Nucleic Acids Research, Vol. 18, No. 18 5559

©-:) 1990 Oxford University Press

Nucleotide sequence of the cellobiohydrolase Trichoderma viride

from

gene

Cheng Cheng, Norihiro Tsukagoshi and Shigezo Udaka* Faculty of Agriculture, Nagoya University, Chikusa, Nagoya 464, Japan Submitted August 9, 1990

EMBL accession

The filamentous fungus, Trichoderma viride, secretes large amounts of several cellulolytic enzymes (1). We report here the nucleotide sequence of the gene coding for exo-cellobiohydrolase (CBH), which accounts for more than 65 % of the total cellulase content. This gene was isolated from a T. viride genomic library using a 30mer oligonucleotide based on the sequence of the reported CBHI gene of T. reesei (2). The amino acid sequence deduced from the nucleotide sequence is highly homologous (95%) to that of the T. reesei CBHI gene. In contrast to the high homology between the enzyme coding regions of the two CBH genes, 67% and 30% homology was found between the 5'-non

GTAGCATTGAGGAACCACTGCTTTTGCCAGGAGCTAGCGTATGCTGTAGGCAAAGCTCTA

AGATAGCCTCATTGAGCGGAAGTCGGCGAACAGTGAAGACCAGAATATCACAT

180 ATATATA 240

TGGCCCAAACGCCGTGTCCCCTTCTCCCTTTCCCCATCTACTCATCAACTCAGATCCTCC

AGAAGACTTCTACATCATCTTTTCGCCCATACCATTCTAGTCGACTACGGACTGCGCATC ATGTATCAAAAGTTGGCCCTCATCTCGGCCTTCTTGGCTACTGCTCGTGCTCAGTCGGCC -17 4

24

44 64 84

104 124 138 142 162

182 202 222

300

REFERENCES 1.

242

262

360 282

TcCACCCTCCAGcGCGAAACTCACCCGCCTCTGACATGGCAGAAATGCTCATCG(iC S S G K C W P L

480

302

ACTTGCACCCAACAGACAGCCTCCCTGGTCATFCGACUCGAACTCGCTGGACTCACGCC H A W T W R A N I D T C T Q Q T G S ACCAACAGCAGCACCAACTGCTACCACGGCAATACTTGCAGCTCAACCCTCTGCCCTCAC D

540

322

600

342

AATGAGACTTGCGCCAAGAACTGCTCCTTCGACGGTGCTGCCTACGCCGTCCACGTACGCA D L C C G A A Y A S T Y G E T C A K GTCACCACGAGCGCTGACAGCCTCTCCATTGCCTTCGTCACTCAGTCTGCGCAAAAGAAC I S S L D G F V T Q S A Q K N A V T T S GTCGGCGCTCGTCTCTACTTGATCGCGAGTCACACGACCTATCAAGAATTCACCCTGCTT

660

362

720

370

M

Y

r

T

T

Q

S

N

L

A

0

A

E T

S

T

N

C

S

I

F

V

V

D

G

N

T

T

A

R

A

QSA

Q

T

P

Y

A

W

S

C

S

T

L

C

G

A

R

L

Y

L

M

A

S

D

T

T

Y

Q

E

F

T

L

E

N

F

S

F

D

V

D

V

S

Q

L

840 900

421

960

441

C

H

1020 461

K

1080 481

V

G

G

I

G

H

G

S

P

H

C

C

S

E

M

D

V

G

I

W

E

A

N

S

TCTGAGGCTCTTACTCCTCATCCTTGCACGACCGTCGGGCAGCAAATTTGCGACGGTGAC D S

E

A

L

T

P

C

T

T

Q

E

I

C

E

G

1140

I

1200

et

on

Enzymc Hydrolysis of Cellulose

81-109.

pp.

al. (1983) BiolTechnology 1, 691-696.

TCTTGCGGTGGAACCTACTCGGGTGACCGATATGGCGGTACTTGCGACCCTGATGGCTGC G C

1260

GATTGGAACCCATATCCCTTGGGCAACACCAGCTTCTATGGCCCCGGCTCCAGCTTCACG P G S S F T N T S F Y D W N P Y R L CTTGACACCACCAAGAACTTCACCCTCGTCACTCAGTTCGAGACTTCGGGTGCCATCAAC T S F E G A I N T Q K L T V T K D T L CCATACTATGTCCAGAATGCCGTCACTTTCCAGCACCCCAACCCCGACCTCCCTCATTAC Y

1320

S

G

C

T

Y

S

R

D

C

Y

G

C

T

D

C

D

P

G

C

C

1380

V

G V T F Q Q P N A E L C D TCTGGCAACTCGCTCCACCATCACTACTGCGCGGCTGAACAGCCCGAGTTTGGCCCCTCC R

Y

Y

V

Q

N

S

C

N

S

L

D

D

D

C

Y

A

A

E

E

E

A

F

C

C

K

C

1500 1560

V

C

C

1440

S

C

401

C

V

al., eds),

CTCCGCTCTTCCTGCTCACCTCGAGTCCAACTCTCCCAACGCCAACGTCGTATACTCCAA G S

P

gATGTGTTGAAC cttgatgccattctcgtattgatttctcgtgactagcttatttaa N L C GGACCTCTTTACTTCGTGTCCATCGACGCGGATCGTGGCCTGACGAAGTATCCCACCAAC T N Y P V T K G D A D S L Y F G A ACTGCCCGTGCCAAGTACGGCACGGCGCTACTCTGACAGCCAGTGCCCTCCTGATCTCAAG T A G A K Y G T G Y C D S Q C P R D L TTCATCAACGGCCAGGCCAATGTTGAGGGCTGGGAGCCGTCCTCTAACAATGCAAACACG E G W E P S S N N A N T F I N G Q A N GGCATTGGCGGACATGGAAGCTGCTGCTCTCAGATGGATATCTGGGAGCCCAATTCCATC

al., (1975) in Symposium

et

381

L

GGCAACGAGTTCTCTTTCGATGATGTTTCGCAGCTGCCgtaagtgaccaactacact G

780

et

(Bailey,M.

TCTTTCTCGCACAAGGGCCCCCCTTACTCAATTCAACAAGGCTACTTCCGGTCCCATCGTC M T S K A Q F L T D K F S S CTGCTCATGACCCTG TGGGATGACgtgagtttcaagaat taacattcacattgtcaacng V M S L W D L aatgactagactgatggagagacgatagTACTACGCCAACATGCTGTGCCTGGACTCTAC N Y A Y M L W L D S CTACCCGACGCACGAGACCTCCTCCACCCCCCGTCCCTCCCTGCAACCTCCTCCACCAG S T G S S

P

N

N

V

L

K

L

Mandels,M.

2. Shoemaker,S.

420

A

X53931

coding and 3'-non coding regions, respectively. Southern-blot and Northern-blot analysis showed the presence of at least one CBH gene on the T. viride chromosome which was transcribed only at the culture condition that cellulase production was induced.

120

GGTGCCACTGCATTTCTGTCGAACATAATGTGATGCTTCGGCAGGCATAATAGCCGCCAA

no.

1620

D

1680

T

Y

P

T

V

S

D

P

E

A

T

Q

S

L

S

E

T S

P

N

C

S

A

P

V N

S

R

A

K

V

V

C

C

C

1860

C

1920

P

C

TACTCACACGCACTATGGCCAGTCCGGTGGAATTGGGTACATCGGGCCCACCGTCTGCGC C G G I G Y I G P T V C Q H Y Q T T GACTCCCACCACTTGCCAGGTCCTCAACCCCTACTACTCTCAGTGCTTCTAAGGTACTCT Q C Y S P Y L N L *1* Q S T C S GGCAACTCCACATACCTAGCTCGAAAGCTTGAGCATCTGCTGGCTTATGGATGAGTTCAT CTCATTATGCACTAGATGGACCATTTACTTTGCTGTATCTACTTCTCACGCTTCCAATAT

1980

A

C.

C

1800

N

Y

CATCAACTTCCCCCCCATCCCCACCACCGGCAACCCTAGCGGCGGAAACCCTCCTGGCGG G T G N P P S P I I F G N P S AAACCCTCCCCCCACCACAACACCGCGCCCGCCTACCTCCACTCCAAGCTCTCCCGGCCC S S P G T G T S P A P R T T T P N P K

1740

2040

V

ATACGCTTATTTCACCTTTGCTCCAATGCTCCCTACCTTGGCAAGCACGGCTTTCGAGAG ACACGCACTGATTCTCTCCTAACTATGCATTATATAAGACTGAAATAGACCAAAAAACGA AAACCTTCCCCACTCCAATTATCTCACGGTGTTCATTTATATGTAGGCCA

2100

2160 2220

2280

2330

of CBH. The deduced amino acid sequence is shown below the coding region of the gene and numbered, on the left, from the amino-terminus (I and II) are shown in lower-case letters. The putative TATA box sequence is underlined. The amino acid sequence of the amino terminal region of the purified enzyme, which was determined chemically, is double underlined. The stop codon is indicated by asterisks.

Nucleotide

sequence

(Gln) of the

*

mature enzyme. Introns

To whom correspondence should be addressed