Gene, 161 (1995) 137 138 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.

50

137

GENE 09006

The cellulase complex of Neurospora crassa: cbh-1 cloning, sequencing and homologies
(Cellobiohydrolase; exoglucanase; signal peptide; binding domain; EC 3.2.1.91)

F. Taleb and A. Radford

Department of Genetics, The University o]'Leeds, Leeds LS2 9J T, UK

Received by J.R. Kinghorn: 20 February 1995; Revised/Accepted: 4 March/16 March 1995; Received at publishers: 20 April 1995

SUMMARY

We describe the isolation, cloning and sequencing of the cellobiohydrolase 1 (EC 3.2.1.91)-encoding gene (cbh-1) of Neurospora crassa. The nucleotide and amino-acid sequences have high homology with the cbh-1 of Trichoderma reesei, Humicola grisea and Phanerochaete chrysosporium, with clear signal, catalytic, hinge and substrate-binding domains in that order. The EMBL database reference for this sequence is $42093.

The best-characterised fungal cellulase complex is from Trichoderma reesei (Tr), where there are isozymes of each class (endoglucanases (EG: EC 3.2.1.4), exoglucanases or cellobiohydrolases (CBH: EC 3.2.1.91) and [3glucosidases (Bgl: EC 3.2.1.21)) within the complex. Cellobiohydrolases of the cbh-l-type from Tr, Phanerochaete chrysosporium (Pc) and Humicola grisea (Hg) have been cloned and sequenced (Teeri et al., 1983; 1987; Shoemaker et al., 1983; Sims et al., 1988; Azevedo and Radford, 1990). The related endogucanase eg-I of Tr has also been sequenced (Penttil/~ et al., 1986). Neurospora crassa (Nc) is able to produce significant quantities of a cellulase complex (Eberhart et al., 1977), and biochemical studies of a cellulase complex in the species similar to that in Tr have been published (Yazdi et al., 1990). The Nc strain used in this investigation was wild-type 74-OR23-1A. The library was constructed in the )~J1 vector (Orbach et al., 1986). It represents 72000 unique
Correspondence to: Dr. A. Radford, Department of Genetics, The University of Leeds, Leeds LS2 9JT, UK. Tel. (44-113) 233-3086; Fax (44-113 t 244-1175; e-mail: a.radford@leeds.ac.uk

Abbreviations: aa, amino acid(s); Bgl, [3-glucosidase; bp, base pair(s); cbh-1, gene encoding cellobiohydrolase-1 or exoglucanase-1; eg-1, gene encoding endoglucanase-1; Hg, Humicola grisea; kb, kilobase(s) or 1000 bp; Nc, Neurospora crassa; nt, nucleotide(s); Pc, Phanerochaete chrysosporium; PCR, polymerase chain reaction; Tr, Trichoderma reesei; tsp, transcription start point(s).
SSDI 0378-1119(95)00288-X

clones. The plasmid vector used for sub-cloning and sequencing was pBluescript. PCR primers were designed from the alignment of the closely related cbh-1 sequences of Tr, Pc, Hg, and the eg-1 sequence of Tr. Following standard PCR with these primers on Nc genomic DNA, of the several fragments amplified, one of 0.8 kb showed strong hybridisation to the cbh-1 clone of Pc. This fragment was subsequently used as a homologous probe. 100000 )~ clones were screened with this probe. Eight pure clones showing an obvious and strong hybridisation to the 0.8-kb PCR fragment were isolated. Restriction enzyme mapping distinguished between two clone types. One, framed and cut by ClaI sites generating two fragments (2.7 and 0.71 kb), was selected for further study. Both ClaI fragments were subcloned, and initial partial sequencing of these fragments identified them as belonging to a cbh-1 gene. Full sequencing of this Nc cbh-1 gene was carried out, using the dideoxy method. Altogether, more than 3714 bp were read. Computer translation of a large open reading frame, together with comparison with cbh-1 genes from related fungi, revealed a coding region of 1551 bp containing two exons separated by a single small intron of 59 nt. The 5' splice junction is 5'-GTTTGT and the 3' splice junction is AAG. The intron internal consensus sequence is AACTAAC. The translation start sequence is TCAAAATGAG. The position of the intron is not identical to those found in the equivalent genes of related

138
gagtctgta~ccaaactctttacc=~tccttsgstccct~tascagtat~tccattgtttcutatataa~ggttagggggt~a~tcccgscsctcatga~ttcqc~ttcttcccttatctqatcgggca~cggaaacca~ttgcactc~
* ,

150 50 300

Suu3A
M R A S L L A F S L A A A V A G G Q Q A G T L T A K R H P S L T W Q K aATGAGC~C CT CG CT CC~C C ~ C T C C C T C G C ~ C C G C C G T G C - C C G G C G G C C A G C A G G C C C ~ C A C T CT C A C C G C C A A G A ~ C A C C C A T C C ~ T~CAG~G~C C A~LI D A N W E W T H A T S G S T K C Y T G N K W Q CGACGCGAA CTGGCGCTGGACTCACGC CACGTCCGGCTCCACGAAGTGCTACACGGGCAACAAGTGGCAGG A T L C P D G K CGACGCTCTGCC CC GATGGC~GT S C A A ~TGCGC~CG~ N C A L D G A D Y ~ C G C G ~ C ~ ~CCGA~A~C T G T G I ~ C A C ~ A C ~ i00 450 T R G G C CA~TGCCCGACC P T ~ L C N T T M V L ~ A T ~ T G ~

Sau3A
T G S G W S L T L Q F V T D N V G A R A Y L M A D D T Q Y Q M L E L L N Q E L W F D V D M -CAC C C ~ G A G C G C ~ C ~ T C C C ~ C A C G C T C C A G T T C G T C A C G G A C ~ C G T C G G C G C C ~ G T G C C T A C C T G A T C G C G G A C G A ~ A ~ G C A G T A ~ C A G A q ~ q ~ A C C T C C ~ C A C ~ A G ~ ` T G q ~ C G A C G T C G A T A T G T ~ G ~ A C A T C C C G T G SicI G L N G A L Y L S A M D A D G G M R K CGGT CTGAA CGG CGCC CTCTAC CT CTCGGCGATGGACGCGGATGG~ATGAGGAAGTACC Y P T N K A G A K CGA CCAA CAAGC~GGCGCT~GTA PCR primer C S E M D C~CTGAGAT~ATAT I W ~ ~ Y ~tII Y I N G I A N C~C~TATC ~ C~ 200 750 S N I P C 150 600

A T G Y C D A Q C P R D L K ~ A C C ~ A ~ G C G A ~ C A G T ~ CCC~AT~C~GTACAT

->

Sau3A
227 900

V E G W T P S T N D A N G I G D H G S C CGTTGAGG~CTGGACCC C~CCAC CAAC GATGCTAACC~TAT~TGAC CA CGGAT C ~

E A N t t t qt t t g c c g a t t t t c c t t t c at c a t t a g c a t c a c a g g t a a c t a a c a c c c a c c t a a q G ~ u % G C G / ~ C

Smu3A
K V S T A F T P H P c AAAGT CT CTACAGCGTT CA CC CCGCACC CC~CA T T I E Q H M C E G D S C G G T Y $ D D R Y G V L C D A D G C CCAC CATCGAACAGCACATGTGCGAGGGTGACTC CTGCGGTGGTAC CTATTC CGACGACCGCTATGGCGTACTTTGCGATGC~T~G~A~G~A ZpnI A ~ D F N S Y C~ R C M G 277 1050

N T T F Y G E G K T V D T S S K F T V V T Q F I K D S A G D L A E I K A A C A CCACCI~C C T A C G G T G A G G G C A A G A C T G T C C ~ T A C C A G C T C C A A G T T C A C C G T T G T C A C C C A G T T C A T C A A G G A C T C C G C T C ~ 3 ~ T ~ G ~ A G A T ~ C Sau3A Sau3A N V D G V S G N AACGTTGA~GAGq~C~CAAC~CCAT S I T Q S F C K S Q CACCCAGTC~C~C~GTCTCAG~GA K T A F G D ~ C G A T A T I

Y V Q N G K ~ACGT C ~ C ~ G T

V C

I A

E N S ~ ~ G T

Q C

327 C 1200

D D F N K K G G L K Q M G K A C ~ T G A ~ C ~ C ~ G ~ C ~ ~ C C ~ C C ~

L T

A ~

Q T

A C

M ~
Nco I

V M S ~T~TGTCC

377 1350

Cl I

I W D D H A A N M L W L D S T Y P V P K V P G A Y R G S G P T T ATCTGC~ACGA CCATGCCGCCAACATC~ CTGGCTCGA CT CCAC CTACCCTGT C CCGAAGGT CC CCGGTGCTTA CCGTGGCAGTGGC CCTAC~C~

S ~

G T

P A E V E C C ~ A ~ T C ~ C

A ~

N C

P N S K V A F C~ ~ C ~ T C ~ ~ C <- P C R S N P S G ~CCC~GC~TACC T

427 1500

S N I K F G H L G I S P F S G G S S G T P P S N P S S S A S TC CAACAT CAAGTT CC~3CCACCTCGGGAT_____~CTCCT~AGCGGCGGCTC~ CCC~CACC CCTC CTT CCAA CC CTTCGA~ CCGC~C pri~er S&u3A SacI G A A H W A Q C G G I G F S G P T T C P E P Y T C A C~TGCTC~CAC~CTCAGTGCc~C~TAq~FGGC~CTC~CCCCACCAC~GCCCAGAGCC~A~GC~GATCACGA~A~CC~G~T~t K D H D

P T C ~ C

S T A K ~C ~ G ~

P C

T S T A ~ C ~ A ~ C

477 1650

513 t act a g c c t g c t a g g g t a a c c t t t t tg g t t c c t c t a c 1 8 0 0 1049

tacggcagctaggtgaactcgac~gcgaagcaaaaaggaacttcgagaa

Fig. 1. The nucleic acid sequence of the Nc cbh-I and aa sequences. Some restriction sites and the PCR primers are indicated. Important consensus sequences are underlined. Intron sequence and non-coding sequences are in lower case. An asterisk (*) marks the stop codon.

fungi. The upstream region of the gene is characterised by a CAAT-like box (CAAAC), a TATA box (TATATAA) and a pyrimidine-rich region. A tsp is found within this pyrimidine-rich region (TCTTC). The Nc cbh-1 gene of Nc was encoding a 516-aa (54 611 Da) protein (see Fig. 1 ). By analogy with other cellulases, the Nc CBHI enzyme sequence could be divided into domains, with a signal peptide (from aa 1-17), a catalytic domain (aa 18-445), a hinge region (aa 446-480) and a binding domain (aa 481-516). This enzyme has an N-terminal catalytic domain and a C-terminal binding domain, and thus has the same arrangement of domains found in CBHI in Tr, Pc and Hg and in Tr EGI. The Nc cbh-1 gene was fully aligned with cellulase genes (cbh-I and eg-1) from related fungi. It is clearly homologous with the other fungal cbh-1 and eg-1 sequences, and closest to cbh-1 of Hg. At the DNA sequence level it shows 69% homology with the cbh-1 of Hg, and 65% homology at protein level.

REFERENCES Azevedo, M.O. and Radford, A.: Sequence of the cbh-1 gene of l-lumicola grisea var thermoidea. Nucleic Acids Res. 18 (1990) 668.

Eberhart, B.M., Beck, R.S. and Goolsby, K.M.: Cellulase of Neurospora crassa. J. Bacteriol. 170 (1977) 181-186. Penttil~, M., Lehtovaara, P., Nevalainen, H., Bhikhabhai, R. and Knowles, J.K.C: Homology between cellulase genes of Trichoderma reesei. Complete nucleotide sequence of the endoglucanase I gene. Gene 45 (1986) 253-263. Saloheimo, M., Lehtovaara, P., Penttil~, M., Teeri, T.T., StShlberg, J., Johansson, G., Pattersson, G., Claeyssens, M., Tomme, P. and Knowles, J.K.C.: EGIII, a new endoglucanase from Trichoderma reesei: the characterization of both gene and enzyme. Gene 63 (1988) 11 22. Shoemaker, S., Schweickart, V., Ladner, H., Gelfands, D., Kwok, S., Myambo, K. and Innis, M.: Molecular cloning of exocellobiohydrolasel derived from Trichoderma reesei strain L27. Bio/ technology 1 (1983) 691-696. Sims, P., James, C. and Broda, P.: The identification, molecular cloning and characterisation of a gene from Phanerochaete chrysosporium that shows strong homology to the exo-cellobiohydrolase I gene of Trichoderma reesei. Gene 74 (1988) 411 422. Teeri, T.T., Lehtovaara, P., Kauppinen, S., Salovuori, I. and Knowles, J.: Homologous domain in Trichoderma reesei cellulolytic genes: gene sequence and expression of cellobiohydrolase II. Gene 51 (1987) 43--52. Teeri, T.T., Salovuori, I. and Knowles, J.K.C.: The molecular cloning of the major cellulase gene from Trichoderma reesei. Bio/Technology 1 (1983) 696-699. Yazdi, M.T., Woodward, J.R. and Radford, A.: Cellulose production by Neurospora crassa: the enzymes of the complex and their regulation. Enz. Microb. Technol. 12 (1990) 116-199.