ala, Arg, Asn, Glu, Gln and Gly, improved both biomass and xylanase production, whereas several other individual amino acids inhibited biomass and/or xylanase production. Heterologous protein production by the recombinant yeast was also improved by supplementing the medium with succinate. The production of heterologous xylanase during growth on ethanol or glucose could thus be improved by supplementing metabolic precursors in the carbon- or nitrogen-metabolism.

Comparison of three expression systems for heterologous xylanase production byS. cerevisiaein defined medium

The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated. Heterologous xylanase production by two prototrophic S. cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium. Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell. Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain. Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids. Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations. The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome. No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression. Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated.

The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium.

Five recombinant S. cerevisiae strains were cultivated under identical conditions to quantify the molecular basis of the metabolic burden of heterologous gene expression, and to evaluate mechanisms for the metabolic burden. Two recombinant S. cerevisiae strains, producing Trichoderma reesei xylanase II under control of either the PGK1 or ADH2 promoters, were compared quantitatively with three references strains, where either the heterologous xylanase II (XYN2) gene, or the heterologous gene and the promoter and terminator were omitted from the recombinant plasmid. Neither the replication of multiple copies of the 2-microm-based YEp352 plasmid nor the replication the foreign XYN2 gene represented a metabolic burden to the cell, as the growth of the host organism was not affected. The inclusion of a glycolytic promoter on the recombinant plasmid, however, reduced the maximum specific growth rate (12% to 15%), biomass yield on glucose (8% to 11%), and specific glucose consumption rate (6% to 10%) of the recombinant strains. The presence of the heterologous XYN2 gene on the recombinant plasmid caused a further reduction in the maximum specific growth rate (11% to 14%), biomass yield (4%), and specific glucose consumption rate (12%) of the host strain during active gene expression, which was dictated by the regulatory characteristics of the promoter utilized. The metabolic effect of foreign gene expression was disproportionally large, with respect to on the amount of heterologous protein produced. This was most likely due to an increased energetic demand for the expression of a foreign gene and/or a competition for limiting amounts of transcription or translation factors, biosynthetic precursors or metabolic energy.

Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens.

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.

Expression of Aureobasidium pullulans xynA in, and secretion of the xylanase from, Saccharomyces cerevisiae

A previous report dealt with the cloning in Escherichia coli and sequencing of both the cDNA and genomic DNA encoding a highly active xylanase (XynA) of Aureobasidium pullulans (X.-L. Li and L. G. Ljungdahl, Appl. Environ. Microbiol. 60:3160-3166, 1994). Now we show that the gene was expressed in Saccharomyces cerevisiae under the GAL1 promoter in pYES2 and that its product was secreted into the culture medium. S. cerevisiae clone pCE4 with the whole open reading frame of xynA, including the part coding for the signal peptide, had xylanase activity levels of 6.7 U ml-1 in the cell-associated fraction and 26.2 U ml-1 in the culture medium 4 h after galactose induction. Two protein bands with sizes of 25 and 27 kDa and N-terminal amino acid sequences identical to that of APX-II accounted for 82% of the total proteins in the culture medium of pCE4. These proteins were recognized by anti-APX-II antibody. The results suggest that the XynA signal peptide supported the posttranslational processing of xynA product and the efficient secretion of the active xylanase from S. cerevisiae. Clones pCE3 and pGE3 with inserts of cDNA and genomic DNA, respectively, containing only the mature enzyme region attached by a Met codon had low levels of xylanase activity in the cell-associated fractions (1.6 U ml-1) but no activity in the culture media. No xylanase activity was detected in clone pGE4, which was the same as pCE4, except that pGE4 had a 59-bp intron in the signal peptide region. A comparison of the A. pullulans and S. cerevisiae signal peptides demonstrated that the XynA signal peptide was at least three times more efficient than those of S. cerevisiae invertase or mating alpha-factor pheromone in secreting the heterologous xylanase from S. cerevisiae cells.