Purified scytalidium laccases and nucleic acids encoding same

The present invention relates to isolated nucleic acid constructs containing a sequence encoding a Scytalidium laccase, and the laccase proteins encoded thereby.

FIELD OF THE INVENTION 
The present invention relates to isolated nucleic acid fragments encoding a 
fungal oxidoreductase enzyme and the purified enzymes produced thereby. 
More particularly, the invention relates to nucleic acid fragments 
encoding a phenol oxidase, specifically a laccase, of a thermophilic 
fungus, Scytalidium. 
BACKGROUND OF THE INVENTION 
Laccases (benzenediol:oxygen oxidoreductases) are multi-copper containing 
enzymes that catalyze the oxidation of phenolics. Laccase-mediated 
oxidations result in the production of aryloxy-radical intermediates from 
suitable phenolic substrate; the ultimate coupling of the intermediates so 
produced provides a combination of dimeric, oligomeric, and polymeric 
reaction products. Such reactions are important in nature in biosynthetic 
pathways which lead to the formation of melanin, alkaloids, toxins, 
lignins, and humic acids. Laccases are produced by a wide variety of 
fungi, including ascomycetes such as Aspergillus, Neurospora, and 
Podospora, the deuteromycete Botrytis, and basidiomycetes such as 
Collybia, Fomes, Lentinus, Pleurotus, Trametes, and perfect forms of 
Rhizoctonia. Laccase exhibits a wide range of substrate specificity, and 
each different fungal laccase usually differs only quantitatively from 
others in its ability to oxidize phenolic substrates. Because of the 
substrate diversity, laccases generally have found many potential 
industrial applications. Among these are lignin modification, paper 
strengthening, dye transfer inhibition in detergents, phenol 
polymerization, juice manufacture, phenol resin production, and waste 
water treatment. 
Although the catalytic capabilities are similar, laccases made by different 
fungal species do have different temperature and pH optima, and these may 
also differ depending on the specific substrate. A number of these fungal 
laccases have been isolated, and the genes for several of these have been 
cloned. For example, Choi et al. (Mol. Plant-Microbe Interactions 5: 
119-128, 1992) describe the molecular characterization and cloning of the 
gene encoding the laccase of the chestnut blight fungus, Cryphonectria 
parasitica. Kojima et al. (J. Biol. Chem. 265: 15224-15230, 1990; JP 
2-238885) provide a description of two allelic forms of the laccase of the 
white-rot basidiomycete Coriolus hirsutus. Germann and Lerch (Experientia 
41: 801,1985; PNAS USA 83: 8854-8858, 1986) have reported the cloning and 
partial sequencing of the Neurospora crassa laccase gene. Saloheimo et al. 
(J. Gen. Microbiol. 137: 1537-1544, 1985; WO 92/01046) have disclosed a 
structural analysis of the laccase gene from the fungus Phlebia radiata. 
Attempts to express laccase genes in heterologous fungal systems frequently 
give very low yields (Kojima et al., supra; Saloheimo et al., Bio/Technol. 
9: 987-990, 1991). For example, heterologous expression of Phlebia radiata 
laccase in Trichoderma reesei gave only 20 mg per liter of active enzyme 
(Saloheimo, 1991, supra). Although laccases have great commercial 
potential, the ability to express the enzyme in significant quantities is 
critical to their commercial utility. At the present time there are no 
laccases which are expressed at high levels in commercially utilized hosts 
such as Aspergillus. Thus, the need exists for a laccase which can be 
produced in commercially useful (i.e., gram per liter or more) quantities. 
The present invention fulfills such a need. 
SUMMARY OF THE INVENTION 
The present invention relates to a DNA construct containing a nucleic acid 
sequence encoding a Scytalidium laccase. The invention also relates to an 
isolated laccase encoded by the nucleic acid sequence. Preferably, the 
laccase is substantially pure. By "substantially pure" is meant a laccase 
which is essentially (i.e.,.gtoreq.90%) free of other non-laccase 
proteins. 
In order to facilitate production of the novel laccase, the invention also 
provides vectors and host cells comprising the claimed nucleic acid 
fragment, which vectors and host cells are useful in recombinant 
production of the laccase. The nucleic acid fragment is operably linked to 
transcription and translation signals capable of directing expression of 
the laccase protein in the host cell of choice. A preferred host cell is a 
fungal cell, most preferably of the genus Aspergillus. Recombinant 
production of the laccase of the invention is achieved by culturing a host 
cell transformed or transfected with the nucleic acid fragment of the 
invention, or progeny thereof, under conditions suitable for expression of 
the laccase protein, and recovering the laccase protein from the culture. 
The laccases of the present invention are useful in a number of industrial 
processes in which oxidation of phenolics is required. These processes 
include lignin manipulation, juice manufacture, phenol polymerization and 
phenol resin production.

DETAILED DESCRIPTION OF THE INVENTION 
Scytalidium thermophilum is a thermophilic deuteromycete, and a member of 
the Torula-Humicola complex which are recognized as dominant species in 
mushroom compost. Other members of the complex include Humicola grisea 
Traaen var. thermoidea Cooney & Emerson, H. insolens Cooney & Emerson, and 
Torula thermophila Cooney & Emerson, the latter of which has been 
reassigned to Scytalidium thermophilum by Austwick (N. Z. J. Agric. Res. 
19: 25-33, 1976). Straatsma and Samson (Mycol. Res. 97: 321-328, 1993) 
have recently determined that both H. grisea var. thermoides and H. 
insolens should be considered as examples of the species Scytalidium 
thermophilum as well. S. indonesiacum (Hedger et al., Trans. Brit Mycol. 
Soc. 78: 366-366, 1982) may also be synonymous with S. thermophilum. 
Members of the complex are known to be producers of thermostable cellulase 
and .beta.-glucosidase enzymes (Rao and Murthy, Ind. J. Biochem. Biophys. 
25: 687-694, 1988; Hayashida and Yoshioka, Agric. Biol. Chem. 44: 
1721-1728, 1980). However, there have been no previous reports of the 
production of a laccase by Scytalidium, or any of the noted synonymous 
species. It has now been determined that not only does Scytalidium produce 
a laccase, but the gene encoding this laccase can be used to produce large 
yields of the enzyme in convenient host systems such as Aspergillus. 
To identify the presence of a laccase gene in Scytalidium, a 5' portion of 
the Neurospora crassa laccase gene (lcc1) is used as a probe, under 
conditions of mild stringency, in southern hybridization of total genomic 
DNA of different fungal species. An approximately 3 kb laccase specific 
sequence is detected in the Scytalidium DNA. The N. crassa fragment is 
then used to screen about 12,000 plaques of an S. thermophilum genomic DNA 
library in a .lambda. EMBL4 bacteriophage cloning vector. Nine plaques 
strongly hybridize with the probe; from these nine, DNA is isolated from 
four. Each of these clones contains a 3 kb BamHI fragment corresponding to 
the one initially identified in the southern blot of genomic DNA. One of 
the fragments is subcloned into a pBluescript vector; however, DNA 
sequencing shows only a portion of the gene to be on this fragment. A 6 kb 
fragment XhoI fragment from the same phage contains the whole lccS gene, 
and this is then subcloned into pBluescript to derive plasmid pShTh6. A 
restriction map of the 6 kb insert is shown in FIG. 3. 
Once the sequence is determined, the positions of introns and exons within 
the gene is assigned based on alignment of the deduced amino acid sequence 
to the corresponding N. crassa laccase gene product. From this comparison, 
it appears that the gene (lccS) of S. thermophilum is composed of seven 
exons(243, 91, 70, 1054 and 390 nucleotides) punctuated by four small 
introns (63, 58, 55 and 65 nucleotides). The coding region, excluding 
intervening sequences is very GC-rich(60.8% G+C) and encodes a 
preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 
amino acid propeptide. The sequence of the S. thermophilum gene and the 
predicted amino acid sequence is shown in FIG. 1 (SEQ ID NOS: 1 and 2) 
The laccase gene is then used to create an expression vector for 
transformation of Aspergillus host cells. The vector, pShTh15 contains the 
A. oryzae TAKA-amylase promoter and the A. niger glaA terminator regions. 
The construction of pShTh15 is outlined in FIG. 2. Aspergillus cells are 
cotransformed with the expression vector and a plasmid containing the pyrG 
or amdS selectable marker. Transformants are selected on the appropriate 
selective medium containing ABTS. Laccase-producing colonies exhibit a 
green halo and are readily isolatable. Selected transformants are grown up 
in shake flasks and culture broths tested for laccase activity by the 
syringaldazine method. Shake flask cultures are capable of producing 50 or 
more mg/liter of laccase, and in fermentors, yields of over 1.6 g/liter 
are observed. 
According to the invention, a Scytalidium gene encoding a laccase can be 
obtained by methods described above, or any alternative methods known in 
the art, using the information provided herein. The gene can be expressed, 
in active form, using an expression vector. A useful expression vector 
contains an element that permits stable integration of the vector into the 
host cell genome or autonomous replication of the vector in a host cell 
independent of the genome of the host cell, and preferably one or more 
phenotypic markers which permit easy selection of transformed host cells. 
The expression vector may also include control sequences encoding a 
promoter, ribosome binding site, translation initiation signal, and, 
optionally, a repressor gene or various activator genes. To permit the 
secretion of the expressed protein, nucleotides encoding a signal sequence 
may be inserted prior to the coding sequence of the gene. For expression 
under the direction of control sequences, a laccase gene to be used 
according to the invention is operably linked to the control sequences in 
the proper reading frame. Promoter sequences that can be incorporated into 
plasmid vectors, and which can direct the transcription of the laccase 
gene, include but are not limited to the prokaryotic .beta.-lactamase 
promoter (Villa-Kamaroff, et al., 1978, Proc. Natl. Acad. Sci. U.S.A. 
75:3727-3731) and the tac promoter (DeBoer, et al., 1983, Proc. Natl. 
Acad. Sci. U.S.A. 80:21-25). Further references can also be found in 
"Useful proteins from recombinant bacteria", in Scientific American, 1980, 
242:74-94; and in Sambrook et al., Molecular Cloning, 1989. 
The expression vector carrying the DNA construct of the invention may be 
any vector which may conveniently be subjected to recombinant DNA 
procedures, and the choice of vector will typically depend on the host 
cell into which it is to be introduced. Thus, the vector may be an 
autonomously replicating vector, i.e. a vector which exists as an 
extrachromosomal entity, the replication of which is independent of 
chromosomal replication, e.g. a plasmid, or an extrachromosomal element, 
minichromosome or an artificial chromosome. Alternatively, the vector may 
be one which, when introduced into a host cell, is integrated into the 
host cell genome and replicated together with the chromosome(s) into which 
it has been integrated. 
In the vector, the DNA sequence should be operably connected to a suitable 
promoter sequence. The promoter may be any DNA sequence which shows 
transcriptional activity in the host cell of choice and may be derived 
from genes encoding proteins either homologous or heterologous to the host 
cell. Examples of suitable promoters for directing the transcription of 
the DNA construct of the invention, especially in a bacterial host, are 
the promoter of the lac operon of E. coli, the Streptomyces coelicolor 
agarase gene dagA promoters, the promoters of the Bacillus licheniformis 
.alpha.-amylase gene (amyL), the promoters of the Bacillus 
stearothermophilus maltogenic amylase gene (amyM), the promoters of the 
Bacillus amyloliquefaciens .alpha.-amylase (amyQ), or the promoters of the 
Bacillus subtilis xylA and xylB genes. In a yeast host, a useful promoter 
is the eno-1 promoter. For transcription in a fungal host, examples of 
useful promoters are those derived from the gene encoding A. oryzae TAKA 
amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral 
.alpha.-amylase, A. niger acid stable .alpha.-amylase, A. niger or A. 
awamori glucoamylase (glaA), Rhizomucor miehei lipase, A. oryzae alkaline 
protease, A. oryzae triose phosphate isomerase or A. nidulans acetamidase. 
Preferred are the TAKA-amylase and glaA promoters. 
The expression vector of the invention may also comprise a suitable 
transcription terminator and, in eukaryotes, polyadenylation sequences 
operably connected to the DNA sequence encoding the laccase of the 
invention. Termination and polyadenylation sequences may suitably be 
derived from the same sources as the promoter. The vector may further 
comprise a DNA sequence enabling the vector to replicate in the host cell 
in question. Examples of such sequences are the origins of replication of 
plasmids pUC19, pACYC177, pUB110, pE194, pAMB1 and pIJ702. 
The vector may also comprise a selectable marker, e.g. a gene the product 
of which complements a defect in the host cell, such as the dal genes from 
B. subtilis or B. licheniformis, or one which confers antibiotic 
resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline 
resistance. Examples of Aspergillus selection markers include amdS, pyrG, 
argB, niad, sC, and hygB a marker giving rise to hygromycin resistance. 
Preferred for use in an Aspergillus host cell are the amds and pyrg 
markers of A. nidulans or A. oryzae. A frequently used mammalian marker is 
the dihydrofolate reductase (DHFR) gene. Furthermore, selection may be 
accomplished by co-transformation, e.g. as described in WO 91/17243. 
It is generally preferred that the expression gives rise to a product that 
is extracellular. The laccases of the present invention may thus comprise 
a preregion permitting secretion of the expressed protein into the culture 
medium. If desirable, this preregion may be native to the laccase of the 
invention or substituted with a different preregion or signal sequence, 
conveniently accomplished by substitution of the DNA sequences encoding 
the respective preregions. For example, the preregion may be derived from 
a glucoamylase or an amylase gene from an Aspergillus species, an amylase 
gene from a Bacillus species, a lipase or proteinase gene from Rhizomucor 
miehei, the gene for the .alpha.-factor from Saccharomyces cerevisiae or 
the calf preprochymosin gene. Particularly preferred, when the host is a 
fungal cell, is the preregion for A. oryzae TAKA amylase, A. niger neutral 
amylase, the maltogenic amylase form Bacillus NCIB 11837, B. 
stearothermophilus .alpha.-amylase, or Bacillus licheniformis subtilisin. 
An effective signal sequence is the A. oryzae TAKA amylase signal, the 
Rhizomucor miehei aspartic proteinase signal and the Rhizomucor miehei 
lipase signal. 
The procedures used to ligate the DNA construct of the invention, the 
promoter, terminator and other elements, respectively, and to insert them 
into suitable vectors containing the information necessary for 
replication, are well known to persons skilled in the art (cf., for 
instance, Sambrook et al. Nolecular Cloning, 1989). 
The cell of the invention either comprising a DNA construct or an 
expression vector of the invention as defined above is advantageously used 
as a host cell in the recombinant production of a enzyme of the invention. 
The cell may be transformed with the DNA construct of the invention, 
conveniently by integrating the DNA construct in the host chromosome. This 
integration is generally considered to be an advantage as the DNA sequence 
is more likely to be stably maintained in the cell. Integration of the DNA 
constructs into the host chromosome may be performed according to 
conventional methods, e.g. by homologous or heterologous is recombination. 
Alternatively, the cell may be transformed with an expression vector as 
described above in connection with the different types of host cells. 
The host cell may be selected from prokaryotic cells, such as bacterial 
cells. Examples of suitable bacteria are gram positive bacteria such as 
Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus 
brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus 
amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus 
lautus, Bacillus inegaterium, Bacillus thuringiensis, or Streptomyces 
lividans or Streptomyces murinus, or gram negative bacteria such as E. 
coli. The transformation of the bacteria may for instance be effected by 
protoplast transformation or by using competent cells in a manner known 
per se. 
The host cell may also be a eukaryote, such as mammalian cells, insect 
cells, plant cells or preferably fungal cells, including yeast and 
filamentous fungi. For example, useful mammalian cells include CHO or COS 
cells. A yeast host cell may be selected from a species of Saccharomyces 
or Schizosaccharomyces, e.g. Saccharomyces cerevisiae. Useful filamentous 
fungi may selected from a species of Aspergillus, e.g. Aspergillus oryzae 
or Aspergillus niger. Alternatively, a strain of a Fusarium species, e.g. 
F. oxysporum, can be used as a host cell. Fungal cells may be transformed 
by a process involving protoplast formation and transformation of the 
protoplasts followed by regeneration of the cell wall in a manner known 
per se. A suitable procedure for transformation of Aspergillus host cells 
is described in EP 238 023. A suitable method of transforming Fusarium 
species is described by Malardier et al., 1989. 
The present invention thus provides a method of producing a recombinant 
laccase of the invention, which method comprises cultivating a host cell 
as described above under conditions conducive to the production of the 
enzyme and recovering the enzyme from the cells and/or culture medium. The 
medium used to cultivate the cells may be any conventional medium suitable 
for growing the host cell in question and obtaining expression of the 
laccase of the invention. Suitable media are available from commercial 
suppliers or may be prepared according to published formulae (e.g. in 
catalogues of the American Type Culture Collection). 
The resulting enzyme may be recovered from the medium by conventional 
procedures including separating the cells from the medium by 
centrifugation or filtration, precipitating the proteinaceous components 
of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, 
followed by purification by a variety of chromatographic procedures, e.g. 
ion exchange chromatography, gel filtration chromatography, affinity 
chromatography, or the like. Preferably, the isolated protein is about 90% 
pure as determined by SDS-PAGE, purity being most important in food, juice 
or detergent applications. 
In a particularly preferred embodiment, the expression of laccase is 
achieved in a fungal host cell, such as Aspergillus. As described in 
detail in the following examples, the laccase gene is ligated into a 
plasmid containing the Aspergillus oryzae TAKA .alpha.-amylase promoter, 
and the Aspergillus nidulans amds selectable marker. Alternatively, the 
amdS may be on a separate plasmid and used in co-transformation. The 
plasmid (or plasmids) is used to transform an Aspergillus species host 
cell, such as A. oxyzae or A. niger in accordance with methods described 
in Yelton et al. (PNAS USA 81: 1470-1474, 1984). 
Those skilled in the art will recognize that the invention is not limited 
to use of the nucleic acid fragments specifically disclosed herein, for 
example, in FIG. 1. It will also be apparent that the invention 
encompasses those nucleotide sequences that encode the same amino acid 
sequences as depicted in FIG. 1, but which differ from those specifically 
depicted nucleotide sequences by virtue of the degeneracy of the genetic 
code. Also, reference to FIG. 1, in the specification and the claims will 
be understood to encompass both the genomic sequence depicted therein as 
well as the corresponding CDNA and RNA sequences, and the phrases "DNA 
construct" and "nucleic acid sequences" as used herein will be understood 
to encompass all such variations. "DNA construct" shall generally be 
understood to mean a DNA molecule, either single- or double-stranded, 
which may be isolated in partial form from a naturally occurring gene or 
which has been modified to contain segments of DNA which are combined and 
juxtaposed in a manner which would not otherwise exist in nature. 
In addition, the invention also encompasses other Scytalidium laccases, 
including alternate forms of laccase which may be found in S. thermophilum 
and as well as laccases which may be found in other fungi which are 
synonyms or fall within the definition of Scytalidium thermophilum as 
defined by Straatsma and Samson, 1993, supra. These include S. 
indonesiacum, Torula thermophila, Humicola brevis var. thermoidea, 
Humicola brevispora, H. grisea var. thermoidea, Humicola insolens, and 
Humicola lanuginosa (also known as Thermomyces lanuginosus). The invention 
also provides the means for isolation of laccase genes from other species 
of Scytalidium, such as S. acidophilum, S. album, S. aurantiacum, S. 
circinatum, S. flaveobrunneum, S. hyalinum, S. lignicola, and S. 
uredinicolum. Identification and isolation of laccase genes from sources 
other than those specifically exemplified herein can be achieved by 
utilization of the methodology described in the present examples, with 
publicly available Scytalidium strains. Alternately, the sequence 
disclosed herein can be used to design primers and/or probes useful in 
isolating laccase genes by standard PCR or southern hybridization 
techniques, using the same publicly available strains. Examples of such 
publicly available strains include, from the American Type Culture 
Collection, ATCC 16463, 28085, 36346, 48409, 66938 (S. thermophilum); 
24569 (S. acidophilum); 16675 (S. album); 22477 (S. aurantiacum); 66463 
(S. circinatum); 13212 (S. flavobrunneum); 52297 (S. fulvum); 38906 (S. 
hyalinum); 46858 (S. indonesiacum); 18984 (S. indonesiacum); 32382 (S. 
uredinaolum); from the International Mycological Institute (IMI; United 
Kingdom), IMI 243 118 (S. thermophilum); from Centraalbureau voor 
Schimmelcultures (CBS; Netherlands) CBS 183.81, 671.88 (S. thermophilum) 
367.72 (S. acidophilum); 372.65 (S. album); 374.65 (S. aurantiacum); 
654.89 (S. circinatum); 244.59 (S. flavo-brunneum); 145.78 (S. hyalinum); 
259.81 (S. indonesiacum); 233.57 (S. lignicola); 171.40 (S. terminale); 
616.84(S. muscorum); from Deutsche Sammlung von Mikroorganismenn und 
Zellkulturen (DSM; Germany) DSM 2842 (S thermophilum); DSM 2695 (S. 
lignicola). The invention also encompasses any variant nucleotide 
sequence, and the protein encoded thereby, which protein retains at least 
about an 80%, preferably about 85%, and most preferably at least about 
90-95% homology with the amino acid sequence depicted in FIG. 1, and which 
qualitatively retains the laccase activity of the sequence described 
herein. Useful variants within the categories defined above include, for 
example, ones in which conservative amino acid substitutions have been 
made, which substitutions do not significantly affect the activity of the 
protein. By conservative substitution is meant that amino acids of the 
same class may be substituted by any other of that class. For example, the 
nonpolar aliphatic residues Ala, Val, Leu, and Ile may be interchanged, as 
may be the basic residues Lys and Arg, or the acidic residues Asp and Glu. 
Similarly, Ser and Thr are conservative substitutions for each other, as 
are Asn and Gln. It will be apparent to the skilled artisan that such 
substitutions can be made outside the regions critical to the function of 
the molecule and still result in an active enzyme. Retention of the 
desired activity can readily be determined by conducting a standard ABTS 
oxidation method, such as is described in the present examples. 
The protein can be used in number of different industrial processes. These 
processes include polymerization of lignin, both Kraft and lignosulfates, 
in solution, in order to produce a lignin with a higher molecular weight. 
A neutral/alkaline laccase is a particular advantage in that Kraft lignin 
is more soluble at higher pHs. Such methods are described in, for example, 
Jin et al., Holzforschung 45(6): 467-468, 1991; U.S. Pat. No. 4,432,921; 
EP 0 275 544; PCT/DK93/00217, 1992. Laccase is also useful in the 
copolymerization of lignin with low molecular weight compounds, such as is 
described in Appl. Microbiol. Biotechnol. 40: 760-767. 
The laccase of the present invention can also be used for in-situ 
depolymerization of lignin in Kraft pulp, thereby producing a pulp with 
lower lignin content. This use of laccase is an improvement over the 
current use of chlorine for depolymerization of lignin, which leads to the 
production of chlorinated aromatic compounds, which are an environmentally 
undesirable by-product of paper mills. Such uses are described in, for 
example, Current opinion in Biotechnology 3: 261-266, 1992; J. Biotechnol. 
25: 333-339, 1992; Hiroi et al., Svensk papperstidning 5: 162-166, 1976. 
Since the environment in a paper mill is typically alkaline, the present 
laccase is more useful for this purpose than other known laccases, which 
function best under acidic conditions. 
Oxidation of dyes or dye precursors and other chromophoric compounds leads 
to decolorization of the compounds. Laccase can be used for this purpose, 
which can be particularly advantageous in a situation in which a dye 
transfer between fabrics is undesirable, e.g., in the textile industry and 
in the detergent industry. Methods for dye transfer inhibition and dye 
oxidation can be found in WO 92/01406; WO 92/18683; EP 0495836; Calvo, 
Mededelingen van de Faculteit Landbouw-wetenschappen/Rijiksuniversitet 
Gent.56: 1565-1567, 1991; Tsujino et al., J. Soc. Chem. 42: 273-282, 1991. 
The present laccase can also be used for the polymerization or oxidation of 
phenolic compounds present in liquids. An example of such utility is the 
treatment of juices, such as apple juice, so that the laccase will 
accelerate a precipitation of the phenolic compounds present in the juice, 
thereby producing a more stable juice. Such applications have been 
described in Stutz, Fruit processing 7/93, 248-252, 1993; Maier et al., 
Dt. Lebensmittel-rindschau 86(5): 137-142, 1990; Dietrich et al., Fluss. 
Obst 57(2): 67-73, 1990. 
Laccases such as the Scytalidium laccase are also useful in soil 
detoxification (Nannipieri et al., J. Environ. Qual. 20: 510-517, 1991; 
Dec and Bollag, Arch. Environ. Contam. Toxicol. 19: 543-550, 1990). 
The invention is further illustrated by the following non-limiting 
examples. 
EXAMPLES 
I. Isolation of Scytalidum thermophilum Laccase Gene 
A. Materials and Methods 
1. DNA Extraction and Hybridization analysis 
Total cellular DNA is extracted from fungal cells of Scytalidium 
thermophila strain E421 grown 24 hours in 25 ml of YEG medium (0.5% yeast 
extract, 2% glucose) using the following protocol: mycelia are collected 
by filtration through Miracloth (Calbiochem) and washed once with 25 ml of 
TE buffer. Excess buffer is drained from the mycelia which are 
subsequently frozen in liquid nitrogen. Frozen mycelia are ground to a 
fine powder in an electric coffee grinder, and the powder added to 20 ml 
of TE buffer and 5 ml of 20% SDS (w/v) in a disposable plastic centrifuge 
tube. The mixture is gently inverted several times to ensure mixing, and 
extracted twice with an equal volume of phenol:chloroform:isoamyl alcohol 
(25:24:1). Sodium acetate (3M solution) is added to give a final 
concentration of 0.3M and the nucleic acids are precipitated with 2.5 
volumes of ice cold ethanol. The tubes are centrifuged at 15,000.times.g 
for 30 minutes and the pellet is allowed to air-dry for 30 minutes before 
resuspending in 0.5 ml of TE buffer. DNase-free ribonuclease A is added to 
a concentration of 100 .mu.g/ml and the mixture is incubated at 37.degree. 
C. for 30 minutes. Proteinase K (200 .mu.g/ml) is added and each tube is 
incubated an additional one hour at 37.degree. C. Finally, each sample is 
extracted twice with phenol:chloroform:isoamyl alcohol before 
precipitating the DNA with sodium acetate and ethanol. DNA pellets are 
dried under vacuum, resuspended in TE buffer, and stored at 4.degree. C. 
Total cellular DNA samples are analyzed by Southern hybridization. 
Approximately 5 .mu.g of DNA is digested with EcoRI and fractionated by 
size on a 1% agarose gel. The gel is photographed under short wavelength 
UV and soaked for 15 minutes in 0.5M NaOH, 1.5M NaCl followed by 15 
minutes in 1 M Tris-HCl, pH 8, 1.5M NaCl. DNA in the gel is transferred 
onto Zeta-Probe.TM. hybridization membrane (BioRad Laboratories) by 
capillary blotting in 20.times.SSPE (R. W. Davis et al., Advanced 
Bacterial Genetics, A Manual for Genetic Engineering. Cold Spring Harbor 
Press. 1980) Membranes are baked for 2 hours at 80.degree. C. under vacuum 
and soaked for 2 hours in the following hybridization buffer at 45.degree. 
C. with gentle agitation: 5.times.SSPE, 35% formamide (v/v), 0.3% SCS, 200 
.mu.g/ml denatured and sheared salmon testes DNA. The laccase-specific 
probe fragment (approx. 1.5 kb) encoding the 5'-portion of the N. crassa 
lcc1 gene is amplified from N. crassa genomic DNA using standard PCR 
conditions (Perkin-Elmer Cetus, Emeryville, Calif.) with the following 
pair of primers: forward primer, 5' CGAGACTGATAACTGGCTTGG 3' (SEQ ID 
NO:3); reverse primer, 5' ACGGCGCATTGTCAGGGAAGT 3' (SEQ ID NO:4). The 
amplified DNA segment is first cloned into a TA-cloning vector 
(Invitrogen, Inc., San Diego, Calif.), then purified by agarose gel 
electrophoresis following digestion with EcoRI. The purified probe 
fragment is radiolabeled by nick translation with .alpha..sup.32 
P!dCTP(Amersham) and added to the hybridization buffer at an activity of 
approximately 1.times.10.sup.6 cpm per ml of buffer. The mixture is 
incubated overnight at 45.degree. C. in a shaking water bath. Following 
incubation, the membranes are washed once in 0.2.times.SSPE with 0.1% SDS 
at 45.degree. C. followed by two washes in 0.2.times.SSPE(no SDS) at the 
same temperature. The membranes are allowed to dry on paper towels for 15 
minutes, then wrapped in Saran Wrap.TM. and exposed to x-ray film 
overnight at -70.degree. C. with intensifying screens (Kodak). 
2. DNA Libraries and Identification of Laccase Clones 
Genomic DNA libraries are constructed in the bacteriophage cloning vector 
.lambda.-EMBL4 (J. A. Sorge, in Vectors, A Survey of Molecular Cloning 
Vectors and Their Uses, Rodriguez et al., eds, pp. 43-60, Butterworths, 
Boston, 1988). Briefly, total cellular DNA is partially digested with 
Sau3A and size-fractionated on low-melting point agarose gels. DNA 
fragments migrating between 9 kb and 23 kb are excised and eluted from the 
gel using .beta.-agarase (New England Biolabs, Beverly Mass.). The eluted 
DNA fragments are ligated with BamHI-cleaved and dephosphorylated 
.lambda.-EMBL4 vector arms, and the ligation mixtures are packaged using 
commercial packaging extracts (Stratagene, Lajolla, Calif.). The packaged 
DNA libraries are plated and amplified on Escherichia coli K802 cells. 
Approximately 10,000-20,000 plaques from each library are screened by 
plaque-hybridization with the radiolabeled lcc1 DNA fragment using the 
conditions described above. Plaques which give hybridization signals with 
the probe are purified twice on E. coli K802 cells, and DNA from the 
corresponding phage is purified from high titer lysates using a Qiagen 
Lambda kit (Qiagen, Inc., Chatsworth, Calif.). 
3. Analysis of Laccase Genes 
Restriction mapping of laccase clones is done using standard methods 
(Lewin, Genes. 2d ed., Wiley & Sons, 1985, New York). DNA sequencing is 
done with an Applied Biosystems Model 373A automated DNA Sequencer 
(Applied Biosystems, Inc., Foster City, Calif.) using the primer walking 
technique with dye-terminator chemistry (H. Giesecke et al., J. Virol. 
Methods 38: 47-60, 1992). Oligonucleotide sequencing primers are 
synthesized on an Applied Biosystems model 394 DNA/RNA Synthesizer. 
B. Results and Discussion 
1. Identification of Laccase Gene Sequence 
Total cellular DNA samples are prepared from the species Neurospora crassa, 
Botrytis cinerea, and Scytalidium. Aliquots of these DNA preparations are 
digested with BamHI and fractionated by agarose gel electrophoresis. DNA 
in the gel is blotted to a Zeta-Probe.TM. membrane filter (BioRad 
Laboratories, Hercules, Calif.) and probed under conditions of mild 
stringency with a radiolabeled fragment encoding a portion of the N. 
crassa lcc1 gene, as described above. Laccase-specific sequences are 
detected in the genomes of S. thermophilum and the N. crassa control, but 
not in the B. cinerea genomic DNA with this probe. 
2. Cloning and Characterization of Scytalidium thermophila Laccase (StL) 
Gene 
The S. thermophilum laccase gene is isolated using plaque hybridization to 
screen the genomic DNA library made in .lambda.-EMBL4. The library 
contains approximately 250,000 independent clones before amplification, 
and 12,000 plaques are screened by hybridization with a radiolabeled N. 
crassa laccase gene fragment as described above. Nine plaques are 
identified which hybridize strongly to the probe. DNA is isolated from 
four of these clones and analyzed by restriction mapping. All four contain 
a 3 kb BamHI fragment that is originally identified in southern blotting 
with genomic DNA as described above. This fragment is isolated from one 
clone and inserted into a pBluescript vector (Stratagene Cloning Systems, 
La Jolla, Calif.). However, DNA sequence analysis indicates that only a 
portion of the gene is located on this segment. Consequently, a 6 kb XhoI 
fragment which contains the entire lccS gene is subcloned into pbluescript 
to derive the plasmid pShTh6. A restriction map of the 6 kb insert in this 
plasmid is shown in FIG. 3. The nucleic acid sequence is shown in FIG. 1 
and SEQ ID NO:1. The deduced amino acid sequence of StL is obtained on the 
basis of amino acid sequence homology with N. crassa laccase. StL shares 
approximately 58% amino acid sequence identity with NcL, and this sequence 
similarity is highest among those amino residues that are involved in the 
formation of the active site copper center. StL, like NcL appears to be 
synthesized as a preproenzyme (616 amino acids with a 21 amino acid signal 
peptide and a propeptide of 24 amino acids). However, since the amino 
terminal sequence of the mature StL protein is not yet determined, the 
exact length of the propeptide is not certain. There are five potential 
sites for N-linked glycosylation in StL. A potential C-terminal processing 
signal with homology to N. crassa laccase also exists in StL 
(Asp-Ser-Gly-Leu*Lys.sub.564 (SEQ ID NO:5)) which may result in the 
proteolytic removal of the last seven amino acids from the primary 
translation product. 
The presence of four small introns (63, 58, 55 and 65 nucleotides) is 
determined by comparing the open reading frames within the coding region 
of lccS to the primary structure of NcL. Excluding these intervening 
sequences, the coding region contains 60.8% G+C. The base composition of 
lccS reflects a bias for codons ending in G or C. 
II. Expression of Scytalidium Laccase in Aspergillus 
A. Materials and Methods 
1. Bacterial and Fungal Host Strains 
Escherichia coli JM101 (Messing et al., Nucl. Acids Res. 9:309-321, 1981) 
is used as a host for construction and routine propagation of laccase 
expression vectors in this study. Fungal hosts for laccase expression 
included the Aspergillus niger strain Bo-1, as well as a 
uridine-requiring(pyrG) mutant of the .alpha.-amylase-deficient 
Aspergillus oryzae strain HowB104. 
2. Plasmids 
Plasmid pSHTh5 is a pBluescript(Stratagene Cloning Systems, LaJolla, 
Calif.) derivative which contains a 6 kb XhoI fragment of S. thermophilum 
DNA encoding StL. Plasmid pToC68(WO 91/17243) contains the A. oryzae 
TAKA-amylase promoter and A. niger glaa terminator, and pToC90(WO 
91/17243) carries the A. nidulans amdS gene. 
3. Construction of Laccase Expression Vectors 
The construction strategy for the laccase expression vector pShTh15 is 
outlined in FIG. 2. The promoter directing transcription of the laccase 
gene is obtained from the A. oryzae .alpha.-amylase (TAKA-amylase) gene 
(Christensen et al., supra), and terminator from the A. niger glaA 
(glucoamylase) terminator region. The expression vector is constructed as 
follows. A 60 basepair synthetic DNA linker, 
##STR1## 
including the region from start codon to an ApaI site, is inserted into 
XhoI- and ApaI-digested pBluescriptSK-(Stratagene, LaJolla, Calif.) to 
produce an intermediate termed pShTh11.5. This vector is digested with 
ApaI and Asp718 and ligated with a 662 base pair ApaI-Asp7l8 fragment 
encoding a portion of StL from pShTh5, generating a second intermediate 
called pShTh13.1. An XbaI site is introduced immediately downstream of the 
stop codon using pShTh5 as a template for a PCR reaction with the 
following primers:forward: 5'GTCATGAACAATGACCT 3'(SEQ ID NO:8); reverse: 
5'AGAGAGTCTAGATTAAACAATCCGCCCAACTAC3'(SEQ ID NO:9). The amplified fragment 
is digested with NsiI and XbaI and subcloned into pUC518 to create the 
intermediate called pShTh12.8. The pShTh12.8 vector is digested with EcoRI 
and Asp718 and ligated with a 700 base pair EcoRI-Asp718 fragment from 
pShTh13.1 to generate pShTh13.1 to generate pShTh13.2. An 800 base pair 
NsiI-Asp718 fragment containing the final portion of the laccase coding 
region is obtained from pShTh5 and inserted into NsiI- and Asp718-cleaved 
pShTh13.2 to give pShTh14. Lastly, the 2.2 kb laccase coding region in 
pShTh14 is removed by cleavage with XhoI and XbaI and inserted between the 
XhoI and XbaI sites of pToC68 to generate the expression vector pShTh15. 
4. Transformation of Aspergillus host cells 
Methods for co-transformation of Aspergillus strains are as described in 
Christensen et al., supra. For introduction of the laccase expression 
vectors into A. oryzae HowB 104 pyrG, equal amounts (approximately 5 .mu.g 
each) of laccase expression vector and pPyrG, which harbors the cloned A. 
nidulans pyrG gene, are used. Protrophic(Pyr.sup.+) transformants are 
selected on Aspergillus minimal medium (Rowlands and Turner, Mol. Gen. 
Genet. 126: 201-216, 1973), and the transformants are screened for the 
ability to produce laccase on minimal medium containing 1 mM 
2,2'-azinobis(3-ethylbenzthiazolinesulfonic acid) ABTS!. Cells which 
secrete active laccase oxidize the ABTS, producing a green halo 
surrounding the colony. A. niger Bo-1 protoplasts are cotransformed using 
equal amounts (approximately 5 .mu.g each) of laccase expression vector 
and pToC90 which contains the A. nidulans amdS (acetamidase) gene (Hynes 
et al., Mol. Cell Biol. 3: 1430-1439, 1983. AmdS.sup.+ transformants are 
selected on Cove minimal medium (Cove, Biochim. Biophys. Acta 113: 51-56, 
1966) with 1% glucose as the carbon source and acetamide as the sole 
nitrogen source and screened for laccase expression on Cove medium with 1 
mM ABTS. 
5. Analysis of Laccase-Producing Transformants 
Transformants which produce laccase activity on agar plates are purified 
twice through conidiospores and spore suspensions in sterile 0.01% 
Tween-80 are made from each. The density of spores in each suspension is 
estimated spectrophotometrically (A.sub.595 nm). Approximately 0.5 
absorbance units of spores are used to inoculate 25 ml of ASPO4 or MY50 
medium in 125 ml plastic flasks. The cultures are incubated at 37.degree. 
C. with vigorous aeration (approximately 200 rpm) for four to five days. 
Culture broths are harvested by centrifugation and the amount of laccase 
activity in the supernatant is determined using syringaldazine as a 
substrate. Briefly, 800 .mu.l of assay buffer (25 mM sodium acetate, pH 
5.5, 40 .mu.M CuSo.sub.4) is mixed with 20 .mu.l of culture supernatant 
and 60 .mu.l of 0.28 mM syringaldazine stock solution (Sigma Chemical is 
Co., St. Louis, Mo.) in 50% ethanol. The absorbance at 530 nm is measured 
over time in a Genesys 5 UV-vis spectrophotometer (Milton-Roy). One 
laccase unit (LACU) is defined as the amount of enzyme which oxidizes one 
.mu.mole of substrate per minute at room temperature. SDS-polyacrylamide 
gel electrophoresis (PAGE) is done using precast 10-27% gradient gels from 
Novex (San Diego, Calif.). Protein bands are developed using Coomassie 
Brilliant Blue(Sigma). 
B. Results and Discussion 
1. Expression of Scytalidium laccase 
The expression vector pShTh15 is used in conjunction with pPyrG (A. 
nidulans pyrG) or pToC90(A. nidulans amdS) plasmids to generate A. oryzae 
and A. niger co-transformants which express StL. The number of 
laccase-producing co-transformants obtained in A. oryzae HowB104pyrG is 
small (3.7% of Pyr.sup.+ transformants) compared to the number obtained 
in A. niger Bo-1 using amdS selection (71.5% of AmdS.sup.+ 
transformants). It is unknown whether this is due to an abnormally low 
co-transformation(i.e., integration) frequency or extremely low expression 
or laccase degradation in many A. oryzae transformants. Expression levels 
of StL range from about 50 mg/l in shake flasks and 1-2 g/l in a 
fermentor. 
III. Purification and Characterization of Recombinant Scytalidum Laccase 
A. Materials and Methods 
1. Materials 
Chemicals used as buffers and substrates are commercial products of at 
least reagent grade. Chromatography is performed on either a Pharmacia 
FPLC. Spectroscopic assays are conducted on either a spectrophotometer 
(Shimadzu PC160) or a microplate reader (Molecular Devices). Britton & 
Robinson (B&R) buffers are prepared according to the protocol described in 
Quelle, Biochemisches Taschenbuch, H. M. Raven, II. Teil, S.93 u. 102, 
1964. 
2. Fermentation 
A 1 ml aliquot of a spore suspension of Aspergillus oryzae transformant 
HowB104-pShTh15-2(approximately 10.sup.9 spores/ml) is added aseptically 
to a 500 ml shake flask containing 100 ml of sterile shake flask medium 
(maltose, 50 g/l; MgSO.sub.4.7H.sub.2 O, 2 g/l; KH.sub.2 PO.sub.4, 10 g/l; 
K.sub.2 SO.sub.4, 2 g/l; CaCl.sub.2.2H.sub.2 O 0.5 g/l; Citric acid, 2 
g/l; yeast extract, 10 g/l; trace metals ZnSO.sub.4.7H.sub.2 O, 14.3 g/l; 
CuSO.sub.4.5H.sub.2 O, 2.5 g/l; NiCl.sub.2.6H.sub.2 O, 0.5 g/l; 
FeSO.sub.4.7H.sub.2 O, 13.8 g/l, MnSO.sub.4.H.sub.2 O, 8.5 g/l; citric 
acid, 3.0 g/l!, 0.5 ml/l; urea, 2 g/l, made with tap water and adjusted to 
pH 6.0 before autoclaving), and incubated at 37.degree. C. on a rotary 
shaker at 200 rpm for 18 hours. 50 ml of this culture is aseptically 
transferred to a 3 liter fermentor containing 1.8 liters of the fermentor 
media (MgSO.sub.4.7H.sub.2 O, 2 g/l; KH.sub.2 PO.sub.4, 2 g/l; citric acid 
4 g/l; K.sub.2 SO.sub.4, 3 g/l; CaCl.sub.2.2H.sub.2 O, 2 g/l; trace 
metals, 0.5 ml/l; pluronic antifoam, 1 ml/l). The fermentor temperature is 
maintained at 34.degree. C. by the circulation of cooling water through 
the fermentor jacket. Sterile air is sparged through the fermentor at a 
rate of 1.8 liter/min (1 v/v/m). The agitation rate is maintained between 
600 and 1300 rpm at approximately the minimum level required to maintain 
the dissolved oxygen level in the culture above 20%. Sterile feed 
(Nutriose 725maltose syrup!, 225 g/l; urea, 30 g/l; yeast extract, 15 
g/l; pluronic antifoam, 1.5 ml/l, made up with distilled water and 
autoclaved) is added to the fermentor by use of a peristaltic pump. The 
feed rate profile during the fermentation is as follows: 30 g of feed is 
added initially before inoculation; 0-24 h, 2 g/l h; 24-48 h, 4 g/l h; 48 
h-end, 6 g/l. 
Copper(in the form of CuCl.sub.2, CuSO4 or other soluble salt) is made as a 
400.times. stock in water or a suitable buffer, filter sterilized and 
added aseptically to the tank to a final level of 0.5 mM. 
Samples for enzyme activity determination are withdrawn and filtered 
through Miracloth to remove mycelia. These samples are assayed for laccase 
activity by the LACU assay described above. Laccase activity is found to 
increase continuously during the course of the fermentation, with a value 
of approximately 3.6 LACU/ml achieved after 115 hours in the fermentation 
containing excess copper. At a specific activity of 1.9 LACU/mg, this 
corresponds to over 1.8 g/l recombinant laccase expressed by this 
transformant. 
3. Enzymatic Assay 
Laccase activity is determined by syringaldazine oxidation at 30.degree. C. 
in a 1-cm quartz cuvette. 60 .mu.l syringaldazine stock solution (0.28 mM 
in 50% ethanol) and 20 gl sample are mixed with 0.8 ml preheated buffer 
solution. The oxidation is monitored at 530 nm over 5 minutes. The 
activity is expressed as pnole substrate oxidized per minute. B&R buffers 
with various pHs are used. The activity unit is referred to here as "SOU". 
A buffer of 25 mM sodium acetate, 40 .mu.M CuSO.sub.4, pH 5.5, is also 
used to determine the activity, which is referred to as LACU, as defined 
above. 2,2'-azinobis(3-ethylbenzo thiazoline-6-sulfonic acid) (ABTS) 
oxidation assays are done using 0.4 mM ABTS, B&R buffer, pH 4.1, at room 
temperature by monitoring .DELTA.A.sub.405. An ABTS oxidase activity 
overlay assay is performed by pouring cooled ABTS-agarose(0.05 g ABTS, 1 g 
agarose, 50 ml H.sub.2 O, heated to dissolve agarose) over a native-IEF 
gel and incubating at room temperature. Thermostability analysis is 
performed using samples that have .about.3 .mu.M enzyme preincubated for 
one hour in B&R buffer, at pH 2.7, 6.1, and 9.0, and various temperatures. 
Samples are assayed after a 44-fold dilution into B & R buffer, pH 4.1, at 
room temperature. 
3. Purification from a fermentor broth 
1.2 liters of cheese-cloth filtered broth (pH 7.9, 13 mS) is filtered 
through Whatman #2 filter paper and concentrated on a Spiral Concentrator 
(Amicon) with a S1Y100 membrane (MWCO:100) to 200 ml. The concentrate is 
adjusted to 0.86 mS by diluting it in water and reconcentrated on S1Y100 
to 324 ml. The washed and concentrated broth has a dense greenish color. 
The broth is frozen overnight at -.degree.20.degree. C., thawed the next 
day(without any loss of activity) and loaded onto a Q-Sepharose XK26 
column (120 ml), preequilibrated with 10 mM Tris, pH 7.7, 0.9 mS. The blue 
laccase band is eluted during a linear gradient with 2M NaCl. 
Pooled laccase fractions(44 ml), dialyzed in 3.5 liters of 10 mM NaAc, pH 
5.5, 0.8 mS at 4.degree. C. overnight, are loaded onto a Mono-Q 16/10 (40 
ml), preequilibrated with 10 mM MES, pH 5.3, 0.8 mS. The laccase eluted 
during a linear gradient with 1M NaCl shows apparent homogeneity on 
SDS-PAGE. 
4. Analysis of amino acid content and N-terminus 
N-terminal sequencing is performed on an ABI 476A sequencer; and total 
amino acid analysis, from which the extinction coefficient of laccase is 
determined, is performed on a HP AminoQuant instrument. 
B. Results and Discussion 
1. Purification 
From 1200 ml fermentor broth, about 0.6 g of laccase are isolated. Initial 
concentration using a membrane with MWCO of 100 kDa removes significant 
amounts of brown material and small contaminant proteins. The low affinity 
of the laccase toward Q-Sepharose matrix equilibrated with 10 mM Tris, pH 
7.7, facilitates its separation from other impurities. The enriched 
fractions are further purified by Mono-Q at pH 5.3. Although it has a pI 
of 5.1, the laccase migrates slowly on Mono-Q and is separated from 
impurities during the washing by 10 mM MES, pH 5.3. An overall 15-fold 
purification and a recovery of 60% are achieved. 
2. Characterization 
The purified laccase shows a MW of 75-80 kDa on SDS-PAGE. The difference 
between the MW derived from DNA sequence(63 kDa) and the observed MW is 
attributable to glycosylation. Native IEF shows 3 bands near pI of about 
5.1, which are active in ABTS overlay assay. 
3. N-terminal Sequencing 
Directly sequencing the N-terminus of the purified laccase from samples 
either in desalted solution or on PVDF membrane are unsuccessful. This 
result suggests a blocked N-terminus, likely a pyroglutamate site based on 
the gene sequence. 
The spectrum of the blue laccase has absorption maxima at 276 and 602 nm; 
with AbS.sub.280 /Abs.sub.600 =23 and AbS.sub.330 /Abs.sub.589 =2.1. The 
extinction coefficient determined by amino acid analysis is 1.9 l/(g*cm). 
The activity is tested by using either syringaldazine or ABTS as 
substrates. Expressed as per AbS.sub.280 or per mg, the laccase has a 
value of 2.2 or 4.2 units for SOU at pH 7, respectively. 
The pH profiles of laccase activity has optimal pH of 7 and 4, for 
syringaldazine and ABTS oxidation, respectively (FIG. 4). Thermostability 
analysis at three pHs is shown in FIG. 5. The laccase is more stable at 
neutral to alkaline pH than at acidic pH. Thermoactivation is also 
observed in neutral-alkaline pH range. 
Deposit of Biological Materials 
The following biological material has been deposited under the terms of the 
Budapest Treaty with the Agricultural Research Service Patent Culture 
Collection, Northern Regional Research Center, 1815 University Street, 
Peoria, Ill., 61604 and given the following accession number. 
______________________________________ 
Deposit Accession Number 
______________________________________ 
E. coli JM101 containing 
NRRL B-21262 
pShTh15 
______________________________________ 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 9 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2476 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: DNA (genomic) 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Scytalidium thermophilum 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 349..411 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 502..559 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 632..686 
(ix) FEATURE: 
(A) NAME/KEY: intron 
(B) LOCATION: 1739..1804 
(ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: join (106..348, 412..501, 560..631, 687..1738, 
1805..2194) 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: 
CTGAATTTAAATACAGGAAGATCGCATTCAATCCAGCCTAGACTGCACAATGGTTCTGCA60 
CGACCGTCGCACACCTGCCAATAGTGTTAATAACGGCCTAATACCATGAAGCGCTTC117 
MetLysArgPhe 
TTCATTAATAGCCTTCTGCTTCTCGCAGGGCTCCTCAACTCAGGGGCC165 
PheIleAsnSerLeuLeuLeuLeuAlaGlyLeuLeuAsnSerGlyAla 
5101520 
CTCGCGGCTCCGTCTACACATCCCAGATCAAACCCCGACATACTGCTT213 
LeuAlaAlaProSerThrHisProArgSerAsnProAspIleLeuLeu 
253035 
GAAAGAGATGACCACTCCCTTACGTCTCGGCAAGGTAGCTGTCATTCT261 
GluArgAspAspHisSerLeuThrSerArgGlnGlySerCysHisSer 
404550 
CCAAGCAACCGCGCCTGTTGGTGCTCTGGCTTCGATATCAACACGGAT309 
ProSerAsnArgAlaCysTrpCysSerGlyPheAspIleAsnThrAsp 
556065 
TATGAGACCAAGACTCCAAACACCGGAGTGGTGCGGCGGGTTAGTATCC358 
TyrGluThrLysThrProAsnThrGlyValValArgArg 
707580 
CAAGTTACGTTTGACCAAGAAATGGACGTGAAGTGTGCTGACTCTCCCGCTAG411 
TACACCTTTGATATCACCGAAGTCGACAACCGCCCCGGTCCCGATGGG459 
TyrThrPheAspIleThrGluValAspAsnArgProGlyProAspGly 
859095 
GTCATCAAGGAGAAGCTCATGCTTATCAACGACAAACTCCTGGTAGG506 
ValIleLysGluLysLeuMetLeuIleAsnAspLysLeuLeu 
100105110 
GTCCTCTCGAACGCCTGCGTCTGCCACACAGCGTAAAACTAACGAACCGCTAG559 
GGCCCGACAGTCTTCGCAAACTGGGGCGACACCATCGAGGTGACCGTC607 
GlyProThrValPheAlaAsnTrpGlyAspThrIleGluValThrVal 
115120125 
AACAACCACCTGAGAACCAACGGAGTAAGCGTTCGGACACAAAGCCCAGCAACC661 
AsnAsnHisLeuArgThrAsnGly 
130135 
TAGACACACTCAACTGACCAAGTAGACCTCCATCCACTGGCACGGCTTGCACCAA716 
ThrSerIleHisTrpHisGlyLeuHisGln 
140145 
AAAGGAACCAACTACCACGACGGCGCCAACGGCGTGACCGAGTGTCCC764 
LysGlyThrAsnTyrHisAspGlyAlaAsnGlyValThrGluCysPro 
150155160 
ATCCCGCCCGGTGGCTCCCGAGTCTACAGCTTCCGAGCGCGCCAATAT812 
IleProProGlyGlySerArgValTyrSerPheArgAlaArgGlnTyr 
165170175 
GGAACGTCATGGTACCACTCCCACTTCTCCGCCCAGTATGGCAACGGC860 
GlyThrSerTrpTyrHisSerHisPheSerAlaGlnTyrGlyAsnGly 
180185190 
GTGAGCGGCGCCATCCAGATCAACGGACCCGCCTCCCTGCCCTACGAC908 
ValSerGlyAlaIleGlnIleAsnGlyProAlaSerLeuProTyrAsp 
195200205 
ATCGACCTCGGCGTCCTCCCGCTGCAGGACTGGTACTACAAGTCCGCC956 
IleAspLeuGlyValLeuProLeuGlnAspTrpTyrTyrLysSerAla 
210215220225 
GACCAGCTCGTCATCGAGACCCTGGCCAAGGGCAACGCTCCGTTCAGC1004 
AspGlnLeuValIleGluThrLeuAlaLysGlyAsnAlaProPheSer 
230235240 
GACAACGTCCTCATCAACGGCACCGCAAAGCACCCCACCACTGGCGAA1052 
AspAsnValLeuIleAsnGlyThrAlaLysHisProThrThrGlyGlu 
245250255 
GGGGAGTACGCCATCGTGAAGCTCACCCCGGGCAAACGCCATCGCCTG1100 
GlyGluTyrAlaIleValLysLeuThrProAspLysArgHisArgLeu 
260265270 
CGGCTCATCAACATGTCGGTGGAGAACCACTTCCAGGTCTCGCTGGCG1148 
ArgLeuIleAsnMetSerValGluAsnHisPheGlnValSerLeuAla 
275280285 
AAGCACACCATGACGGTCATCGCGGCGGACATGGTCCCCGTCAACGCC1196 
LysHisThrMetThrValIleAlaAlaAspMetValProValAsnAla 
290295300305 
ATGACCGTCGACAGCCTGTTTATGGCCGTCGGGCAGCGGTATGATGTT1244 
MetThrValAspSerLeuPheMetAlaValGlyGlnArgTyrAspVal 
310315320 
ACCATCGACGCGAGCCAGGCGGTGGGGAATTACTGGTTCAACATCACC1292 
ThrIleAspAlaSerGlnAlaValGlyAsnTyrTrpPheAsnIleThr 
325330335 
TTTGGAGGGCAGCAGAAGTGCGGCTTCTCGCACAATCCGGCGCCGGCA1340 
PheGlyGlyGlnGlnLysCysGlyPheSerHisAsnProAlaProAla 
340345350 
GCCATCTTTCGCTACGAGGGCGCTCCTGACGCTCTGCCGACGGATCCT1388 
AlaIlePheArgTyrGluGlyAlaProAspAlaLeuProThrAspPro 
355360365 
GGCGCTGCGCCAAAGGATCATCAGTGCCTGGACACTTTGGATCTTTCA1436 
GlyAlaAlaProLysAspHisGlnCysLeuAspThrLeuAspLeuSer 
370375380385 
CCGGTGGTGCAAAAGAACGTGCCGGTTGACGGGTTCGTCAAAGAGCCT1484 
ProValValGlnLysAsnValProValAspGlyPheValLysGluPro 
390395400 
GGCAATACGCTGCCGGTGACGCTCCATGTTGACCAGGCCGCGGCTCCA1532 
GlyAsnThrLeuProValThrLeuHisValAspGlnAlaAlaAlaPro 
405410415 
CACGTGTTTACGTGGAAGATCAACGGGAGCGCTGCGGACGTGGACTGG1580 
HisValPheThrTrpLysIleAsnGlySerAlaAlaAspValAspTrp 
420425430 
GACAGGCCGGTGCTGGAGTATGTCATGAACAATGACCTGTCTAGCATT1628 
AspArgProValLeuGluTyrValMetAsnAsnAspLeuSerSerIle 
435440445 
CCGGTCAAGAACAACATTGTGAGGGTGGACGGAGTCAACGAGTGGACG1676 
ProValLysAsnAsnIleValArgValAspGlyValAsnGluTrpThr 
450455460465 
TACTGGCTCGTCGAAAACGACCCGGAGGGCCGCCTCAGTTTGCCGCAT1724 
TyrTrpLeuValGluAsnAspProGluGlyArgLeuSerLeuProHis 
470475470 
CCGATGCATCTACACGTAAGTCACATCCCCCACTACCATTCGGAATGACCACCAG1779 
ProMetHisLeuHis 
475 
GTACTGACACCCTCCTCCTCAATAGGGACACGATTTCTTTGTCCTAGGCCGC1831 
GlyHisAspPhePheValLeuGlyArg 
480485 
TCCCCCGACGTCTCGCCCGATTCAGAAACCCGCTTCGTCTTTGACCCG1879 
SerProAspValSerProAspSerGluThrArgPheValPheAspPro 
490495500 
GCCGTCGACCTCCCCCGTCTGCGCGGACACAACCCCGTCCGGCGCGAC1927 
AlaValAspLeuProArgLeuArgGlyHisAsnProValArgArgAsp 
505510515 
GTCACCATGCTTCCCGCGCGCGGCTGGCTGCTGCTGGCCTTCCGCACG1975 
ValThrMetLeuProAlaArgGluTrpLeuLeuLeuAlaPheArgThr 
520525530 
GACAACCCGGGCGCGTGGTTGTTCCACTGCCACATCGCGTGRCACGTG2023 
AspAsnProGlyAlaTrpLeuPheHisCysHisIleAlaTrpHisVal 
535540545 
TCGGGCGGGTTAAGCGTCGACTTTCTGGAGCGGCCGGACGAGCTGCGC2071 
SerGlyGlyLeuSerValAspPheLeuGluArgProAspGluLeuArg 
550555560565 
GGGCAGCTGACGGGAGAGAGCAAGGCGGAGTTGGAGCGTGTTTGTCGC2119 
GlyGlnLeuThrGlyGluSerLysAlaGluLeuGluArgValCysArg 
570575580 
GAGTGGAAGGATTGGGAGGCGAAGAGCCCGCATGGGAAGATCGATTCG2167 
GluTrpLysAspTrpGluAlaLysSerProHisGlyLysIleAspSer 
585590595 
GGGTTGAAGCAGCGGCGATGGGATGCGTGAGGTAGTTGGGCGGATTG2214 
GlyLeuLysGlnArgArgTrpAspAla 
600605 
TTTAACACGTAGTGGGTAAGGTTGGGGCGGGTTTGTTTGGCGTTTTCAGGGGTTGGGGTG2274 
CGGATGCTGGTCATCCGGGAAACGGCTCTACAACTGGTGTCAATAGACTAATATAGAGTG2334 
ATCAAAGAACTGAGGTTCTGAAAGAGGCGTGGAAGTCGCGTTGTGACTCCCTTTGCCATG2394 
TTGGGAAGTGTGGCTCAACATTGTGTTCAGGTTTGCTCAGGGTGATNTCGAACTGACGTN2454 
TTGATGAGGGTTATTGCNTAGA2476 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 616 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Scytalidium thermophilum 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
MetLysArgPhePheIleAsnSerLeuLeuLeuLeuAlaGlyLeuLeu 
151015 
AsnSerGlyAlaLeuAlaAlaProSerThrHisProArgSerAsnPro 
202530 
AspIleLeuLeuGluArgAspAspHisSerLeuThrSerArgGlnGly 
354045 
SerCysHisSerProSerAsnArgAlaCysTrpCysSerGlyPheAsp 
505560 
IleAsnThrAspTyrGluThrLysThrProAsnThrGlyValValArg 
65707580 
ArgTyrThrPheAspIleThrGluValAspAsnArgProGlyProAsp 
859095 
GlyValIleLysGluLysLeuMetLeuIleAsnAspLysLeuLeuGly 
100105110 
ProThrValPheAlaAsnTrpGlyAspThrIleGluValThrValAsn 
115120125 
AsnHisLeuArgThrAsnGlyThrSerIleHisTrpHisGlyLeuHis 
130135140 
GlnLysGlyThrAsnTyrHisAspGlyAlaAsnGlyValThrGluCys 
145150155160 
ProIleProProGlyGlySerArgValTyrSerPheArgAlaArgGln 
165170175 
TyrGlyThrSerTrpTyrHisSerHisPheSerAlaGlnTyrGlyAsn 
180185190 
GlyValSerGlyAlaIleGlnIleAsnGlyProAlaSerLeuProTyr 
195200205 
AspIleAspLeuGlyValLeuProLeuGlnAspTrpTyrTyrLysSer 
210215220 
AlaAspGlnLeuValIleGluThrLeuAlaLysGlyAsnAlaProPhe 
225230235240 
SerAspAsnValLeuIleAsnGlyThrAlaLysHisProThrThrGly 
245250255 
GluGlyGluTyrAlaIleValLysLeuThrProAspLysArgHisArg 
260265270 
LeuArgLeuIleAsnMetSerValGluAsnHisPheGlnValSerLeu 
275280285 
AlaLysHisThrMetThrValIleAlaAlaAspMetValProValAsn 
290295300 
AlaMetThrValAspSerLeuPheMetAlaValGlyGlnArgTyrAsp 
305310315320 
ValThrIleAspAlaSerGlnAlaValGlyAsnTyrTrpPheAsnIle 
325330335 
ThrPheGlyGlyGlnGlnLysCysGlyPheSerHisAsnProAlaPro 
340345350 
AlaAlaIlePheArgTyrGluGlyAlaProAspAlaLeuProThrAsp 
355360365 
ProGlyAlaAlaProLysAspHisGlnCysLeuAspThrLeuAspLeu 
370375380 
SerProValValGlnLysAsnValProValAspGlyPheValLysGlu 
385390395400 
ProGlyAsnThrLeuProValThrLeuHisValAspGlnAlaAlaAla 
405410415 
ProHisValPheThrTrpLysIleAsnGlySerAlaAlaAspValAsp 
420425430 
TrpAspArgProValLeuGluTyrValMetAsnAsnAspLeuSerSer 
435440445 
IleProValLysAsnAsnIleValArgValAspGlyValAsnGluTrp 
450455460 
ThrTyrTrpLeuValGluAsnAspProGluGlyArgLeuSerLeuPro 
465470475480 
HisProMetHisLeuHisGlyHisAspPhePheValLeuGlyArgSer 
485490495 
ProAspValSerProAspSerGluThrArgPheValPheAspProAla 
500505510 
ValAspLeuProArgLeuArgGlyHisAsnProValArgArgAspVal 
515520525 
ThrMetLeuProAlaArgGlyTrpLeuLeuLeuAlaPheArgThrAsp 
530535540 
AsnProGlyAlaTrpLeuPheHisCysHisIleAlaTrpHisValSer 
545550555560 
GlyGlyLeuSerValAspPheLeuGluArgProAspGluLeuArgGly 
565570575 
GlnLeuThrGlyGluSerLysAlaGluLeuGluArgValCysArgGlu 
580585590 
TrpLysAspTrpGluAlaLysSerProHisGlyLysIleAspSerGly 
595600605 
LeuLysGlnArgArgTrpAspAla 
610615 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CGAGACTGATAACTGGCTTGG21 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
ACGGCGCATTGTCAGGGAAGT21 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 5 amino acids 
(B) TYPE: amino acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
AspSerGlyLeuLys 
15 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 65 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
TCGAGATGAAGCGCTTCTTCATTAATAGCCTTCTGCTTCTCGCAGGGCTCCTCAACTCAG60 
GGGCC65 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 57 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
CCTGAGTTGAGGAGCCCTGCGAGAAGCAGAAGGCTATTAATGAAGAAGCGCTTCATC57 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 17 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
GTCATGAACAATGACCT17 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: cDNA 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
AGAGAGTCTAGATTAAACAATCCGCCCAACTAC33 
__________________________________________________________________________