Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC2 in and...

BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS

Expression of an AT-rich xylanase gene from the anaerobicfungus Orpinomyces sp. strain PC-2 in and secretionof the heterologous enzyme by Hypocrea jecorina

Xin-Liang Li & Christopher D. Skory &

Eduardo A. Ximenes & Douglas B. Jordan &

Bruce S. Dien & Stephen R. Hughes & Michael A. Cotta

Received: 13 September 2006 /Revised: 28 November 2006 /Accepted: 30 November 2006 / Published online: 16 January 2007# Springer-Verlag 2007

Abstract The catalytic domain encoded by an adenine–thymine (AT)-rich xylanase gene (xynA) of the anaerobicfungus Orpinomyces was expressed in Hypocrea jecorinaunder the control of the cel7A promoter and terminator.No XynA protein was detected in H. jecorina culturesupernatants when the original sequence was fused to theH. jecorina cel5A region coding for its signal peptide,carbohydrate-binding module, and hinge. Replacing thexynA (56% AT content) with a synthetic sequence contain-ing lower AT content (39%) supported the extracellularproduction (150 mg l−1) of the fusion xylanase byH. jecorina. Northern analysis revealed that successfulproduction after the decrease in AT content was related tohigher levels of the xylanase-specific mRNA. Anotherconstruct with an RDKR-coding sequence inserted between

the cel5A linker and the xynA catalytic domain allowedproduction of the fully processed active xylanase catalyticdomain. Both the fusion (40 kDa) and the fully processed(28 kDa) forms displayed enzymatic properties of family 11xylanases. Both the R and the Kex2-like KR sites wererecognized during secretion, resulting in a mixture of twoamino termini for the 28-kDa xylanase. The work demon-strated for the first time that glycoside hydrolases derivedfrom anaerobic fungi can be produced by H. jecorina.

Keywords Orpinomyces . Xylanase . Trichoderma .

Hypocrea . Hemicellulose

Introduction

The US fuel ethanol production passed the 4.0 billiongallons per year milestone in 2005, and the increase inproduction is accelerating. Ethanol in the USA is currentlyproduced primarily from cornstarch, which consumesabout 20% of the corn harvest. Further expansion ofethanol production may be limited by the corn supply,necessitating the use of alternative carbohydrate-richfeedstock (Gray et al. 2006; Somerville 2006). Lignocellu-losic biomass, such as corn stover, switchgrass, and forestryby-products, could supply alternative feedstock for futureexpansion of ethanol production (Somerville 2006). Thesugars in these biomass materials exist as polysaccharides,primarily, in the form of cellulose and hemicelluloses,which are physically and chemically associated togetherand with lignin, proteins, and starch. To make the sugarsavailable to fermenting microorganisms, the lignocellulosemust be converted into mono-sugars through pretreatmentand enzymatic saccharification (Dien et al. 2006). Amongthe many hemicelluloses, arabinoxylan represents the most

Appl Microbiol Biotechnol (2007) 74:1264–1275DOI 10.1007/s00253-006-0787-6

The mention of firm names or trade products does not imply that theyare endorsed or recommended by the US Department of Agricultureover other firms or similar products not mentioned.

X.-L. Li (*) : E. A. Ximenes :D. B. Jordan :B. S. Dien :M. A. CottaFermentation Biotechnology Research Unit, National Center forAgricultural Utilization Research, United States Department ofAgriculture—Agricultural Research Service,1815 N. University Street,Peoria, IL, USAe-mail: [email protected]

C. D. Skory : S. R. HughesBioproducts and Biocatalysis Research Unit, National Center forAgricultural Utilization Research, United States Department ofAgriculture—Agricultural Research Service,1815 N. University Street,Peoria, IL, USA

Present address:E. A. XimenesDepartment of Microbiology, University of Georgia,Athens, GA, USA

abundant form found in agricultural lignocellulosic materi-als, and a key enzyme for its degradation is a xylanase,hydrolyzing the glycosidic bonds of the backbone. Otherenzymes required for the complete degradation of arabi-noxylan include β-xylosidase, α-arabinofuranosidase,α-glucuronidase, acetyl xylan esterase, phenolic acidesterase, and glucuronoyl esterase (Biely 1985; Shallomand Shoham 2003; Špániková and Biely 2006). Currently,xylanases are used commercially in animal feed, pulp andpaper pre-bleaching, and bakery processes (Bhat 2000).

The ascomycete Hypocrea jecorina (anamorph: Tricho-derma reesei) is a fungus currently used for commercialmanufacturing of cellulase and hemicellulase products.Recently, the fungus has been singled out as the mostpromising organism for the production of enzymes forlignocellulose conversion to fermentable sugars (Potera2006). In addition to a long history of safe commercialuse, the fungus is capable of secreting significantquantities of enzymes and has well-developed geneticsystems (Penttila et al. 1987a; Hazell et al. 2000). Usingthe strong inducible promoter of the cel7A gene coding forthe cellobiohydrolase I (CBHI; Shoemaker et al. 1983), anumber of homologous and heterologous proteins havealready been successfully produced by the fungus, usuallyas extracellular enzymes (Paloheimo and Suominen 1998;Penttila 1998). Although continued progress has beenreported on increasing the titer and effectiveness of H.jecorina cellulase system (Gray et al. 2006), much lesswork has been reported on improving its ability to degradehemicelluloses.

Anaerobic fungi, first discovered by Orpin (1975), arenatural inhabitants of the digestive tracts of herbivores andare intimately involved in the symbiotic digestion ofherbaceous materials in the animals. Some secrete numerouslignocellulose-degrading enzymes (Borneman et al. 1989).The fungal hydrolytic enzymes usually have high specificactivities (Gilbert et al. 1992; Chen et al. 1997). Genomes ofanaerobic fungi are unusually adenine–thymine (AT) rich(Brownlee 1989; Nicholson et al. 2005). From the polycen-tric isolate Orpinomyces PC-2, a number of hydrolyticenzymes and their genes have been studied. A xylanasecDNA (xynA) was previously isolated from the fungus codesfor an amino-terminal glycoside hydrolase family 11catalytic domain (CD), a Thr-rich linker, and two carboxyterminal non-catalytic docking domains (Li et al. 1997;Steenbakkers et al. 2001). The cDNA has an AT content of56% for the open reading frame (ORF) and 89.2% for thenoncoding regions (Li et al. 1997).

The present paper describes heterologous production ofthe Orpinomyces xylanase A by the industrially relevantfungus H. jecorina. We show that the nucleotide composi-tion of foreign genes can play a critical role when used forheterologous protein production by H. jecorina. Successful

development of heterologous production technologies forthe enzymes from anaerobic fungi constitutes a significantadvance in applying this promising source of genes towardslignocellulose bioconversion.

Materials and methods

Organisms and vectors H. jecorina (anamorph T. reesei)NRRL 11460 (RUT C-30), purchased from American TypeCulture Collection, was used throughout the project.Escherichia coli strain DH5α (Invitrogen, Carlsbad, CA)and XL-Blue MRA (Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 gyrA96. relA1 lac; Stratagene, LaJolla, CA) were used for plasmid propagation and 1 libraryscreening, respectively. The pBluescript KS(−) served asthe starting plasmid for the expression cassette for H.jecorina transformation, whereas 1 phage was the vectorfor constructing a H. jecorina genomic 1-DASH library.Both were purchased from Stratagene. Plasmid pOX8harboring the Orpinomyces PC-2 xynA cDNA (accessionnumber, U57819; Li et al. 1997) served as a template forpolymerase chain reaction (PCR) amplification.

Cloning and sequencing of the H. jecorina cel7A locus Wetmycelium (100 mg) of H. jecorina grown on potatodextrose (PD) broth for 3 days was used for genomic DNAextraction using the Easy DNA extraction kit (Invitrogen).The integrity and purity of the extracted DNA wereassessed by restriction digestion and 1.0% (w/v) agarosegel electrophoresis according the method of Sambrook andRussell (2001). The DNA sample was partially digested byEcoRI and separated by electrophoresis on an agarose(1.0%, w/v) gel. After staining with ethidium bromide, theDNA smear was visualized under UV light. A broad bandcorresponding to 5.0–10.0 kb was excised, and the DNA inthe agarose was extracted using the DNA Gel Extraction kit(Qiagen, Valencia, CA). The purified DNA fragments wereligated into the ready-to-use 1-DASH DNA by the T4ligase (Stratagene). Packaging, preparation of host cells,infection, plaque-lifting, and screening were performedaccording to the manufacturer’s instructions (Stratagene).A 661-bp cel7A probe was generated by PCR usingCBHIF1 and CBHIR1 (Table 1) as primers, 10 ng H.jecorina genomic DNA as template, and Taq polymerase.The probe was labeled with digoxigenin (Dig; RocheDiagnostics, Indianapolis, IN) during PCR by adding 2 μlDig dUTP (Roche) per 100-μl reaction. Conditions forhybridization, washing stringency, and detection were asdirected in the Dig Hybridization and Detection kit(Roche). Initial positive clones were purified after asecondary screening with fewer plaques forming units perplate. Purified positive 1 was prepared in liquid cultures,

Appl Microbiol Biotechnol (2007) 74:1264–1275 1265

and large scale 1 samples (>50 μg) were purified using the1 Purification Kit (Qiagen). Sequencing the regions ofinterest of the 1 clones was done by walking upstream anddownstream of cel7A using oligonucleotide primers syn-thesized according to known sequences. Sequence datawere analyzed using the DNAStar software (DNAstar,Madison, WI).

Construction of a fungal expression plasmid A fungalexpression vector pT3C (accession number, EF127523)constructed in pBluescript KS(−) contains a 2.2-kb cel7Aupstream and promoter region, a 0.3-kb eg2/cel5A (formerlyegl3, Saloheimo et al. 1988, Foreman et al. 2003) regioncoding for amino acid resides −21 to 70, a 0.7-kb cel7A 3′terminator sequence, the hygromycin B resistance gene(Cullen et al. 1987; Staben et al. 1989), and a 2.8-kb cel7A3′ region (Fig. 1). The cel5A sequence codes for a signalpeptide, a carbohydrate-binding module (CBM), and a linkersequence, followed by SpeI and PstI sites that facilitateinsertion of the genes of interest.

Cloning of the Orpinomyces xynA into the fungalexpression vector pT3C A 700-bp DNA fragment codingfor amino acid residues 29–255 of xynAwas amplified bythe KOD polymerase using OXF1 and OXR1 (Table 1) asprimers and the pOX8 plasmid as a template (Li et al.1997). This region codes for the CD (Li et al. 1997). ASpeI site in OXF1 and a PstI site in OXR1 were added tofacilitate cloning of the fragment into pT3C, whereas aTAA codon (AUU in RNA) was incorporated into OXR1.The insert was sequenced. Comparison of the sequence datarevealed the lack of mutations except that codons TCC andCCC coding for Ser213 and Pro241, respectively, werechanged to GCC and ACC. Sequencing of the originalclone pOX8 revealed that the errors were not caused byPCR or cloning, but rather by sequencing errors in pOX8.Thus Ser213 and Pro241 should be in fact A213 and T241,respectively. The plasmid was named pOX (Fig. 1).

A DNA fragment coding the identical amino acidsequence, but with much higher guanine–cytosine (GC)content (Fig. 2), was synthesized by Genescript (Piscat-away, NJ). SpeI, PstI, and TAA sites were added at thesame locations. The replacement plasmid was named pSOX(Fig. 1). A SpeI site was inserted after bp 330 of cel5A(Saloheimo et al. 1988) by site-directed mutagenesis usingthe QuickChange Site-Directed Mutagenesis Kit (Strata-gene), SPEIF, and SPEIR (Table 1) as primers, and pSOXas template. Digestion of the mutagenized pSOX with SpeIresulted in 11.0- and 200-bp fragments. The 11.0-kbfragment was isolated and ligated to generate pSOXS thatis devoid of the cel5A sequence (Fig. 1).

To engineer a RDKR-coding region (Paloheimo et al.2003) between the cel5A linker and xynA-coding regions,site-directed mutagenesis was performed with KexF andKexR (Table 1) as primers and pSOX as template. Theplasmids containing the correct sequence were namedpKSOX, as illustrated in Fig. 1. All plasmids were digestedovernight with EcoRI, and the larger band (around 8.0 kb)was excised using clean razor blades after agarose gelelectrophoresis. DNA was extracted using the DNA GelExtraction kit (Qiagen), dried in a speed vacuum, andreconstituted to 0.5 μg/μl in TE buffer.

Cel7A promoter

Cel5A sp

Cel7A terminator

HygB

Cel7A 3’ region

pSOX

pOX

pSOXS

pKSOX

EcoRIEcoRI SacII SalI SphIPstI

SpeI

Fig. 1 Illustration of a H. jecorina expression vector pT3C andvarious Orpinomyces PC-2 xynA sequences amplified and cloned forexpression. Striped bars represent DNA sequences of the cel7A locus;gray bars, that of cel5A; dotted bar, bacterial hygromycin B resistancegene (HygB) and fungal transcription signals. Other symbols: blackand white bars, original and GC-riched Orpinomyces xynA catalyticdomain coding region, respectively; horizontal line, cel5A linker-coding sequence; short strip with horizontal lines in pKSOX, asequence coding for RDKR

Table 1 Oligonucleotides used for this work

Name Nucleotide sequence Utility

CBHIF1 CATGGACGCGGATGGTGGC Cel7A probe generationCBHIR1 ATCCCACAGACTCATGACCA Cel7A probe generationOXF1 AGCCACTAGTGGTCAAAGATTAAGCGTT XynA amplificationOXR1 ATGATACTCTGCAGTTAACGAGGAGCAGAACCTTGTTTGG XynA amplificationSPEIF CGTCGCACAGCAGACTAGTTGGGGCCAGTGTGGAGG Cel5A CBM/linker removalSPEIR CCTCCACACTGGCCCCAACTAGTCTGCTGTGCGACG Cel5A CBM/linker removalKexF CCACCCACTAGTTCTCGCGACAAGCGGGGCCAGAGGTTGAGCGTCG Cleavage site creationKexR CGACGCTCAACCTCTGGCCCCGCTTGTCGCGAGAACTAGTGGGTGG Cleavage site creation

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Fungal transformation and xylanase detection Transfor-mation of H. jecorina Rut C-30 was done according toHazell et al. 2000. To detect expression, spores wereinoculated into 50-ml cellulase induction medium in a250-ml shake flask and shaken at 28°C for 5 days in anInnova 4230 shaker/incubator (New Brunswick Scientific,Edison, NJ). The medium (per liter) contained 20 g lactose,15 g corn steep liquor, 10 g KH2PO3, 1.0 g (NH4)2SO4,0.5 g MgSO4·7H2O, 1.0 ml Tween 80, and 1 ml tracemineral solution. The medium was adjusted to pH 4.8 with1 M K2HPO3 before autoclaving (120°C, 15 min). For pH-controlled cultures, medium was the same except that250 mM potassium phosphate buffer, pH 6.0, instead of10 g KH2PO3 per liter was used. Before assays or sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis, mycelia were removed by centrifugation(3,750×g, 15 min) at 4°C. Culture supernatants were

subjected to xylanase assay (see below) and SDS-PAGE,as well as Western blot analysis for the detection of theproduction of the CBHI (Li et al. 2005) and XynA (Li et al.1997) polypeptides. To extract RNA samples, myceliumwas collected by centrifugation, rinsed with MilliQ water,frozen under liquid nitrogen, and disrupted using the BeatMill MM 301 (Retsch, Newtown, PA). Total RNA wasextracted using the Qiagen RNeasy Plant kit. Northernanalysis was done as described (Li et al. 1997), and anothergel with identical loading as the one for Northern analysiswas stained with ethidium bromide.

Fermentation and enzyme purification Conidial spores(1.0×107) were inoculated into 40-ml PD broth in a 250-mlErlenmeyer flask that was shaken at 250 rpm and 28°C for3 days. The fungal mycelia (200 ml) were then transferredto a 2.8-l baffled Erlenmeyer flask containing 1.0-l PD

* * * * * * * * * * * * * * * *XynA GGT CAA AGA TTA AGC GTT GGT GGT GGT CAA AAC CAA CAT AAA GGT GTT TTT GAT GGC TTC AGT TAT GAA 240OxynA GGC CAG AGG TTG AGC GTC GGT GGT GGC CAG AAC CAG CAC AAG GGC GTC TTC GAT GGC TTC AGC TAC GAG AA Gly Gln Arg Leu Ser Val Gly Gly Gly Gln Asn Gln His Lys Gly Val Phe Asp Gly Phe Ser Tyr Glu 51

*******XynA ATC TGG TTA GAT AAC ACC GGT GGT AGT GGT TCC ATG ACC CTT GGT AAA GGA GCT ACC TTC AAG GCT GAA 309OxynA ATC TGG CTG GAT AAC ACC GGC GGT AGC GGT TCC ATG ACC CTG GGT AAG GGA GCT ACC TTC AAG GCT GAGAA Ile Trp Leu Asp Asn Thr Gly Gly Ser Gly Ser Met Thr Leu Gly Lys Gly Ala Thr Phe Lys Ala Glu 74

* * * * * * * * * * XynA TGG AGT GCA GCT GTT AAC CGT GGT AAC TTC CTT GCC CGT CGT GGT CTT GAT TTC GGT TCT ACC AAA AAG 378OxynA TGG AGC GCA GCT GTC AAC CGC GGT AAC TTC CTG GCC CGC CGC GGT CTG GAT TTC GGC TCC ACC AAG AAGAA Trp Asn Ala Ala Val Asn Arg Gly Asn Phe Leu Ala Arg Arg Gly Leu Asp Phe Gly Ser Thr Lys Lys 97

* * * * * * * * * * XynA GCA ACC GCT TAC GAA TAC ATC GGA TTG GAT TAT GAA GCA AGT TAC AGA CAA ACT GCC AGC GCA AGT GGT 447OxynA GCC ACC GCT TAC GAG TAC ATC GGC CTG GAT TAC GAG GCA AGC TAC AGG CAG ACT GCC AGC GCA AGC GGTAA Ala Thr Asp Tyr Glu Tyr Ile Gly Leu Asp Tyr Glu Ala Ser Tyr Lys Gln Thr Ala Ser Ala Ser Gly 120

** * * * * * * * * * * * * * XynA AAC TCC CGT CTT TGT GTA TAC GGC TGG TTC CAA AAC CGT GGA GTT CAA GGC GTA CCT TTG GTA GAA TAC 516OxynA AAC AGC CGC CTC TGC GTC TAC GGC TGG TTC CAG AAC CGC GGA GTG CAG GGC GTC CCC CTG GTC GAG TACAA Asn Ser Arg Leu Cys Val Tyr Gly Trp Phe Gln Asn Arg Gly Val Gln Gly Val Pro Leu Val Glu Tyr 143

* * * * * * * * * * * *XynA TAC ATC ATT GAA GAT TGG GTT GAT TGG GTA CCA GAT GCA CAA GGA AAA ATG GTA ACC ATC GAT GGT GCA 585OxynA TAC ATC ATC GAG GAT TGG GTC GAC TGG GTC CCG GAT GCG CAG GGA AAG ATG GTC ACC ATC GAT GGC GCCAA Tyr Ile Ile Glu Asp Trp Val Asp Trp Val Pro Asp Ala Gln Gly Lys Met Val Thr Ile Asp Gly Ala 166

* * * * * * * * * * * * * XynA CAA TAT AAG ATT TTC CAA ATG GAT CAC ACT GGT CCA ACT ATC AAT GGT GGT AAT GAA ACC TTT AAG CAA 654OxynA CAG TAC AAG ATC TTC CAG ATG GAT CAC ACT GGC CCG ACC ATC AAC GGC GGT AAC GAG ACC TTC AAG CAGAA Gln Tyr Lys Ile Phe Gln Met Asp His Thr Gly Pro Thr Ile Asn Gly Gly Asn Glu Thr Phe Lys Gln 189

* * * * * * *** * * * * * *** * * XynA TAC TTC AGT GTC CGT CAA CAA AAG AGA ACT TCT GGT CAT ATT ACT GTA TCA GAT CAC TTT AAG GCA TGG 723OxynA TAC TTC AGC GTC CGC CAG CAG AAG CGC ACT AGC GGC CAC ATC ACC GTC AGC GAT CAC TTC AAG GCG TGGAA Tyr Phe Ser Val Arg Gln Gln Lys Arg Thr Ser Gly His Ile Thr Val Ser Asp His Phe Lys Ala Trp 212

* * * * * * * * * * * * * XynA GCC AAT CAA GGT TGG GGT ATT GGA AAC CTC TAT GAA GTT GCA TTG AAC GCA GAA GGT TGG CAA AGT AGT 792OxynA GCC AAC CAG GGC TGG GGC ATC GGC AAC CTC TAC GAG GTC GCA CTG AAC GCA GAG GGT TGG CAG AGC AGTAA Ala Asn Gln Gly Trp Gly Ile Gly Asn Leu Tyr Glu Val Ala Leu Asn Ala Glu Gly Trp Gln Ser Ser 235

* * * * * * *XynA GGT GTC GCT GAC GTC ACC AAG TTG GAT GTC TAC ACC ACC AAA CAA GGT TCT GCT CCT CGT 852OxynA GGT GTC GCC GAC GTC ACC AAG CTG GAT GTC TAC ACC ACC AAG CAG GGT TCG GCC CCT CGCAA Gly Val Ala Asp Val Thr Lys Leu Asp Val Tyr Thr Thr Lys Gln Gly Ser Ser Pro Arg 255

Fig. 2 Nucleotide and amino acid sequences of the OrpinomycesxynA catalytic domain. Original (xynA) and optimized (OxynA)sequences are shown. The altered nucleotides are by the asterisksymbols on top. Amino acid sequence is shown as AA and the regions

underlined were identified by LC/MS/MS after the purified 28-kDaxylanase was subjected to in-gel trypsin digestion and peptideextraction. Numbering of nucleotides and amino acids is shown onthe right and is consistent with that reported (Li et al. 1997)


broth. The flask was shaken under the same conditions for2 days, and the fungal mycelia were used to inoculate a5.0-l bench-top bioreactor controlled by a Biostat B Unit(B. Braun Biotech International, Melsungen, Germany).Bioreactor medium (3.5-l working volume) contained (perliter) 4.0 g KH2PO4, 4.0 g (NH4)2SO4, 1.0 g MgSO4·7H2O,20.0 g lactose, 20.0 g corn steep liquor, 1.0 ml Tween 80,and 1.0 ml trace mineral solution. Temperature wascontrolled at 28°C and pH was held at 4.8 with 4 M NH4OHand 4 N H3PO4. Airflow was set at 3.5 l/min. Dissolved O2

was maintained between 20 and 95% saturation by varyingthe agitation rate between 300 and 1050 rpm. Cultivationwas terminated after 168 h, and fungal mycelia and othersolids were removed by centrifugation (6000×g, 30 min,4.0°C) using a Sorvall RC5 centrifuge (Thermo Electron,Asheville, NC).

Ammonium sulfate crystals were added to the clarifiedculture supernatant (1.0 l) to 65% saturation. The suspen-sion was left at 4°C for 4 h and then centrifuged for 30 minat 11,000×g. Supernatant after centrifugation was dis-carded, and the pellet was dissolved in 20 mM sodiumphosphate buffer (SPB), pH 6.5. Saturated ammoniumsulfate solution was slowly added to the supernatant to30% saturation while stirring. Precipitated proteins wereremoved by centrifugation as described above, and the clearsupernatant was loaded onto a Phenyl Sepharose column(5×15 cm). The column was controlled using an AKTA-basic System (GE Life Science USA, Piscataway, NJ).Starting buffer was 30% saturated ammonium sulfate in20 mM SPB, pH 6.5, whereas the elution buffer was20 mM SPB. Flow rate was set at 5.0 ml/min, and fractionsof 5.0 ml each were collected. The presence of the specificxylanase was detected by Western blot (see above).Fractions containing the xylanase were pooled and concen-trated using Centricon Plus tubes (Millipore, Bellerica,MA). The 40-kDa fusion xylanase was further purified byanion exchange with a MonoQ HR 16/10 (GE Biotech)using 50 mM Tris/HCl, pH 7.5, for binding, and proteinswere eluted by a gradient of 0–0.5 M NaCl in 50 mM Tris/HCl, pH 7.5. Flow rate was 3.0 ml/min, and fractions of1.5 ml each were collected. The 28-kDa xylanase waspurified further with gel filtration using a prepackedSuperdex 75 column (GE Biotech) with a mobile phase of50 mM Tris/HCl, pH 7.5. Fractions containing the xylanasewere pooled and concentrated using a Centricon Plus tube.Purified proteins were quantified using extinction coeffi-cients of 75,120 and 60,940 at 280 nm for the 40- and 28-kDa xylanase forms, respectively, and stored at −80°C.

Enzyme assay Xylanase assays were performed in 250-μlreaction volumes containing 200 μl 1.0% (w/v) oat speltxylan (Sigma) and 50 μl of appropriately diluted enzymepreparations, both in 50 mM SPB, pH 6.5. After the

substrate was equilibrated to 50°C for 5 min in a waterbath, the reactions were initiated by the addition of thediluted enzyme sample, and the incubation continued for15 min. Reactions were terminated by the addition of1.0 ml dinitrosalicylic acid reagent (Miller 1959). The testtubes were then boiled for 5.0 min and then chilled in icywater for 10 min. The absorbance values at 540 nm of 200-μl samples were measured on a Benchmark Plus micro-titerplate spectrophotometer (Bio-Rad). D-Xylose (Sigma) wasused as the standard for the assays of the reducing sugar.Other polysaccharide substrates were used under similarconditions except that mixing was applied when insolublesubstrates were used and that glucose was used as thestandard when using cellulose or mannan substrates. Oneunit of enzyme activity is defined as the amount of enzymerequired to generate one micromole of xylose or glucoseequivalent per minute. kcat Values were determined based onprotein molar concentrations for the purified proteinscalculated from the extinction coefficient values and theOD280 readings of the homogenous polypeptides.

Proteomic analysis of enzyme samples Amino acid se-quencing was done with protein samples obtained fromSDS-PAGE gels. The proteins were transferred ontopolyvinylidene difluoride membranes in the CriterionSystem (Bio-Rad). Transferred proteins were visualizedby amido black staining (Sigma) and excised with a razorblade. N-terminal amino acid sequencing was performed atthe Wistar Institute Proteomics Facility (WIPF, Philadel-phia, PA) on an Applied Biosystems model 477A gas-phasesequencer equipped with an automatic online phenyl-thiohydantoin analyzer. For obtaining mass data of theprotein sequence, proteins after SDS-PAGE were visual-ized by Coomassie Brilliant Blue staining, and proteinbands were excised from the gel. The gel pieces weresmashed and subjected to trypsin digestion. The extractedpeptides were analyzed at WIPF using microcapillaryreverse phase high-performance liquid chromatographynano-spray tandem mass spectrometry on a ThermoFinni-gan LTQ quadrupole ion trap mass spectrometer.

Results

Construction of a H. jecorina cel7A replacement vector TheH. jecorina cel7A chromosomal sequences of 5–20 kbwere cloned from a genomic DNA library. Five purified 1

clones were obtained after screening 5.0×104 plaqueforming units. Restriction digestion with EcoRI andSouthern blot analysis revealed that they all possessed a3.8-kb band. Sub-cloning of a 9.0-kb region (2.5 kbupstream and 4.0 kb downstream) of the cel7A locus was


achieved. Comparison between the cel7A reported nucleo-tide sequence (Shoemaker et al. 1983) and thecorresponding region of the 9.0-kb region revealed thatthey were identical except that a cytidine, 6 nucleotidesupstream of the cel7A ORF, was missing from the reportedsequence. Besides two internal EcoRI sites, there are twoadditional EcoRI sites, one 2,207 bp upstream and the other3,219 bp downstream of the ORF. The expression vectornamed pT3C consists of 2.2 kb 5′, 0.7-kb terminator, and2.8 kb 3′ regions of cel7A (Fig. 1). A 0.3-kb cel5A regioncoding for the EGII (formerly EGIII, Saloheimo et al. 1988)signal peptide, CBM, and linker sequences was inserted,followed by SpeI and PstI sites that were used to facilitatethe cloning of genes of interest into the vector (Fig. 1).

Evidence that low GC content genes are not compatiblewith high level protein production by H. jecorina The CD-coding sequence of the Orpinomyces xynAwas amplified andinserted into pT3C, resulting in pOX (Fig. 1). Eighty-ninetransformants were obtained and purified after transforma-tion with EcoRI linearized plasmid (Table 2). Western andSouthern analyses revealed that less than 10% of thetransformants (8 out of 89) were devoid of CBHI production(Table 2), and about one-third of these CBHI negativemutants were single copy gene replacement mutants througha double crossover event at the cel7A locus (data not shown).Xylanase activity assay and Western blot analysis for theculture samples collected at days 3 and 5 failed to detecteither the XynA band with the specific antibodies onWestern blot analysis or significantly elevated xylanaseactivity when compared to the host strain. Analysis of thexynA coding sequence revealed that the GC content of44.4% is similar to those of the terminator sequences butmuch lower than those (above 52.0%) of the coding regionsof some well-expressed H. jecorina genes of extracellularenzymes (Shoemaker et al. 1983; Penttila et al. 1987b; Chenet al. 1987; Saloheimo et al. 1994; and Torreonen et al.1992). Therefore, the plasmid pSOX (Fig. 1) with a syntheticxynA CD sequence of higher GC content (61.4%, Figs. 1, 2)replacing xynA was used for the transformation. Fifty-twopure transformants were obtained, and among them, threehad the cel7A locus replaced (Table 2). Again, no signifi-

cantly elevated xylanase activity was detected in culturesupernatants under cellulase induction conditions without pHcontrol. However, about one-third of the transformants (6 out18) produced XynA specific bands, but sizes of the bands(between 10 and 15 kDa) were smaller than that (32.0 kDa)calculated for the fusion protein (data not shown). Theintensity of the bands varied from barely visible to heavybands. It was noticed that the pH of the cultures dropped tobetween 3.5–3.8 after day 3 under the culturing conditions,and the low pH was shown to be a factor triggeringdegradation of extracellular proteins (Karlsson et al. 2001;Haakana et al. 2004). When pH was kept above 5.5 for theentire period of 5 days, xylanase activity of the untrans-formed control culture was 23.0 U ml−1, whereas elevatedxylanase activity levels (50.0–360 U ml−1) were obtained for24 of the 52 transformants at day 5. Western analysisrevealed that about one-third of transformants gave a broadband of about 40 kDa (Fig. 6), larger than that calculated(32 kDa) for the fusion protein. The intensity of the bandappeared to correlate well with the levels of the xylanaseactivity among the transformants. The size increase is likelydue to glycosylation, as shown previously for proteinsproduced and secreted by the fungus (Saloheimo et al.1988; Eriksson et al. 2004).

Low level of transcript was responsible for the lack of theAT-rich xynA expression To further assess why the originalxynA sequence was not expressed in H. jecorina, singlecel7A replacement transformant OX-S3 and SOX-S5strains, obtained using pOX and pSOX, respectively, alongwith the original Rut-C 30 host strain were subjected toSouthern and Northern analyses. As shown in Fig. 3, thegenomic DNA samples after EcoRI digestion of both OX-S3 and SOX-S5 strains each had one copy of the xynAexpression cassette band (8.5 kb) that replaced cel7A locus,but not present in Rut-C 30. The amount of xynA transcriptat day 2 cellulase induction cultures was high for SOX-S5but undetectable for OX-S3 or Rut C-30 cultures (Fig. 3b),although similar levels of total RNA were loaded for eachof the three cultures (Fig. 3c). Thus, these data indicate thatthe lack of xylanase A production in the strain OX-S3 wasdue to low steady-state levels of xylanase-specific mRNA.

Carrier-polypeptide fusion aided the secretion of heterologousproteins by H. jecorina To examine the effect of the carrierpolypeptide, the cel5A CBM and linker region wasremoved, resulting in the construction of pSOXS (Fig. 1).Using this plasmid, a total of 67 and 6 cel7A replacementtransformants were obtained (Table 2). Regardless whethercel7A was deleted, no transformant grown under thecellulase induction conditions produced higher levels ofxylanase activity in comparison to the host. Furthermore,Western blot analysis using the XynA-specific antibodies

Table 2 Analysis of transformants with anti-CBHI antibodies

Plasmid Total transformantspurified and analyzed

Cel7A presentmutants

Cel7A absentmutants

pOX 89 81 8pSOX 52 49 3pSOXS 67 61 6pKSOX 45 42 3


for the transformants failed to detect any bands for theculture supernatant samples. These results suggest that acarrier polypeptide is necessary for the transport of theheterologous xylanase through the secretory pathway.

Recognition of protease cleavage sites and secretion of amature xylanase Site-directed mutagenesis was used tointroduce a RDKR-coding insert into pSOX between thecel5A linker and XynA CD sequence, resulting in con-struction of pKSOX (Fig. 1). Forty-five stable transform-ants were obtained, of which three were cel7A replacementmutants (Table 2). Ten of the random integration and allthree cel7A replacement transformants produced between40–350 U ml−1 xylanase after 5 days of growth in shake-flask cultures. SDS-PAGE and Western blot analysesrevealed that the cultures produced a band with molecularmass of 28 kDa that was absent in the control cultures andrecognized by the XynA CD-specific antibodies. None ofthe cultures produced the 40-kDa band when analyzed byWestern. Moreover, the levels of xylanase activity in theculture supernatants appeared to correlate with the intensityof the 28-kDa band.

Production of the heterologous xylanase by H. jecorina infermenter Batch fermentations were carried out by bio-reactors using H. jecorina Rut C-30 and the pSOX andpKSOX single gene replacement strains SOX-S5 andKSOX-S1. Xylanase activity was detected after 1 day ofgrowth, and it rapidly increased between days 2 and 4(Fig. 4). Between days 5 and 6, both SOX-S5 and KSOX-S1 cultures reached the highest levels (865 and 605 U ml−1,respectively). Based upon a specific activity of 4,500 Umg−1 for the homogenous xylanase (unpublished data), thelevels of xylanase A production under this condition wasestimated to be around 150 mg l−1. The H. jecorina controlstrain produced substantial but much lower levels ofactivity than that of the transformants tested. The low

Time (Day)

Acti

vit

y (

U m

l-1)

0

200

400

600

800

1000

0 42 6

Fig. 4 Production of extracellular xylanase activity by H. jecorinaRut C-30 (filled circle), its Orpinomyces xylanase A single genereplacement transformants SOX-S5 (open circle), and KSOX-S1(inverted triangle) growing under cellulase-induction conditions in5-l fermenters. Values were means of three replicated activitymeasurements of one single culture

4.0

3.0

2.5

2.0

1.5

4.0

3.0

2.5

2.0

1.5

1.0

23

9.4

6.5

4.3

2.3

kb

a

b

c

1 2 3

Fig. 3 Southern and Northern blot analyses of the single genereplacement transformants of pOX and pSOX using the originalxylanase A CD-coding sequence as a hybridization probe labeled withdigoxigenin. Purified genomic DNA was analyzed with Southern blotusing a xynA hybridization probe (a). Total RNA were analyzed withNorthern blot using the xynA probe (b) and stained with ethidiumbromide (c). Lanes 1, 2, and 3 were loaded with DNA and RNAsamples of H. jecorina Rut C-30, and OX-S3 (pOX), and SOX-S5(pSOX), respectively


levels of xylanase activity could be due to the presence ofmany endogenous xylanase genes (Torronen et al. 1992;Ogasawara et al. 2006). Western blot analysis revealed thatSOX-S5 produced a broad band of 40 kDa, detectable asearly as day 2. At later stage, lower molecular mass bands,possibly degradative products of the fusion protein, werealso visible (data not shown). The KSOX-S1 cultureproduced a 28-kDa band detected after day 3, and again,minor low molecular mass bands were also found at laterstage (Fig. 5). No band larger than 28 kDa was obtained,suggesting that the protease cleavage at the RDKR siteduring the secretion was efficient.

Purification and characterization of the 40-kDa and 28-kDa xylanase forms Of the 40- and 28-kDa xylanase forms,69 and 52 mg were purified from 1 l of fermentation brothfrom 7-day-old fermenter cultures of SOX-S5 and KSOX-S1, respectively. Both were homogenous as analyzed onSDS-PAGE (Fig. 6a) and recognized by the XynA CDantibodies (Fig. 6b). Both the xylanase forms were able to

hydrolyze arabinoxylan extracted from birch wood, wheat,and oat spelt with apparent kcat values in the range of1,250–1,700 s−1 at 50°C (Table 3). Most interestingly, theseproteins had high levels of the activity against insolublewheat xylan. However, neither the 40-kDa nor the 28-kDaenzyme hydrolyzed the corn fiber arabinoxylan to adetectable level. As anticipated, they did not hydrolyze β-glucan, carboxymethyl cellulose, galactomannan, pNP-β-glucopyranoside, or pNP-β-xyloside (Table 3).

The 28-kDa xylanase was subjected to amino-terminalsequencing, and its first ten amino acid residues weredetermined. The amino acid residue signals clearly revealedthat two sequences, DKRGQRLSVG and GQRLSVGGGQ,were present in approximately 60 and 40%, respectively, inthe xylanase sample based on the amino acid peak heights(Fig. 7). These data suggest that both the first monobasicresidue R and the dibasic site KR in RDKR wererecognized by proteases during secretion. The 40-kDa formof the xylanase was not sequenced because the secretedEGII is blocked by a pyroglutamyl residue as the N-terminal residue (Saloheimo et al. 1988). The 28-kDaxylanase band in SDS-PAGE gel was also digested withtrypsin, and the digested peptides were subjected to liquidchromatography/mass spectrometry/mass spectrometry(LC/MS/MS) analysis. Eight mass signals matched themolecular masses of the regions found within the XynAamino acid sequence (Fig. 2), further suggesting that thepurified enzyme was encoded by xynA and that other

M MkDa

50

75

25

20

15

10

100

150

37

1

a

21 2

b

Fig. 6 SDS-PAGE (a) and Western blot (b) analyses of the 40-kDaand the 28-kDa forms of the Orpinomyces xylanase A purified fromH. jecorina SOX-S5 (lane 1) and KSOX (lane 2) culture supernatants.Of the purified enzymes, 4 and 0.2 μg were loaded for the analyses ofSDS-PAGE and Western blot, respectively. Lane M, Precision PlusProtein Standards (Bio-Rad)kDa

50

75

25

37

20

15

10

M 6 7543210

Fig. 5 Western blot analysis of culture supernatant samples of theOrpinomyces xynA single gene replacement H. jecorina transformantKSOX-S1 grown under cellulase-induction conditions in a 5-l fermen-ter. Lanes 0 to 7 of the gels were loaded with days 0 to 7 supernatantsamples, respectively. Lane M, Bio-Rad Precision Plus ProteinStandards


regions of the XynA might be glycosylated, as suggestedby the mass discrepancy between that calculated and thatdetermined by SDS-PAGE.

Discussion

The Orpinomyces PC-2 XynA CD was produced asfunctionally active xylanases in fusion and fully processedforms by the industrially important fungus H. jecorina. Theheterologously produced enzymes are secreted into theculture broth. To our knowledge, this work presents the firstsuccessful production of hydrolytic enzymes from anaerobicfungi by an aerobic filamentous fungus. One of the well-documented differences between genes of aerobic andanaerobic fungi is that the genes of the latter are unusuallyAT-rich (Brownlee 1989; Nicholson et al. 2005). Anotherdifference is that most hydrolytic enzymes of anaerobicfungi have higher-specific activities when compared withtheir aerobic counterparts (Gilbert et al. 1992; Chen et al.1997). Although they produce high-specific activity

enzymes, anaerobic fungi will unlikely serve as productionhosts for industrial enzyme manufacturing because of theirslow and strictly anaerobic growth requirement as well astheir low yields of protein secretion. Alternatively, geneticengineering to introduce the hydrolytic enzyme-codinggenes into more robust industrial fungi may overcome thischallenge. H. jecorina has been used for decades to produceenzymes marketed to food, feed, textile, detergent, and pulpand paper industries and has shown great promise for theproduction of enzymes for the cost-effective conversion oflignocellulosic biomass to fermentable sugars to produceliquid fuels and bio-based products (Gray et al. 2006).Lignocellulosic materials contain various percentages ofcellulose, hemicelluloses (usually xylan), and lignin.Depending on the pretreatment methods used (Wyman etal. 2005), hemicelluloses are left intact or just partiallyhydrolyzed. Therefore, there is a need for enzymatichydrolysis of hemicellulose polysaccharides along with thehydrolysis of cellulose by enzymes. As demonstrated in thisstudy, the enzyme preparations of the Orpinomyces PC-2XynA are capable of hydrolyzing several forms of arabinox-ylan. These preparations may prove suitable for the biomassconversion where lignocellulose is pretreated under mildconditions (Wyman et al. 2005). Such pretreatment technol-ogies have been viewed favorably because loss of sugars tothe formation of furfural and hydroxymethylfurfural isminimal. In addition to sugar loss, these compounds cancause severe inhibition to fermentation microbes.

H. jecorina has been shown to produce extracellularenzymes in high yields. Previous research has demonstratedthat homologous proteins can be overexpressed andsecreted when fused downstream of the cel7A promoter(Paloheimo and Suominen 1998). For heterologous pro-teins, the high yields are not always a certainty, and reasonsfor that have been found to be related to problems duringposttranslational processing of secreted proteins, e.g.unfolded protein response (UPR; Saloheimo et al. 2003)and low levels of transcription (Paloheimo et al. 2003). Tominimize UPR and other factors that destroy the unfoldedheterologous proteins, a carrier polypeptide sequence isusually fused in front of the gene of interest (Kiiskinen etal. 2004; Paloheimo et al. 2003). For the H. jecorinasystem, whole proteins, such as hydrophobin (Kiiskinen etal. 2004), mannanase (Paloheimo et al. 2003), as well asother small domains (Paloheimo et al. 2003), have servedas carrier polypeptides. In this work, the region coding forthe H. jecorina cel5A CBM and linker polypeptideapparently worked effectively in this regard. However,secreted levels of both the fusion (40 kDa) and fullyprocessed (28 kDa) forms of the xylanase by the singlecopy cel7A replacement transformants were about 200 mgl−1 and accounted only about 10% of the total secretedenzymes, still much lower than the CBHI level (about 50%)

SpeI

...CCACCCACTAGTTCTCGCGACAAGCGGGGCCAGAGGTTGAGCGTCGGTGGTGGCCAGAAC...

... P P T S S R D K R G Q R L S V G G G Q N ...

60% D K R G Q R L S V G...

40% G Q R L S V G G G Q...

Cel5A Linker

Fig. 7 Partial nucleotide sequence of pKSOX with its coded aminoacid sequence and the amino terminal sequences of the 28-kDaxylanase. The signal strength of each sequence expressed inpercentage was calculated based on those of the average for all theresidues obtained during the Edman sequencing

Table 3 Substrate specificity of the two forms of the Orpinomycesxylanase A produced by and purified from H. jecorina cultures

Substratea Activity (kcat, s−1)

40 kDa 28 kDa

Corn fiber arabinoxylan NDb NDWheat soluble xylan, high viscosity 1,420 1,260Wheat soluble xylan, low viscosity 1,350 1,550Wheat insoluble xylan 1,620 1,710Oat spelt xylan 1,540 1,490Birch wood xylan 1,590 1,620β-1,3-1,4-Glucan (barley) ND NDCarboxymethyl cellulose, low viscosity ND NDGalactomannan ND NDp-Nitrophenyl β-xylosidase ND NDp-Nitrophenyl β-gluconopyranoside ND ND

aActivities were measured using reactions containing 0.8% (w/v)substrate at 50°C and pH 6.5

b ND refers to activity less than 1.0 s−1


when the host strain was used. The lower yields could bedue to lower transcript levels, activation of UPR, incom-pleteness of secretion, and/or proteolysis in the culturemedia. Proteolysis in the pH-uncontrolled, and evencontrolled, cultures was evident (Fig. 5). In work withAspergillus oryzae (Oda et al. 2006), trapping of an α-amylase within cell walls in submerged cultures wasshown. The fully processed form of the Orpinomycesxylanase seemed more vulnerable to proteolysis, as thefusion construct transformants achieved higher levels ofxylanase activity during the late stage of the fermentation.To further improve the yield of the fully processedOrpinomyces xylanase by H. jecorina, future strategiesmay include (1) developing transformants with increasedcopy number of the expression cassette (Paloheimo et al.2003), (2) mutagenesis and screening for mutant trans-formants with improved secretion and decreased proteoly-sis, (3) establishing a fed-batch fermentation scheme, and(4) deletion of other cellulase-coding genes in the genome(Paloheimo and Suominen 1998).

The most interesting observation was that no XynA proteinwas synthesized until the original AT-rich gene was enrichedfor a higher GC content (61.4%). The lack of expression withthe original sequence could be due to low steady-state levelsof the specific mRNA, lack of efficient translation due toinsufficient levels of high AT codon tRNAs, or both.Comparison of xynA-specific mRNA by Northern analysisclearly demonstrates that the lack of stable xynA transcriptmay serve as the determining and/or as the first obstacle. TheNorthern data are in general agreement with the comparisonof GC content between the ORF and terminator regions ofthe genes coding for some H. jecorina extracellular enzymes.We do not know what GC content is ideal for geneexpression in H. jecorina. Furthermore, this work could notrule out the possibility that insufficient tRNAs to certaincodons may also be an important factor, as replacing 20codons of a bacterial xylanase gene with H. jecorinapreferred codons improved the expression and productionof a heterologous xylanase (Te’o et al. 2000).

Amino terminal sequencing of the 28-kDa xylanaserevealed that the monobasic R and the dibasic KR sitesinserted as RDKR at the junction of fusion between Cel5Aand XynA CD were processed by endopeptidases (Goller etal. 1998). We used the residues for producing the fullyprocessed xylanase because the residues were previouslyfound to support the highest secreted level of a bacterialxylanase by the fungus (Paloheimo et al. 2003). It wasbelieved that either a monobasic or a dibasic proteaserecognition site supports the cleavage as long as there is anopen region such as a hinge adjacent to the cleavage sites.(Paloheimo et al. 2003). The fact that the fully processedxylanase is a mixture of two amino termini, which resultedfrom cleavage at the two sites, indicates that the two

systems work independently and that only one cleavageoccurs. What is not known is whether the adjacent sites aresynergistic or interfering during secretion of the heterolo-gous protein. Future studies comparing xylanase yields inculture supernatants and in mycelia between single cel7Areplacement strains including KSOX-S1 and transformantswith a monobasic R and dibasic KR at the location mayprovide answers to the above questions.

Both the 40- and 28-kDa forms of xylanases hydrolyzedxylans of birch wood, wheat, and oat spelt, but thehydrolysis of corn pericarp arabinoxylan was negligible.The specific activities on oat spelt xylan hydrolysis(1,500 s−1) were comparable to that (6,000 U/mg or2500 s−1) of a Neocallimastix patricarcium xylanase Aproduced by E. coli (Gilbert et al. 1992) but higher thanthose of other xylanases (Ratanakhanokchai et al. 1999;Saha 2001; Leskinen et al. 2005; Wu et al. 2006). The lackof hydrolysis against corn fiber arabinoxylan by either formlikely owes to the high percentage of side chains present onthe backbone (Saulnier et al. 1995) sterically hindering theaccess by GH family 11 xylanases, as was found to be thecase for two previously tested xylanases, one purifiedfrom Fusarium verticillioides (Saha 2001) and the otherfrom F. proliferatum (Saha 2002). The extremely highlevels of activity observed toward wheat insoluble xylanmight be a very attractive attribute for applying the enzymeto biomass conversion and animal feed applications.

In conclusion, several strategies were employed tosuccessfully express the highly AT-rich xylanase gene fromthe anaerobic fungus Orpinomyces PC-2 by H. jecorina.These strategies include increasing GC content for thewhole sequence, fusion to a carrier region, and engineeringprotease cleavage sites for the removal of the fusionpeptide during secretion. The work also demonstrated therecombinant Orpinomyces xylanase is able to hydrolyzexylans extracted from many sources and, thus, mighthave utility in biomass conversion and other industrialapplications.

Acknowledgment The authors thank Patrick M. Kane, Patricia J.O’Bryan, Kristina Glenzinski, Jay Braker, and Joy Danniel fortechnical support and WIPF for the N-terminal sequencing and LC/MS/MS analyses.

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Expression of an AT-rich xylanase gene from the anaerobic fungus Orpinomyces sp. strain PC2 in and...

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