Dictyoglomus Research Articles

Making a Bacterial Thermophilic Enzyme in a Fungal Expression System.

This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei. Successful expression of a gene of interest in a heterologous host involves front-end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled-up fermentation. The thermophilic xylanase B (XynB) enzyme from the bacterium Dictyoglomus thermophilum discussed here can be easily purified by heat-precipitation from the culture supernatant of the mesophilic host. A functional XynB can also be produced in Escherichia coli, but at a lower yield compared to that obtained in T. reesei. The protocol provided here can be adapted to various other proteins and filamentous fungal hosts. © 2018 by John Wiley & Sons, Inc.

Enhanced features of Dictyoglomus turgidum Cellulase A engineered with carbohydrate binding module 11 from Clostridium thermocellum

Lignocellulosic biomass (LCB) is a low-cost and abundant source of fermentable sugars. Enzymatic hydrolysis is one of the main ways to obtain sugars from biomass, but most of the polysaccharide-degrading enzymes are poorly efficient on LCB and cellulases with higher performances are required. In this study, we designed a chimeric protein by adding the carbohydrate binding module (CBM) of the cellulosomal enzyme CtLic26A-Cel5E (endoglucanase H or CelH) from Clostridium (Ruminiclostridium) thermocellum to the C-terminus of Dtur CelA, an interesting hyperthermostable endoglucanase from Dictyoglomus turgidum. The activity and binding rate of both native and chimeric enzyme were evaluated on soluble and insoluble polysaccharides. The addition of a CBM resulted in a cellulase with enhanced stability at extreme pHs, higher affinity and activity on insoluble cellulose.

Biochemical characterization of a novel thermostable β-glucosidase from Dictyoglomus turgidum

Dtur_0462 gene from the hypertermophilic bacterium Dictyoglomus turgidum, encoding a β-glucosidase, was synthetically produced and expressed in Escherichia coli BL21(DE3)-RIL. DturβGlu was purified to homogeneity by affinity chromatography and its homotetrameric structure was determined by gel filtration. The monomer is composed by 418 amino acidic residues and showed high sequence similarity with Glycoside Hydrolases (GHs) belonging to GH1 family. The maximum activity of DturβGlu was observed at 80°C and at pH5.4. DturβGlu was stable in the range of pH5–8 and retained 70% of its activity after 2h of incubation at 70°C. Metal ions and chemical reagents differently influenced the β-glucosidase activity; furthermore, DturβGlu displays a good ethanol and glucose tolerance (Ki 750mM). The enzyme is active on p-nitrophenyl-β-d-glucopyranoside (pNPGlu) (Km 0.84mM) and p-nitrophenyl-β-d-galactopyranoside (pNPGal) (Km 1.36mM) and shows a broad substrate specificity towards natural compounds as salicin, cellobiose and genistin. The ability to hydrolyze different substrates, the activation in the presence of surfactants, the good thermal resistance, and finally the high glucose and ethanol tolerance make this enzyme a good candidate for industrial applications.

Complete Biotransformation of Protopanaxatriol-Type Ginsenosides in Panax ginseng Leaf Extract to Aglycon Protopanaxatriol by β-Glycosidases from Dictyoglomus turgidum and Pyrococcus furiosus.

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as memory enhancement and tumor inhibition. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues linked to APPT, but not other glycoside residues. β-Glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer sugar at C-6 but not the inner glucose at C-6 or the glucose at C-20. Thus, the combined use of DT-bgl and PF-bgl is expected to increase the biotransformation of PPT-type ginsenosides to APPT. We optimized the ratio of PF-bgl to DT-bgl, the concentrations of substrate and enzyme, and the reaction time to increase the biotransformation of ginsenoside Re and PPT-type ginsenosides in Panax ginseng leaf extract to APPT. DT-bgl combined with PF-bgl converted 1.0 mg/ml PPT-type ginsenosides in ginseng leaf extract to 0.58 mg/ml APPT without other ginsenosides, with a molar conversion of 100%. We achieved the complete biotransformation of ginsenoside Re and PPT-type ginsenosides in ginseng leaf extract to APPT by the combined use of two β-glycosidases, suggesting that discarded ginseng leaves can be used as a source of the valuable ginsenoside APPT. To the best of our knowledge, this is the first quantitative production of APPT using ginsenoside Re, and we report the highest concentration and productivity of APPT from ginseng extract to date.

Complete conversion of all typical glycosylated protopanaxatriol ginsenosides to aglycon protopanaxatriol by combined bacterial \u03b2-glycosidases

Aglycon protopanaxatriol (APPT) has valuable pharmacological effects such as anti-inflammatory and anti-stress activities. However, the complete conversion of all typical glycosylated protopanaxatriol ginsenosides to APPT has not been achieved to date. β-Glycosidase from the hyperthermophilic bacterium Dictyoglomus turgidum (DT-bgl) hydrolyzes the glucose residues at C-6 and the inner glucose at C-20 in protopanaxatriol (PPT), but not the outer rhamnose residues at C-6. In contrast, β-glycosidase from the hyperthermophilic bacterium Pyrococcus furiosus (PF-bgl) hydrolyzes the outer rhamnose residue at C-6 but not the inner glucose residues at C-6 and C-20 in PPT. Thus, the combined use of DT-bgl and PF-bgl resulted in the complete the conversion of all typical glycosylated PPT ginsenosides, including R1, R2, Re, Rg1, Rg2, Rh1, Rf, F1, F3, and F5, to APPT. DT-bgl combined with PF-bgl completely hydrolyzed 1.0 mg ml−1 R1 and 1.0 mg ml−1 total PPT-type ginsenosides in Panax notoginseng root extract to 0.5 and 0.63 mg ml−1 APPT for 4 and 3 h, with molar conversions of 100% and productivities of 125 and 210 mg l−1 h−1, respectively. To the best of our knowledge, this is the first report of the complete conversion of all typical glycosylated PPT ginsenosides to APPT and the highest productivity of APPT obtained from ginseng extract achieved to date.

The novel thermostable cellulose-degrading enzyme DtCel5H from Dictyoglomus thermophilum: crystallization and X-ray crystallographic analysis.

Cellulose-based products constitute the great majority of municipal waste, and applications of cellulases in the conversion of waste biomass to biofuels will be a key technology in future biorefineries. Currently, multi-enzymatic pre-treatment of biomass is a crucial step in making carbohydrates more accessible for subsequent fermentation. Using bioinformatics analysis, endo-β-(1,4)-glucanase from Dictyoglomus thermophilum (DtCel5H) was identified as a new member of glycosyl hydrolase family 5. The gene encoding DtCel5H was cloned and the recombinant protein was overexpressed for crystallization and biophysical studies. Here, it is shown that this enzyme is active on cellulose substrates and is highly thermostable. Crystals suitable for crystallographic investigations were also obtained in different crystallization conditions. In particular, ordered crystals of DtCel5H were obtained using either ammonium sulfate or polyethylene glycol (PEG) as a precipitant agent. The crystals obtained in the presence of ammonium sulfate belonged to space group P32, with unit-cell parameters a = 73.1, b = 73.1, 73.1, c = 127.8 Å, and diffracted to 1.5 Å resolution, whereas the second crystal form belonged to the orthorhombic space group P212121, with unit-cell parameters a = 49.3, b = 67.9, c = 103.7 Å, and diffracted to 1.6 Å resolution. The crystal structure was solved in both space groups using molecular-replacement methods. Structure-activity and structure-stability studies of DtCel5H will provide insights for the design of high-performance enzymes.

A thermophilic enzymatic cocktail for galactomannans degradation.

The full utilization of hemicellulose sugars (pentose and exose) present in lignocellulosic material, is required for an efficient bio-based fuels and chemicals production. Two recombinant thermophilic enzymes, an endo-1,4-β-mannanase from Dictyoglomus turgidum (DturCelB) and an α-galactosidase from Thermus thermophilus (TtGalA), were assayed at 80 °C, to assess their heterosynergystic association on galactomannans degradation, particularly abundant in hemicellulose. The enzymes were tested under various combinations simultaneously and sequentially, in order to estimate the optimal conditions for the release of reducing sugars. The results showed that the most efficient degree of synergy was obtained in simultaneous assay with a protein ratio of 25% of DturCelB and 75% of TtGalA, using Locust bean gum as substrate. On the other hand, the mechanism of action was demonstrated through the sequential assays, i.e. when TtGalA acting as first to enhance the subsequent hydrolysis performed by DturCelB. The synergistic association between the thermophilic enzymes herein described has an high potential application to pre-hydrolyse the lignocellulosic biomasses right after the pretreatment, prior to the conventional saccharification step.

Synergistic production of 20(S)-protopanaxadiol from protopanaxadiol-type ginsenosides by \u03b2-glycosidases from Dictyoglomus turgidum and Caldicellulosiruptor bescii

20(S)-Protopanaxadiol (APPD) has potential uses in the pharmaceutical, cosmetic, and food industries because of its anti-stress, anti-fatigue, anti-cancer, anti-inflammatory, and anti-wrinkle properties. However, APPD production is difficult because β-glycosidases that convert the protopanaxadiol (PPD)-type ginsenoside compound K to APPD are rare. β-Glycosidase from Dictyoglomus turgidum (DT-bgl) has the highest specific activity for converting compound K to APPD, but exhibits no activity towards the α-l-arabinopyranoside moiety in compound Y. Therefore, β-glycosidase from Caldicellulosiruptor bescii (CB-bgl), which has a strong α-l-arabinopyranosidase activity, was used along with DT-bgl. The volumetric and specific productivities of the two-enzyme system for APPD using ginseng root extract were 38.4- and 38.7-fold higher, respectively, than those of β-glycosidase from Pyrococcus furiosus, which had the highest volumetric productivity previously reported, at the same enzyme and substrate concentrations. Thus, DT-bgl combined with CB-bgl completely converted PPD-type ginsenosides to APPD with the highest volumetric and specific productivities reported thus far.

Biochemical characterization of a thermostable endomannanase/endoglucanase from Dictyoglomus turgidum.

Dictyoglomus turgidum is a hyperthermophilic, anaerobic, gram-negative bacterium that shows an array of putative glycoside hydrolases (GHs) encoded by its genome, a feature that makes this microorganism very interesting for biotechnological applications. The aim of this work is the characterization of a hyperthermophilic GH5, Dtur_0671, of D. turgidum, annotated as endoglucanase and herein named DturCelB in agreement to DturCelA, which was previously characterized. The synthetic gene was expressed in Escherichia coli. The purified recombinant enzyme is active as a monomer (40kDa) and CD structural studies showed a conserved α/β structure at different temperatures (25 and 70°C) and high thermoresistance (Tm of 88°C). Interestingly, the enzyme showed high endo-β-1,4-mannanase activity vs various mannans, but low endo-β-1,4 glucanase activity towards carboxymethylcellulose. The K M and V max of DturCelB were determined for both glucomannan and CMC: they were 4.70mg/ml and 473.1μmol/minmg and 1.83mg/ml and 1.349μmol/minmg, respectively. Its optimal activity towards temperature and pH resulted to be 70°C and pH 5.4, respectively. Further characterization highlighted good thermal stability (~50% of enzymatic activity after 2h at 70°C) and pH stability over a broad range (>90% of activity after 1h in buffer, ranging pH 5-9); resistance to chemicals was also observed.

Chemo-enzymatic synthesis of 3-O- (β-d-glycopyranosyl)-sn-glycerols and their evaluation as preservative in cosmetics

Abstract d-Glycopyranosyl glycerols are common natural products and exhibit strong biological properties, notably as moisturizing agents in cosmetics. Their chemical synthesis remains tedious thus decreasing their potential industrial and economic development, as well as the study of their structure-function relationships. In this work, the chemo-enzymatic synthesis of three enantiopure 3-O-(β-d-glycopyranosyl)-sn-glycerols was efficiently performed using an original glycosidase from Dictyoglomus thermophilum and their preservatives properties were assessed using a challenge test method. Amongst them, the 3-O-(β-d-glucopyranosyl)-sn-glycerol exhibited a specific anti-fungus activity.

Is the acid/base catalytic residue mutation in β-d-mannosidase DtMan from Dictyoglomus thermophilum sufficient enough to provide thioglycoligase activity?

Glycoside hydrolases can be turned into thioglycoligase by mutation of the acid/base catalytic carboxylate residue. These mutants have proven valuable to generate S-glycosides, however, few examples in literature have described efficient thioglycoligase activity, and even fewer the underlying molecular mechanism. DtMan, a GH2 family β-d-mannosidase from the thermophilic Dictyoglomus thermophilum was cloned and expressed in E. coli. The recombinant protein is highly specific for β-d-mannosides, and exhibits efficient catalysis constants coupled to thermostability. However, seven variants bearing mutated acid/base residue could not be turned into efficient thioligases. Crystal structure of DtMan Glu425Cys mutant and molecular modeling calculations have demonstrated that unlike other GH2 thioligase reported, active site accessibility of thiol acceptor may be impaired by entrance loop rigidity. This structural feature may explain why DtMan mutants do not exhibit thioglycoligase activity.

The Complete Genome Sequence of Hyperthermophile Dictyoglomus turgidum DSM 6724™ Reveals a Specialized Carbohydrate Fermentor.

Here we report the complete genome sequence of the chemoorganotrophic, extremely thermophilic bacterium, Dictyoglomus turgidum, which is a Gram negative, strictly anaerobic bacterium. D. turgidum and D. thermophilum together form the Dictyoglomi phylum. The two Dictyoglomus genomes are highly syntenic, and both are distantly related to Caldicellulosiruptor spp. D. turgidum is able to grow on a wide variety of polysaccharide substrates due to significant genomic commitment to glycosyl hydrolases, 16 of which were cloned and expressed in our study. The GH5, GH10, and GH42 enzymes characterized in this study suggest that D. turgidum can utilize most plant-based polysaccharides except crystalline cellulose. The DNA polymerase I enzyme was also expressed and characterized. The pure enzyme showed improved amplification of long PCR targets compared to Taq polymerase. The genome contains a full complement of DNA modifying enzymes, and an unusually high copy number (4) of a new, ancestral family of polB type nucleotidyltransferases designated as MNT (minimal nucleotidyltransferases). Considering its optimal growth at 72°C, D. turgidum has an anomalously low G+C content of 39.9% that may account for the presence of reverse gyrase, usually associated with hyperthermophiles.

Characterization of two novel heat-active α-galactosidases from thermophilic bacteria.

Two genes (agal1 and agal2) encoding α-galactosidases were identified by sequence-based screening approaches. The gene agal1 was identified from a data set of a sequenced hot spring metagenome, and the deduced amino-acid sequence exhibited 99% identity to an α-galactosidase from the thermophilic bacterium Dictyoglomus thermophilum. The gene agal2 was identified from the whole genome sequence of the thermophile Meiothermus ruber. The amino-acid sequences exhibited structural motifs typical for glycoside hydrolase (GH) family 36 members and were also differentiated into different subgroups of this family. Recombinant production of the heat-active GH36b enzyme Agal1 (87kDa) and GH36bt enzyme Agal2 (57kDa) was carried out in E. coli. Agal1 exhibited a specific activity of 1502.3U/mg at 80°C, pH 6.5, and Agal2 225.4U/mg at 60-70°C, pH 6.5. Half-lives of 14h (Agal1) and 39h (Agal2) were obtained at 50°C, and Agal1 showed half-lives of 4 and 2h at 70 and 80°C, respectively. In addition to the natural substrates melibiose, raffinose, and stachyose, 4NP α-D-galactopyranoside was hydrolyzed. Galactose was also liberated from locust bean gum. Both heat-active enzymes are attractive candidates for application in food and feed industry for high-temperature processes for the degradation of raffinose family oligosaccharides.

Stability and activity of Dictyoglomus thermophilum GH11 xylanase and its disulphide mutant at high pressure and temperature

The functional properties of extremophilic Dictyoglomus thermophilum xylanase (XYNB) and the N-terminal disulphide-bridge mutant (XYNB-DS) were studied at high pressure and temperature. The enzymes were quite stable even at the pressure of 500MPa at 80°C. The half-life of inactivation in these conditions was over 30h. The inactivation at 80°C in atmospheric pressure was only 3-times slower. The increase of pressure up to 500MPa at 80°C decreased only slightly the enzyme's stability, whereas in 500MPa the increase of temperature from 22 to 80°C decreased significantly more the enzyme's stability. While the high temperature (80–100°C) decreased the enzyme reaction with short xylooligosaccharides (xylotetraose and xylotriose), the high pressure (100–300MPa) had an opposite effect. The temperature of 100°C strongly increased the Km but did not affect the kcat to the same extent, thus indicating that the interaction of the substrate with the active site suffers before the catalytic reaction begins to decrease as the temperature rises. Circular dichroism spectroscopy showed the high structural stability of XYNB and XYNB-DS at 93°C.

Effect of Trichoderma reesei proteinases on the affinity of an inorganic-binding peptide.

An inorganic-binding peptide sequence with high affinity to silica-containing materials was fused to a glycoside hydrolase GH26 mannanase, ManA, from the extremely thermophilic bacterium Dictyoglomus thermophilum. The resulting recombinant enzyme produced in Escherichia coli, ManA-Linker, displayed high binding affinity towards synthetic zeolite while retaining its catalytic activity at 80 °C. ManA-Linker was able to bind to the zeolite at different pH levels, indicating a true pH-independent binding. However, complete degradation of the peptide linker was observed when the recombinant ManA-Linker was exposed to the supernatant from the filamentous fungus Trichoderma reesei. This degradation was caused by extracellular proteinases produced by T. reesei during its growth phase. Several derivatives of ManA-Linker were designed and expressed in E. coli. All the derivatives carrying a single sequence of the linker were still susceptible to T. reesei proteinase degradation. Complete substitution of the linker sequence by (GGGGS)16 resulted in a proteinase-resistant ManA derivative, ManA-Linker-(GGGGS)16, which was able to bind to zeolite in a pH-dependent manner.

Performance of a New Thermostable Mannanase in Breaking Guar-Based Fracturing Fluids at High Temperatures with Little Premature Degradation

A new thermostable β-1,4-mannanase (DtManB) cloned from Dictyoglomus thermophilum CGMCC 7283 showed the maximum activity towards hydroxypropyl guar gum at 80 °C, with a half-life of 46 h. DtManB exhibited good compatibility with various additives of fracturing fluid, retaining more than 50 % activity in all the cases tested. More importantly, premature degradation could be alleviated significantly when using DtManB as breaker, because at 27 and 50 °C it displayed merely 3.7 and 18.5 % activities compared to those at 80 °C. In a static test, 0.48 mg DtManB could break 200 mL borax cross-linked fracturing fluid dramatically at 80 °C, and merely 18 mPa s of the viscosity was detected even after the broken fluid was cooled down and only 161.4 mg L(-1) of the residue was left after the enzymatic reaction. All these positive features demonstrate the great potential of this mannanase as a new enzyme breaker for application in enhanced recovery of petroleum oil.

Production of aglycone protopanaxatriol from ginseng root extract using Dictyoglomus turgidum β-glycosidase that specifically hydrolyzes the xylose at the C-6 position and the glucose in protopanaxatriol-type ginsenosides

The hydrolytic activity of a recombinant β-glycosidase from Dictyoglomus turgidum that specifically hydrolyzed the xylose at the C-6 position and the glucose in protopanaxatriol (PPT)-type ginsenosides followed the order Rf > Rg₁ > Re > R₁ > Rh₁ > R₂. The production of aglycone protopanaxatriol (APPT) from ginsenoside Rf was optimal at pH 6.0, 80 °C, 1 mg ml⁻¹ Rf, and 10.6 U ml⁻¹ enzyme. Under these conditions, D. turgidum β-glycosidase converted ginsenoside R₁ to APPT with a molar conversion yield of 75.6 % and a productivity of 15 mg l⁻¹ h⁻¹ after 24 h by the transformation pathway of R₁ → R₂ → Rh₁ → APPT, whereas the complete conversion of ginsenosides Rf and Rg₁ to APPT was achieved with a productivity of 1,515 mg l⁻¹ h⁻¹ after 6.6 h by the pathways of Rf → Rh₁ → APPT and Rg₁ → Rh₁ → APPT, respectively. In addition, D. turgidum β-glycosidase produced 0.54 mg ml⁻¹ APPT from 2.29 mg ml⁻¹ PPT-type ginsenosides of Panax ginseng root extract after 24 h, with a molar conversion yield of 43.2 % and a productivity of 23 mg l⁻¹ h⁻¹, and 0.62 mg ml⁻¹ APPT from 1.35 mg ml⁻¹ PPT-type ginsenosides of Panax notoginseng root extract after 20 h, with a molar conversion yield of 81.2 % and a productivity of 31 mg l⁻¹ h⁻¹. This is the first report on the APPT production from ginseng root extract. Moreover, the concentrations, yields, and productivities of APPT achieved in the present study are the highest reported to date.

Expression, crystallization and preliminary X-ray crystallographic analysis of cellobiose 2-epimerase from Dictyoglomus turgidum DSM 6724.

Cellobiose 2-epimerase epimerizes and isomerizes β-1,4- and α-1,4-gluco-oligosaccharides. N-Acyl-D-glucosamine 2-epimerase (DT_epimerase) from Dictyoglomus turgidum has an unusually high catalytic activity towards its substrate cellobiose. DT_epimerase was expressed, purified and crystallized. Crystals were obtained of both His-tagged DT_epimerase and untagged DT_epimerase. The crystals of His-tagged DT_epimerase diffracted to 2.6 Å resolution and belonged to the monoclinic space group P2₁, with unit-cell parameters a=63.9, b=85.1, c=79.8 Å, β=110.8°. With a Matthews coefficient VM of 2.18 Å3 Da(-1), two protomers were expected to be present in the asymmetric unit with a solvent content of 43.74%. The crystals of untagged DT_epimerase diffracted to 1.85 Å resolution and belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a=55.9, b=80.0, c=93.7 Å. One protomer in the asymmetric unit was expected, with a corresponding VM of 2.26 Å3 Da(-1) and a solvent content of 45.6%.

Thermostabilization of extremophilic Dictyoglomus thermophilum GH11 xylanase by an N-terminal disulfide bridge and the effect of ionic liquid [emim]OAc on the enzymatic performance

In the present study, an extremophilic GH11 xylanase was stabilized by an engineered N-terminal disulphide bridge. The effect of the stabilization was then tested against high temperatures and in the presence of a biomass-dissolving ionic liquid, 1-ethyl-3-methylimidazolium acetate ([emim]OAc). The N-terminal disulfide bridge increased the half-life of a GH11 xylanase (XYNB) from the hyperthermophilic bacterium Dictyoglomus thermophilum by 10-fold at 100°C. The apparent temperature optimum increased only by ∼5°C, which is less than the corresponding increase in mesophilic (∼15°C) and moderately thermophilic (∼10°C) xylanases. The performance of the enzyme was increased significantly at 100–110°C. The increasing concentration of [emim]OAc almost linearly increased the inactivation level of the enzyme activity and 25% [emim]OAc inactivated the enzyme almost fully. On the contrary, the apparent temperature optimum did not decrease to a similar extent, and the degree of denaturation of the enzyme was also much lower according to the residual activity assays. Also, 5% [emim]OAc largely counteracted the benefit obtained by the stabilizing disulfide bridge in the temperature-dependent activity assays, but not in the stability assays. Km was increased in the presence of [emim]OAc, indicating that [emim]OAc interfered the substrate–enzyme interactions. These results indicate that the effect of [emim]OAc is targeted more to the functioning of the enzyme than the basic stability of the hyperthermophilic GH11 xylanase.

Simultaneous expression of the bacterial Dictyoglomus thermophilum xynB gene under three different Trichoderma reesei promoters

Multiple copies of expression cassettes driven by the Trichoderma reesei xylanase 2 (xyn2) and cellobiohydrolase 2 (cbh2) promoters were introduced into the recombinant T. reesei EC-21 generated to express a thermostable Dictyoglomus thermophilum xylanase (XynB) under the egl2 promoter for further improvement of the enzyme yield. The transformants were screened based on increased XynB activity only. Multiple promoter transformant MPP-4 expressing the xynB gene under all the three promoters was found to be the highest producer of XynB, giving a 65% increase in yield compared to the parental single-promoter recombinant EC-21. The multiple-promoter transformant strains harboured six to nine copies of the xynB gene. Amongst the three promoters, egl2 seemed to have the strongest effect on XynB expression. The shotgun approach we used proved to be effective for rapid enhancement of protein expression using three promoters active at the near-neutral pH of the cultivation medium.