Curr Genet (1995) 29:73-80

9 SpringerLVerlag 1995

S h o k o Yoshino 9 M e g u m i Oishi 9 Ryuichi M o r i y a m a Masashi Kato 9 N o r i h i r o Tsukagoshi

Two family G xylanase genes from Chaetomiumgracile and their expression in Aspergillus nidulans

Received: 12 April / 12 June 1995

With otigonucleotides based on the amino-terminal and internal amino-acid sequences of a xylanase, two xylanase genes, cgxA and cgxB, were isolated and sequenced from Chaetomium gracile wild and mutant strains. Each gene isolated from both strains was essentially the same as far as nucleotide sequences were compared. The mature CgXA and CgXB xylanases comprise 189 and 211 amino acids, respectively, and share 68.5% homology. The CgXA was found to be the major enzyme in the mutant strain. Comparison of these amino-acid sequences with xylanase sequences from other origins showed that they have a high degree of identity to the family G xylanases. The cgxA and cgxB genes were introduced into AspergilIus nidulans and found to be expressed with their own promoters. Abstract

Key w o r d s

Chaetomium gracile

9

Xylanase genes (cgxA and cgxB) 9Family G xylanases 9 Expression in A. niduIans

Introduction Xylan, the major hemicellulose component, is a [3-1,41inked polymer of xylose substituted with side chains of other pentoses, hexoses and uronic acid, depending on its botanical origin. Because of this heterogeneity of the sugar polymer, xylanolytic microorganisms produce a complex spectrum of different activities that are required to degrade xylan completely and which have different functions in this degradation process. The major component of the complex is formed by endo-l~-1,4-xylanases (1,4-[~-D-xylan-xylanohydroiase, E.C. 3.2.1.8), the so called xylanases (Biely 1985). Many xylanases have been characterized and a number of xylanase-encoding genes have been cloned. Multiple S. Yoshino 9M. Oishi 9R. Moriyama 9M. Kato. N. Tsukagoshi ([]) Department of Applied Biological Sciences, Faculty of Agriculture, Nagoya University Nagoya 464-01, Japan Communicated by K. Esser

xylanases could be degradation products of a single gene product or else be encoded by specific genes (Wong et al. 1988). That multiple xylanases from the same organism are products from different genes has been reported for eukaryotic microorganisms as well as prokaryotes such as Trichodema reesei (Torronen et al. 1992 ),Aspergillus kawachii (Ito et al. 1992 a,b), Streptomyces lividans (Shareck et al. 1991) and Pseudomonasfluorescens (Gilbert et al. 1988). After extensive screening of xylanolytic fungi, Chaetomium gracile IFO6568, a plant pathogen, was found to secrete xylanases producing xylobiose as the major hydrolytic product. The C. gracile xylanase showed a strong preference for internal linkages and hydrolyzed more than 90% of birchwood xylan under optimal conditions, where xylobiose comprised approximately 80% of the hydrolyzate. C. gracile IFO 6568, however, produced a rather small amount of the enzyme. A mutant strain 1161, whose enzyme productivity increased 115-fold, was isolated after mutagenesis by 6~ y-ray irradiation. The relevant data will be published elsewhere. As a first step to characterize the molecular properties of the C. graciIe xylanases, and to elucidate the xylan-mediating induction mechanisms for gene expression, we have determined the amino-terminal and internal aminoacid sequences of xylanases of the mutant strain, which are responsible for most of the xylanolytic activity formed during growth on xylan. Based on these amino-acid sequences, two xylanase-encoding genes, designated as cgxA and cgxB, were cloned from both wild and mutant strains and sequenced. Comparison with amino-acid sequences from other xylanases revealed that they are members of the family G xylanases. Furthermore, these two genes have been found to be expressed with their own promoters in Asper-

gillus nidulans.

Materials and methods

Strains, plasmids, media and transformation. C. gracile IFO6568 and mutant 1161 were used for isolation of DNA and grown at 30~

74 in Cg medium consisting of 2.8% corn starch, 0.9% polypeptone, 0.075% MgSO 4 97H20, 0.025% KC1, 0.1% CaC12 - 2H20 and 0.05% KH2PO4. A. nidulans GI91 (pyr4, pabaA1, fwA1, uaY9: a generous gift from Dr. G. Turner), a uridine-requiring recipient for transformation, was grown at 30~ in standard A. nidulans media as described by Nagata et al. (1993). Transformants carrying C. gracile genes were grown at 30~ in MM medium (Rowlands and Turner 1973) supplemented with 2 gg/ml of PABA and 1% birchwood xylan (Sigma Chemical Co.). The following strains of Escherichia coIi were used: XLl-blue for DNA manipulation and growth of M13 phages, and MV 1184 for growth of )~ gt 10; pUC plasmids were used to subclone various restriction fragments. Strains of plasmid-carrying E. coli were grown at 37~ in LB medium containing 50 gg/ml of ampicillin. Plasmid pDJB 1, which contains the Neurospora crassa pyr4 gene, was kindly provided by Dr. G. Turner and used for transformation ofA. nidulans. A. nidulans protoplasts were prepared from mycelial cells using Novozyme 234 (NOVO) and transformed as described by Ballance and Turner (1985) with a slight modifications where 0.6 M KC1 was replaced by 0.8 M NaC1. Transformation of E. coli was performed by the method of Lederberg and Cohen (1974).

Construction and screening of genomic DNA libraries. C. gracile IFO6568 and mutant 1161 were used as DNA-donor strains to construct genomic DNA libraries. To produce restriction maps of xylanase genomic genes, Southern-blot analysis was performed with the PCR-amplified xylanase DNA fragments, XA andXB, described below. For the cloning of the cgxA gene, the 1.5-kb PstI-HindIII fragment hybridized to the XA probe was recovered from agarose gels and inserted between the PstI and HindIII sites on pUC 118, followed by selection with the 32p-labelled XA DNA fragment as a probe. For the cloning of the cgxB gene, the 4.4-kb EcoRI fragment hybridized to the XB probe was recovered from agarose gels and cloned into L gtl0 vector, followed by screening with the 32P-labelled XB DNA fragment as a probe. Construction of the expression plasmids for the cgxA and cgxB genes. To determine whether the cgxA and cgxB genes could be expressed in A.nidulans, plasmids, pCgXA and pCgXB, were constructed using pDJB 1 as a vector. For the construction of pCgXA the approximately 3.5-kb PstI cgxA DNA fragment shown in Fig. 2 was subcloned to the PstI site on pUC118, followed by digestion with PvuII which generated an approximately 3.8-kb DNA fragment containing the entire cgxA DNA. Then, EcoRI linker DNA was ligated to the PvulI DNA fragment, followed by digestion with EcoRI, which was inserted into the EcoRI site on pDJB 1. For the construction ofpCgXB the approximately 4.4-kb EcoRI cgxB DNA fragment shown in Fig. 2 was inserted into the EcoRI site on pDJB1. Amino-terminal and internal amino acid sequencing. Xylanase purified from C. gracile 1161 was kindly provided by the Shin Nihon Chemical Co., Ltd, Aichi, Japan, and used for amino-acid sequencing. Sodium dodecylsulphate-electrophoresis (SDS-PAGE) analysis of this preparation revealed the presence of three components with molecular masses of approximately 20 kDa, 12 kDa and 6 kDa, each of which was isolated by using preparative SDS-PAGE, especially suitable for the separation of low-molecular-weight proteins (Hussain et al. t980). Their amino-terminal amino-acid sequences were determined with a gas-phase sequence analyzer (ABI 477A-120A protein sequencer). The sample for sequence analysis was prepared by the method of Matsudaira (1987). The 20-kDa xylanase was digested with lysyl endopeptidase and the resulting peptides were separated by high-performance liquid chromatography (HPLC) on a reversed-phase column (Synchropac RP-8 column, Synchrom Inc.) with a linear gradient of 5-95% CH~CN in 0.05% CF3COOH. Two major peptides, designated P1 and P2, were subjected to amino-acid sequencing as described above. Amplification of xylanase genomic DNA sequences by PCR. Genomic DNA fragments encoding a portion of xylanase genes were amplified by the polymerase chain reaction with chromosomal DNA of C. gracile 1161 as a template. Two oligonucleotideprimers were syn-

thesized based on the amino-acid sequences; the sense primer [5'-GGCACGGGCACGAAT(C)AAT(C)GGCTAT(C)TT-3'] corresponding to the amino-terminal sequence (Gly-Thr-Gly-Thr-AsnAsn-Gly-Tyr-Phe) and the antisense primer [5'-CCCTCA(G)ATGG(C)A(T)CGGT(C)TGA(G)TT-3']to the amino-acid sequence of the P1 peptide (Asn-Gln-Pro-Ser-Ile-Glu-Gly).Oligonucleotides were extended with Vent DNA polymerase in 25 cycles of 1 min at 94~ for denaturation, 1 min at 55~ for annealing and 1 min at 75~ for extension. The amplified product of the approximately 460-bp fragment was purified on agarose gels, ligated to the SmaI site on pUC118 and used to transform E.coli to ampicillin resistance. The identity of PCR products was confirmed by sequencing. The amplified fragment comprised two different sequences, designated as XA and XB.

Other methods. Protein was determined by the standard Bio-Rad protein assay with bovine plasma gamma globulin as a standard. Xylanase activity was assayed by the hydrolysis of Remazol brilliantbluemodified xylan (RBB-xylan; 5.75 mg/ml, Sigma Chemical Co.) in 50 mM acetate buffer (pH 5.4) at 30~ The reaction was terminated by the addition of 2 vol of 96% ethanol. The increase in absorbance at 595 nm was used to express the xylanase activity as described by Biely et al. (1985). One arbitrary unit was defined as an increase of 0.01 per min per ml of culture filtrate. Immunoblot analysis of the products was performed as described by Towbin et al. (1979) after SDS-PAGE (Laemmli 1970). To visualize proteins cross-reactive with the antibody to the 20-kDa xylanase raised in a rabbit, anti-rabbit IgG conjugated with horseradish peroxidase and the chromophore 4-chloro- 1-naphtol were used. Nucleotide sequences were determined by the dideoxy chain-termination method of Sanger et al. (1977) with a DNA sequencer (A.L.F. DNA Sequencer, Pharmacia) after the subcloning of appropriate restriction fragments into either derivatives of bacteriophage M 13 or pUC plasmids. The sequences for the cgxA and cgxB genes have been assigned DDBJ accession numbers D49850 and D49851, respectively.

Results and discussion A m i n o - a c i d s e q u e n c i n g of xylanases and amplification of partial xylanase genes The xylanase purified from C. gracile 1161 comprised three polypeptides with approximate molecular masses of 20 kDa, 14 kDa and 6 kDa (Fig. 1A). The a m i n o - t e r m i n a l a m i n o - a c i d sequences of these polypeptides were exactly the same and f o u n d to be A l a - G l y - T h r - P r o - S e r - G l y - T h r G l y - T h r - A s n - A s n - G l y - T y r - P h e .This clearly indicates that three polypeptides are degradation products of a single gene product, probably the 20-kDa xylanase. Lysyl endopeptidase digestion of the 20-kDa xylanase, followed by HPLC, yielded two major peptides, P1 and P2 (Fig. 1B) ; their a m i n o - t e r m i n a l a m i n o - a c i d sequences were Val-AsnG l n - P r o - S e r - I l e - G l u - G l y - T h rfor the P1 peptide and AsnP r o - G l y - A l a - A l a - A r g - T h r - I l e - A s n - P h e - S e r - G l y - T h r for the P2 peptide. U p o n c o m p a r i s o n of these sequences to those of other xylanases, the a m i n o - t e r m i n a l a m i n o - a c i d sequence of xylanases and the a m i n o - a c i d sequences of the P1 and P2 peptides beared high h o m o l o g y to those of the family G xylanases and were the same as those of the T. reesei xylanase II from a m i n o - a c i d residues 6 to 14, 122 to 130 and 59 to 63, respectively (Torronen et al. 1992). Therefore, two oligonucleotide mixtures were designed based on the a m i n o - t e r m i n a l a m i n o - a c i d sequence from residues 6 to 14 and the P I peptide a m i n o - a c i d sequence.

75 Fig. 1A, B Biochemical characterization of the major xylanase of C. gracile. A SDSpolyacrylamide gel electrophoretic profiles of the xylanase isolated from a C. gracile mutant strain. The enzyme comprised three polypeptides of approximately 20, 12 and 6 kDa (lane 2), each of which was purified to homogeneity (lanes 3, 4 and 5). The gel was stained with Coomassie brilliant blue R-250. The amino-terminal amino-acid sequence determined chemically for each polypeptide is shown at the bottom. B separation of lysyl endopeptidase digests of the 20kDa enzyme. The digests were fractionated on a reversedphase HPLC column. P1 and P2 peptides were subjected to amino-acid sequencing. Their sequences are indicated at the bottom

PstI

B

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cgxB SphI SmaI

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I

5'

I

I

i

10

20

30

I ', ', ~ q 40 50 60 70 80 Retention Time(rain)

P h VNQPSIEGT P2:NPGAARTINFSGT

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EcoRI S~ ""

~0 30 ~ 20 r~ 10

I

3' I XB I f

F

lO00bp

Fig. 2 Restriction maps of the cgxA and cgxB genes. Exons and introns are represented by black and open boxes, respectively. The entire regions sequenced are underlined. XA and XB fragments amplified by PCR are also indicated

Approximately 460-bp fragments were amplified with chromosomal D N A of mutant 1161 as a template, cloned into E. coli and sequenced. Two D N A fragments, designated as XA and XB, differed by 26 bp in size and their sequences also differed significantly from each other as shown in Figs. 3 and 4. The XA fragment encoded a polypeptide composed of exactly the same amino-acid sequence determined chemically for P2, while the XB fragment encoded a polypeptide with only a partial similarity to the P2 sequence. This indicates that C. gracile contains two distinct xylanase genes, designated as cgxA and cgxB.

Isolation and nucleotide sequencing of xylanase genes To clone the cgxA and cgxB genes, their genomic restriction maps were constructed by Southern blotting with the

XA andXB fragments as probe DNAs (Fig. 2). A rather small 1.5-kb PstI-HindIII fragment which hybridized to the XA probe was cloned and sequenced. Then, the 5' noncoding region between the HindIII and PstI sites was further cloned and sequenced for 562 bp upstream of the HindIII site. Although the exact translation-initiation site has not been elucidated, the structural part of the cgxA gene appeared to be 731 bp long and was interrupted by a single intron of 74 bp (Fig. 3). The cgxA gene contained an open reading frame coding for 219 amino-acid residues. The amino-terminal amino-acid sequence determined chemically was found in the deduced amino-acid sequence starting at 31 and up to residue 45. Furthermore, the region consisting of the amino-terminal 30 amino-acid residues was highly hydrophobic. Therefore, the enzyme was synthesized as a precursor with a putative signal peptide of 30 amino-acid residues and the secretory precursor was processed at a specific cleavage site between the Arg and Ala residues, resulting in the formation of mature enzyme with a molecular weight of 20 161. The amino-acid sequences determined chemically for the internal peptides, P1 and P2, were found from amino-acid residues 152-160 and 82-94, respectively (Fig. 3). Therefore, the cgxA gene encodes the major xylanase in the mutant strain. A 4.4-kb EcoRI fragment, hybridized to the XB probe, was cloned by using a ~, gtl0 system, subcloned onto plasmid vectors and sequenced for 2140 bp from the EcoRI site to the SphI site (Figs. 2 and 4). The cgxB structural gene appeared to comprise 774 bp and was interrupted by a single intron of 51 bp. Only a partial amino-acid sequence from residues 7 to 13 of the amino-terminal sequence of xylanase was found in the deduced amino-acid sequence starting at 38 and up to residue 44. Assuming that the number of amino~acids of the CgXB signal pcptide could be the same as that of CgXA, the cleavage site should be between Arg30 and Gln31. This is consistent with several xylanases from the G family, since they are processed at Arg-X; for example, the xylanases of T. reesei (Torronen et al. 1992) and A. kawachii (Ito et al. 1992b). Therefore, the mature CgXB enzyme comprises 211 amino-acid residues with a molecular weight of 22 538. CgXB contained only a partial sequence for P2 peptide.The cgxB gene could

76 GCCGCTCGCTCTTTAC~--~-~ATGGAACAGTCGTCATGACCGCCAGGGATT~GCCAGTGACACTGGCAAGGAAACAATATACAGGAAACATGCCGTCTTGGGAGAGGCCTTGTG

- 703

TTGGCCATGTATTC-GA~ACTC,C T T C ~ C A G C G T T G C ~ - ~ T C ~ G A C ~ C CC~TTCTC-GTGGC-GTATACTCGATCGCTGATCTGTTCCCAGCTTCTAGJ~C~TCTC~-583 GCCAACGCAGGGTCAAGCGCCCAGTTGCGGCCAAGGCTGTCTCTACGCAT~CCCCGTCGATCACTTAAGGGCGGATATTTCAGGCTAAATACTGTATTTTCTTTCATTCGTGC

- 463

CCGACGAGGGAGGCAGCGCTTCCCCAGACCTGACTATGCGACCTCCTGCCGCAAGGCCCCTCGTCGGCTCTGGTTGACAAATCGCC~GC

- 343

C G A C T G C C G A T C C A T A G T G C C C C C G C C A A T C T C C C G G T A A T A G G T G G T G A C G G A C G G C T G T G C 9 • G C A T A G A T G C C G G C G G A A A G C T T G C A G T T C T C T G G C T A G G A C C C A C C C A A G A T A A C - 223 AGCGGGTGAGGCGGTGGGACAGCTCGAACGAAGCCAGGAACCTCCTGGTCCAAGTTAGAGCCGGGTGACGGACTTACGGAAAGGGCGTCGTTGATCTGGACGTTAAAGTGTACTTATACG +i

A~ACGATTCCCTCTC~GTTCCAGCCCT~TCCCTC~TCCTcAcC~C~C~TCACCACCGATCA~GTCTCCTCA~TGTCTA~CC~ACGC~C~T~TTTCCTTc~GGCC M

V

S

F

K

A

~TCCTCCTC~C~C~C~TG~CTT~GTTCC~CTTC~CGT~C~CA~TGAAc~G~TTGT~T~GC~A~C~CCAGC~AC~GGCA~G~C~CGGGTACTTCTATTCC L

L

L

G

A

A

G

A

L

A

F

P

F

N

V

T

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M

N

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G

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N

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- 103 18 6 138 46

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AN,te~ TTCTGGAC~AC~CGGCGGCACCGTC~CTACCAGAACGGCGCC~T~CTCCTACAGCGTCCAGT~CAG~CTGT~C~CTTTGTCG~C~GCTGGT~GT~T~C~T F W T D G G G T V N Y Q N G A G G S Y S V Q W Q N C G N F V G G K G TACCCCTGGATTCA~GAAAAGACCCCGAAGCTGACCAAACGTGACGACGCAGGAAT

258 81

W

CCCGGCGCGGCCCGCACCATCAACTTCTCCGGCACCTTCAGCCCGCAGGGCAACGGCT P G A A R T I N F S G T F S P Q G N G Y

378 102

P2 ACCTGGCCATCTACGGGTGGACCCAGAACCCGCTGGT CGAGTACTACATCGTTGAGTCGTTCGGCACCTACGACCCCTCGTCGCAGGCGTCCAAGTTCGGCACCATCCAGCAGGACGGCA L A I Y G W T Q N P L V [~ Y Y I V E S F G T Y D P S S Q A S K F G T I Q Q D G S

498 142

GCACCTACACCATCGCCAAGACTAC CCGCGTCAACCAACCGTCTATCGAGGGCACCAGTACGTTCGACCAGTTCTGGTC CGTCCGCCAGAACCACCGCAGCTCGGGTTCGGTCAACGTCG T Y T I A K T T R V N Q P S I E G T S T F D Q F W S V R Q N H R S S G S V N V A

618 182

N

P1 CCGCCCATTTCAACGCTTGGGCTCAGGCTGGCCTCAAGCTCGGATCGCACAACTACCAGATCGTTGCTACTGAGGGCTACCAGAGCAGCGGTTC TTCATCCATCACTGTGTCTTAAGTCG A

H

F

N

A

W

A

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A

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L

G

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H

N

Y

Q

I

V

A

T [E] G

Y

Q

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GCAGT TATGTGGGTTTT GAGT GTTGGGCCC T TT G C G G A G G A T C G C A A A C A A A ~ A G G T G G A A G G A ~ T G G A G G A G G G G T G G A G G A G G A G A T G C G A A T G T G A T

T

V

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738 219

TGAC TAAGTGT TGG

858

GCTTGCGCTAGTCTATGTTTTTGTCTGCGTTGCAGTCTTGCAGGCTTTCTGCACGTATATACTGTTTGCGCGTTGCGTTTTTGAATGTACCTCCGATTCCAGTTTTGAAGACCGGCTGTA

978

T T A A T T A C A A C C C C T T G C C T C C C G C C T G A T G C C T G A T T A A T C A A C C C C C A T C T G C C C A T G C A T G T C C A A G T C C T G T G G T C T T G G A G T T G G T G T T T T C C G C T G T A T G G A T A • C G G A G C C G G 1098 CCCGGCCATc43ACATCGTGCGCTAGAAGCCTGGTCGGAGACATTCGAGTCGGcGAAT~GGATGGAGC42GAA~CTAATTAACCCTGCAG

Fig. 3 Nucleotide and deduced amino-acid sequences of the cgxA gene. The putative translation initiation site and the stop codon are indicated by +1 and an asterisk, respectively. The sequence of the XA fragment amplified by PCR is underlined. The intron is shown by a dotted overline. Putative CREA-binding sites are boxed. The 15-, 13- and 9-residue amino-terminal sequences for the 20-kDa enzyme, and the P1 and P2 peptides, respectively are double underlined. The cleavage site of the signal sequence is marked by an upward arrow. Putative catalytic residues,Glu, are also indicated

encode a minor xylanase significantly different from the major one, CgXA. Two xytanase genomic genes were also isolated from the wild-type strain, using XA and XB fragments as probe DNAs, and sequenced. The wild-type cgxA gene revealed exactly the same sequence as that of the mutant in respect of the 1454 bp that were sequenced. The wild-type cgxB gene also had exactly the same coding sequence as that of the mutant, and they contained only one different nucleotide at -281 in the 2208 bp of the 5'-noncoding region that was sequenced (Fig. 4). The intron within the cgxA and cgxB genes resembles other filamentous fungal introns in both size and the conserved sequences. The intron and exonjunctions follow the 'GT-AG' rule. The amino-acid sequences of mature CgXA and CgXB revealed 68.5% homology. However, there was only low homology (26.7%) in their putative signal peptides. Most

1193

noteworthy, the C-terminal portion of CgXB was significantly longer than that of CgXA and contained two continuous repeats of five amino-acids (Ser-Thr- Gly-Gly-

Gly).

Comparison of the amino-acid sequences of the C. gracile xylanases with those of other xylanases The xylanases known so far have been classified by hydrophobic cluster analysis into two distinct groups; families F and G (Henrissat et al. 1989; Gilkes et al. 1991). The amino-acid sequences derived from the cgxA and cgxB genes were compared to the published sequences for other xylanases from fungal and bacterial origins. No clear homology was found at the levels of either DNA or aminoacid sequences with the family F xylanases. However, all of the xylanases showing significant similarities (60-70%) to the CgXA and CgXB have been grouped into the G family. This is a class of low-molecular-mass xylanases ranging from 20 to 25 kDa. An extensive multiple alignment of the amino-acid sequences of 17 members of family G has been provided by Torronen et al. (1993); therefore, an amino-acid sequence comparison has been made (Fig. 5) only for the C. gracile xylanases and those from other filamentous fungi such as Trichoderma viride (Ujiie et al. 1991), T. reesei (Torronen et al. 1992), A. kawachii (Ito et al. 1992b), Aspergitlus tubigensis (de Graaff et al. 1994)

77 GAATTCCCAGACCAGCTGCCAGTGTAAGGAATGGCGATCTTCCCCATATCTATACCGGGTAGTTAGTACGTCTCGGATC~GTACCCCCGTGAGAGTTGTAGAGCGTGGTCTTTGGCTTC

- 667

TTCGGCAGTGATC`GCCTCTGGCAGGCTCGAACCCCGTCCATCAAGGCTCGCTCAAGTAAGGCGTACTGAACTGTCTCCCATTCACCAGTCGGCCC~TCGAAAGTCCCCACGT

- 547

A A G C T G G T A A C A T G T A G G C T G C T G C C C A G T G A C T G C A C G G T G G T C - G T G T T G T G A C CT T C C G T C C C C T T C C A T C C C C C C T C C T T G G A A C A A A T T C T C G G T C G T C G A G A G C C G T G C T T C C T T

- 427

TGTT~CCAA~CGTC~AAGTCGTGGTGGCGCCAGCCCCAGCGCCTAAAGACGTTAGAACCGAGCTAGAAGGTTCCGAAGTGCTTGCCGTGCCAG~CCCTTGCCGTTGGGTTGCACCAG C GCCGAGTTTAGCCGGCAGTGTCGACTAGGGGGTGCTCGATCCCAGCTCCGAGGCTGACGGCACGC~TAACGCACCCGAGCTTC-GACTCAGGGGTAGCACGGGCAGTCCAGAAGGAG

- 307 - 187

G C T A A G A A T A C ~ T T C C C - O : & A A G G G A G A G G A A A C A A G G A T A T C A G CTC CC.~AGCCAGACGCGACT G C G A G C T T G C A G A G T G T G T A T A T A G C C A T C T T G G T C C T C C T C G T C G A G A G C T G C A C +I CCATC TCGCCAGTTGTCACTTGCT CACCCACCAGCAGACTTGCACAGCAGATCACCACTGCCCACCATGGTCAACTTCTCTTCCCTCTTCCTTGCTGCCTCGGCGGCCGTCGTGGCCGTC M V N F S S L F L A A S A A V V A V

-67

GC C G C T C C T G G C G A G C T G C C C G G C A T G C A C A A G C G C C A A A ~ G C T C A C C A G C A G C C A G A C T G G C A C C A A C A A C G G C T A C T A C T A C T C G T T C T G G A C T G A C G G C C A ~ A A C G T C C A G T A C A A P G E L P G M H K R Q T L T S S Q T G T N N G Y Y Y S F W T D G Q G N V Q Y

174 58

54 18

A ACCAACGAGC/2 T G G C ~ T N E A G G

CAGTACAGCGTGACGTGGTCC~AACGGCAATTGGGTCGGCGC/2AAG~TGGAACCCGGGCAGTGC O Y S V T W S G N G N W V G G K G W N P G S

A

TCGGTACGTCTCC TATTACTAGACACCGATCTTC R

~/~}GG(~} ~ . 8 ~ ( ~ C~ } C.~GGACCAT CAACTACACGGCC~CTACAAC CCC~CC4F~AACT CGTACCT C,-GCCGTCT A C ~ C T ~ A C G C ~ C T

I

N

Y

T

A

N

Y

N

P

N

G

N

S

Y

L

A

V

Y

G

W

T

R

N

294 88

CCGTT~TCGAGTATTATGTTGTT ~ G

414

P

L

I []

Y

Y

V

V

E

121

AACTTTGGCACGTACAACCCCTCGACGGGCGcCACCCGGCTC~AGCGTGACGACcGACGGGTCTTGcTACGAcATCTACCGCACGCAGCGCGTCAACCAGCCGTCGATCGA~TACC N F G T Y N P S T G A T R L G S V T T D G S C Y D I Y R T Q R V N Q P S I E G

T

534 161

P1 AGCAC CTTCTACCAATTCTGGTCGGTGCGCCAGAACAAG-CGCAGCGGCGGCAGcGTC~CATC~CGCCCACTTCAACGC~TGGGcCGCCGCCGGCCTGCA~T~A~C~C~CTAC S T F Y Q F W S V R Q N K R S G G S V N M A A H F N A W A A A G L Q L G T H

D

Y

654 201

CAGAT TGT CGCCACCGAGGGC TACTAC TCGAGCGGCTCTGCGACCGTCAACGT CGGCGCCTCGAGCGACGGCTCCACGGGCGGCGGTTCCAC CGGT~TCTACC~CGT~GT~ T [~ G Y Y S S G S A T V N V G A S S D G S T G G G S T G G G S T N V Q A I V

S

F

C

TGACCCTGATTCTCACCCCTAACGTTCTCCATGCTGACTGTCGTTCCAGTGCTCTGCCAAGTC~AATGCGGTGGCCA~AGTAAGTCTTTTCTTTTTCTCATTCCGCAAACAAGT

774 241 894

GAACGGAGGAGACTGACAGAAAACAGTGGACCC-GCCCGACCTGCTGCCAGTCCGGCTCGACCTGCCAGGCCTCCAACCAGTGGTACTCGCAGTGCCTGTAAGCGCGT~CA~TCGA~C

1014

T~TC`C~2GAAGCTCCCTTTGAGAAGTCCAATGACTAAGCC~CAGTGCTATTCAGTTGTTGGTGACAGCAGGAGTCTGCATTCAGGGGTCC~TAGCJ{GATTGGAC~GGTTC

1134

ATATACATACTTACCGTTTTGCATCCATGCCTGGTTGCAGTACAAATACTTGAATTGAACATTGAAGTTGTTCATCTCATAGTTCCCCTAAAAATTCAGGCTTTCCATAGTTGCTTCTTC

1254

T A C - C G T G G G A C G C A T C A T C C A C C A C A C C A T C A C T A T A C C G T A A G G T G C C C A G C C G T C C T G G C C C G G A A C A G A A C A A A C A T G C - C A T G G A C G C T A A C A T A C A A G T T G G T G G T G T T T G T T T C C 1374 1422

TGCGCTGTTTTCTCTCCGATATCCA~GCAACGTTAGACAGCATGC

Fig. 4 Nucleotide and deduced amino-acid sequences of the cgxB gene.The putative translation initiation site and the stop codon are indicated by +1 and an asterisk, respectively. The sequence of the XB fragment amplified by PCR is underlined. The intron is overlined with a dotted line. A putative CREA-binding site is boxed. Partial amino-acid sequences of the 20-kDa enzyme and the P2 peptide, and the amino-acid sequence of the P1 peptide are double underlined. The most favoured signal peptide cleavage site is indicated by an upward arrow. Putative catalytic residues, Glu, are also indicated. One nucleotide of the wild-strain gene different from the mutantstrain cgxB gene is indicated above the sequence

lytic residues on the basis of their three-dimensional structure and site-directed mutagenesis (Ko et al. 1992). The two glutamate residues are completely conserved in the family G xylanases and the corresponding residues are Glu115 and Glu216 for CgXA and Glu116 and Glu217 for CgXB. The C-terminal portions of xylanases which carry the catalytic domain tend to be more highly conserved compared to their respective N-terminal portions (Fig. 5).

Expression of the C. gracile xylanase genes in A. niduIans and Aspergillus awamori (Hessing et al. 1994), Humicola isolens (Dalboge and Heldt-Hansen, 1994). To the G family also belong bacterial xylanases from Bacillus pumilus (Fukusaki et al. 1984), Bacillus circulans (Yang et al. 1988), Bacillus subtilis (Paice et al. 1986), Clostridium acetobuty!icum (Zappe et al. 1990) and Ruminococcus flavefaciens (Zhang and Flint, 1992). An analysis of the secondary structures of CgXA and CgXB by the method of Chou and Fasman(1987) predicts that the polypeptide chains primarily form p-sheets. The amino-acids conserved throughout all xylanases of this type are often in positions of ~3-turns linking two [3-sheets. Recently, two glutamic acid residues (Glu93 and Glu182) of the B. pumilus xylanase have been identified as cata-

Little is known about the mechanisms regulating xylanase gene expression. In several studies, the effect of potential inducers, such as methyl-fl-D-xyloside, xylose and arabinose, has been determined; but in none of those cases was gene expression investigated at the molecular level except for the A. tubigensis xlnA gene (de Graaff et al. 1994). As a first step to elucidate the regulatory mechanisms of C. gracile xylauase gene expression, it was determined whether or not the C. gracile xylanase genes are expressed in A. nidulans. The cgxA and cgxB genes were introduced into A. nidulans G191 using pyr4 as a selection marker. A random selection of five transformants was made and these transformants were then analysed for gene expression af-

78

Fig. 5 Alignment of the amino-acid sequences of the C. gracile xylanases with those of other filamentous fungal enzymes. A.awa, Aspergillus awamori; A.kaw, AspergiIlus kawachii; A.tub, AspergilIus tubigensis; H. iso, Humicola isolens; T.har, Trichoderma harzianum (Accession number, A44593); T.reeII and I, Trichoderma reesei; T.vir, Trichoderma viride; CgXA and CgXB, this paper.The asterisks mark amino-acids which are conserved in all ten sequences compared. Sequences homologous to the Z reesei xylanase II described in the text are indicated by dotted overlines. The catalytic amino-acid residues are shaded. A repeat of five amino-acids is also indicated by a dotted overline

A.awa.

I:M-KVTAAFAGL--LVTAFAAPVP-EP-VLVSRSA-GI-NYV-QNYNGN--LGDFTYD---ESAG--TF--

A.kaw.

I:M-KVTAASAGL--LGHAFAAPVP-QP-VLVSRSA-GI-NYV-QNYNGN--LADFTYD---ESAG--TF--

A.tub.

I:M-KVTAAFAGL--LVTAFAAPAP-EPD-LVSRSA-GI-NYV-QNYNGN--LGDETYD---ESAG--TF--

H.iso.

I:MVSLKSVLAAATAVSSAIAAPFDFVPRDNSTALQARQVTPNAEGWHNG--YFYSWWSDGGGQV--QYTNL

T.har.

l: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

T.ree. II

I:NVSF-TSL--LAASPPSRA$CRPAAEVESVAVEKRQTIQPG-TG~G--~FYSYWNDGHGGV--TYTNG

T.ree. I

I:MVAFSSLICALTSIASTLAMPTGLEPESSVNVTERGMYDFVLGAHNDHRRRASINYDQNYQTGGQVSYSP

T.vir.

i: . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

QTIGPG-TGYSNG--YYYSYWNDGHAGV--TYTNG

QTIGPG-TGFNNG--YFYSYWNDGHGGV--TYTNG

CgXA CgXB

~ : m ~ - s s ~ - - ~ s a a w ~ v ~ a ~ - ~ - ~ ~ s s ~ - - ~ s ~ ~ - - ~

A. awa.

54 :S-M-YWED-GVSSDFWGLGWTTGSSNAITYSAEYSASGSSSYLAVYGWVNYPQANYYIVEDYGDYNP-C

A.kaw.

54 :S - M - Y W E D - G V S S D F W G L G W T T G S S N A I S Y S A E Y S A S G S S S Y L A V Y G W V N Y P Q A ~ Y Y I V E D Y G D Y N P - C

A.tub. H.iso.

54 :S-M-YWED-GVSSDFWGLGWTTGSS-TITYSAEYSASGSASYLAVYGW'V'NYPQA~YYIVEDYGDYNP-C i!iiiili 67 :E G S R Y Q V R W R N T G N F V G G K G W N P G - T G R T I N Y G G Y F N P Q G N G Y L A V Y G W T R N P L V ~ Y Y V I ESYGTYNPGS

T.har.

31 :G G G S F T V N W S N S G N F V G G K G W Q P G T K N K V I N F S G S Y N P N G N S Y L S

T.ree. II 63 :P G G Q F S V N W S N S G N F V G G K G W Q P G T K N K V I N F

IYGWSRNPLI~YYIVENFGTYNPST

S;SYNPNGNSYL SVYGWSRNPL I~YY IVENFGTYNPST

T.ree. I

71 :SNTGFSVIXrWNTQDDFVVGVGWTTGS

T.vir.

3 I: P G G Q F S V N W S N S G N F V G G K G W Q P G T K N K V I N F S G T Y N P N G N S Y L S V Y G W S R N P L

SAPINFGGSFSVNSGTGLLSVYGWSTNPLV~YYIMEDNHNY-

P- -

I~YYIVENFGTYNPST

CgXA

61 :A G G S Y S V Q W Q N C G N F V G G K G W N P G

CgXB

62 :AGGQYSVTWSGNGNWVGGKGWNPGS-ARTINYTANYNPNGNSYLAVYGWTRNPLI~YYVVENFGTYNPST *

*

**

- A A R T ~NF S G T F S P Q G N G Y L A I Y G W T Q N P L V ~ Y Y IVE S F G T Y D P S S *

.

***

*

***

.

A.awa.

120:SSATSLGTVYSDGSTYQVCTDTRTNEPSITGTSTFTQYFSVRESTRTSGTVTVANHFNFWAQHGFGNSDF

A.kaw.

120:SSATSLGTVYSDGSTYQVCTDTRTNEPSITGTSTFTQYFSVRESTRTSGTVTVANHFNFWAQHGFGNSDF

A.tub.

II9:SSATSLGTVYSDGSTYQVCTDTRTNEPSITGTSTFTQYFSVRESTRTSGTVTVANHFNFWAHHGFGNSDF

H.iso.

136:QA-QYKGTFYTDGDQYDIFVSTRYNQPSIDGTRTFQQYWSIRKNKRVGGSVIgMQNHFNAWQQHGMPLGQH

T.har.

101:GATKL-GEVTSDGSVYDIYRTQRbVNQPSIIGTATFYQYWSVRRNHRSSGSVNTANHFNAWASHGLTLGTM

.

*

T.ree.II~33:GATK~GE~T~DG~VYDIYRTQR~QPS~IGTATFYQY~SVRRNHRSSGSVNTANHFNAWAQQGLTLGTM T.ree. I 1 3 8 : A Q G T V K G T V T S D G A T Y T I W E N T R V N E P S I Q G T A T F N Q Y I S V R N S P R T S G T V T V Q N H F N A W A S L G L H L G Q M T.vir.'

101:GATKL-GEVTSDGSVYDIYRTQRVNQPSIIGTSTFYQYWSVRRTHRSSGSVNTAIqHFNAWAQQGLTLGTM

CgXA

130:QASKF-GTIQQDGSTYTIAKTTR~QPSIEG~STFDQEWSVRQNHRSSGSVNVAAHFNAWAQAGLKLGSH

CgXB

131:GATRL-GSVTTDGSCYDIYRTQRVNQPSIEGTSTFYQFWSVRQNKRSGGSVNMAAHFNAWAAAGLQLGTH . ** . . . *** ** ** . . . . . . *** . .

A.awa.

190 : N Y Q V M A V ~ W S G A G S A S V T I S S

A.kaw.

190 :NYQVN.AV~WSGAGSASVTIS$ . . . . . . . . . . . . . . . . . . . .

A.tub. H.iso.

189 : N Y Q V V A V ~ W S G A G S A S V T I S S iiiii[ii 205 : Y Y Q W A T ~ G Y Q S S G E S D I Y V Q T H

T.har.

170 : D Y Q I V A V ~ G Y F S S G S A S I T V S . . . . . . . . . . . . . . . . . . . . .

.................... .................... ...................

T.ree. I I 2 0 2 :DYQIVNV~GYFSSGSNSITVS . . . . . . . . . . . . . . . . . . . . . T.ree. I 208 NYQWNV~GWGGSGSNSQSVSN . . . . . . . . . . . . . . . . . . . . T.vir. CgXA CgXB

170 : D Y Q I V A V ~ G Y F $ S G S N S I T V S . . . . . . . . . . . . . . . . . . . . . iiiiiii! 199 : N Y Q I V A T ~ G Y Q $ S G S $ S I T V $ . . . . . . . . . . . . . . . . . . . . . 200 : D Y Q I V A T ~ G Y Y S S G S A T V N V G A S S D G S T G G G S T G G G S T N V S F ** * * *

ter growth on birchwood xylan or glucose as a carbon source. The expression was analysed by both Western blotting and the xylanase activity of culture filtrates (Fig. 6 and Table 1). All transformants carrying the cgxA gene secreted immunoreactive proteins with almost the same molecular mass, approximately 20 kDa, as the authentic enzyme after growth on birchwood xylan, while the levels of immunoreactive proteins were drastically reduced after growth on glucose (Fig. 6). Essentially the same results were found as for the xylanase activity (Table 1). All transformants had significantly high levels of activity on the xylan medium,

whereas, no or only a very low, activity was found on the glucose medium. These results clearly indicate that cgxA gene expression is repressed in the presence of glucose. Transformants carrying the cgxB gene secreted no immunoreactive proteins even after growth on birchwood xylan (data not shown), but they exhibited a distinct xylanase activity in their culture filtrates which was more than that detected in the cgxA gene-carrying cells (Table 1). The enzyme levels observed after gowth on glucose were comparable to those after growth on xylan. The cgxB gene appeared to be constitutively expressed. The cgxA and cgxB

79 tion initiation site, as shown in Fig. 3. We are currently att e m p t i n g to c o n f i r m these sequence e l e m e n t s b y a D N a s e I footprinting assay using a M A L E - C R E A fusion protein s y n t h e s i z e d in E.coli. As to the induction m e c h a n i s m s , the role o f putative cis-acting r e g u l a t o r y elements in the p r o m o t e r region, and the identity of the trans-acting r e g u l a tory factors controlling them, have yet to be established.

References Fig. 6 Immunoblot analysis of the xylanase secreted by the recipientA. nidulans and transformants #1 and #2 carrying the cgxA gene. A. nidulans G 191 carrying only the vector, pDJB 1, grown on birchwood xylan, was used as a control (lane2) and transformants #1 and #2 were grown with birchwood xylan (lanes 3 and 5) or glucose (lanes 4 and 6). Lanes 3 and 4 and lanes 5 and 6 indicate transformants #t and #2, respectively. All clones were cultivated at 37~ for 4 days. The xylanases in 1 ml of culture filtrates were precipitated with 10% TCA, solubilized in a small volume and used for immunoblotting. Each lane contained protein equivalent to 70 gg. An authentic xylanase was used as a standard (lane 1; 30 ng)

Table 1 Xylanase activity in culture filtrates of transformants. Transformants were grown on xylan or glucose at 30~ for 4 days. The enzyme activity was determined with RBB-xylan as a substrate. The unit represents the activity in 1 ml culture and the average values for two independent experiments Carbon source

Xylan

Glucose

Strain A. nidulans pDJB 1

0.05

0.05

Transformants carrying the cgxA gene #1 #2 #3 #4 #5

2.15 1.80 1.45 2.25 1.55

0.45 0.20 0.20 0.05 0.25

Transformants carrying the cgxB gene #1 #2 #3 #4 #5

5.15 4.95 6.70 3.60 2.55

6.50 3.90 7.40 2.75 3.00

genes had only 45% h o m o l o g y at the D N A level in the 5 ' - n o n c o d i n g region, suggesting that their e x p r e s s i o n could be differently regulated. Since the cgxA gene e n c o d e s the m a j o r x y l a n a s e in C. gracile and is i n d u c i b l y e x p r e s s e d in A. nidulans, we are now a t t e m p t i n g to c h a r a c t e r i z e the r e g u l a t o r y m e c h a nisms o f cgxA gene e x p r e s s i o n using A. nidulans as an i n t e r m e d i a t e host. The r e p r e s s i o n o f cgxA gene e x p r e s s i o n caused b y g l u c o s e c l e a r l y indicates that the cgxA gene is under carbon c a t a b o l i t e r e p r e s s i o n m e d i a t e d via the A. nidulans creA gene. T h e cgxA gene contains three copies of the C R E A - b i n d i n g site, 5 ' - G / C P y G G G G - 3 ' ( K u l m b u r g et al. 1993), within the first 823 bp u p s t r e a m o f the transla-

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