Effective Conversion of Renewable Plant Biomass to Energy by Using

Molecular-Genetically Improved Fungi

Research Coordinator:
Masaaki Kuwahara, Kyoto University
Team Members:
Michael H. Gold, Oregon Graduate Institute of Science and Technology, U. S. A.
Yitzhak Hadar, The Hebrew University of Jerusalem, Israel
Christopher F. Thurston, King’s College London, U. K.
Kazuo Shishido, Tokyo Institute of Technology, Japan
Hiroyuki Wariishi, Kyushu Univeristy, Japan
Yasushi Morikawa, Nagaoka University of Technology, Japan

Duration: April 1998 - March 2000

1. INTRODUCION
Biomass resources derived from plants (lignocellulosic resources) including wood and its residues
and agricultural wastes contain carbohydrate polymers such as cellulose and hemicellulose and a phenolic
polymer, lignin, as their main components. These components are the most abundant renewable and
sustainable resources on the earth, and can be used as the alternatives to petroleum and other fossil
resources. The proposed process to produce these useful materials is summarized in Fig. 1.

Fig. 1 Scheme of the conversion of lignocellulosic materials to alcohol, sugars and chemicals

A group of fungi, wood-rotting fungi, degrades wood and plant resources effectively. These fungi
produce enzymes which degrade component polymers of plants to produce low molecular weight
compounds. The purpose of this study is to produce sugars from carbohydrate polymers which can be
converted to alcohol and other commodity chemicals and to remove lignin which prevents the attack by
enzymes using fungi. This study also aims to enhance enzyme productivity by using molecular-
genetical techniques.
The main subjects of this research are as follows:
1) Development of up-stream technologies
Genetic transformation system of fungi including basidiomycetes will be constructed for the

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enhancement of productivity of cellulase, hemicellulase and lignin-degrading enzymes. Protein
engineering technologies are applied to improve the catalytic activities of these enzymes
2) Development of down-stream technologies
Production of sugars and removal of lignin from plant biomass by using genetically improved fungi
and enzymes will be examined as well as large-scale production of these enzymes by genetically
improved fungi. Finally, practical conditions for the production of sugars, alcohol and other chemicals
will be analyzed based on the results obtained in this project.

2. RESULTS
2.1 Genetics Engineering for the Expression of Ligninolytic Enzymes
It has been well recognized that white-rot fungi produce ligninolytic phenol oxidases such as lignin
peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) extracellularly. Mechanism of oxidative
degradation of lignin and its related aromatic compounds by these enzymes is understood to be the one
electron oxidation of the substrates. In this research, construction of homologous and heterologous
expression systems for ligninolytic enzymes, mainly MnP, was examined.

2.1.1 Construction of expression system in Dichomitus squalens

Dichomitus squalens belongs to a group of white-rot fungi which express manganese peroxidase
(MnP) and laccase but do not express lignin peroxidase (LiP). To understand the mechanism of lignin
degradation by these fungi, we have initiated work on the MnP genes and proteins from D. squalens
Genes encoding two MnP from D. squalens were cloned and sequenced. The mnp1 and mnp2 genes
encode mature proteins of 369 and 365 amino acids, respectively. The amino acids involved in
peroxidase function, those forming the MnII binding site, and those forming the five disulfide bonds in
other MnPs are conserved in these sequences. Both predicted D. squalens proteins contain multiple
acidic residues in their C-terminal sequences. Both genes contain seven small introns, the locations of
which align with each other.
The predicted sequences of the two mature DsMnP proteins are compared with that of MnP1 from P.
chrysosporium in Table 1. The three genes show 69% sequence identity and 80% sequence identity plus
similarity, suggesting they are closely related. Amino acids that are known to be involved in the

Table 1. N-Terminal amino acid sequencing of rDsMnP

a
Conserved amino acids are underlined. The mature protein sequence is indicated in bold with the actual N-
terminal amino acid overlined. *Indicates the computer predicted N terminus.

catalytic cycle of peroxidases--His46, Arg42, Asn80 and Glu74 on the distal side of the heme and His173
and Asp242 on the proximal side--are conserved in the DsMnP sequences as well as in peroxidase
sequences from various other plants and fungi. Sequences surrounding and including the MnII binding

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ligands in PcMnP1--Glu35, Glu39 and Asp179 --are conserved in the predicted DsMnPs as is the position
of Arg177, a residue also involved in MnII binding. The position of the ten Cys residues of PcMnP1
known to participate in disulfide bonds also are conserved in the predicted DsMnP proteins. Finally, the
N-glycosylation sequence NXS/T at position 131 in PcMnP1 is conserved in both DsMnP protein
sequences.
To facilitate structure/function studies of
MnP from D. squalens, we heterologously
expressed the enzyme in the well-studied
basidiomycete, Phanerochaete chrysosporium.
The glyceraldehydes–3 -phosphate-dehydroge-
nase (gpd) promoter of P. chrysosporium was
fused to the coding region of the mnp2 gene of
D. squalens and placed in a vector containing
the ura1 gene as a selectable marker. Extra-
cellular rDsMnP activity was detected in
shaken HCHN (high carbon and high nitrogen)
cultures of both purified pUDGM2 trans-
formants. The time course showed maximal
rDsMnP activity in selected fransformant (T32
and T41) after 72 h. No additional MnP
activity peaks appeared during this period nor
was any MnP activity observed within 9 days
of inoculation in either the wild-type P.
chrysosporium strain or in a Ura- strain trans-
formed with the pUB vector (Fig. 2).
Purified recombinant protein (rDsMnP) was similar in kinetic and spectral characteristics to the wild-
type MnPs from D. squalens and P. chrysosporium (PcMnP). The N-terminal amino acid sequence of the
rDsMnP was determined and was identical to the predicted sequence. However, the protein from D.
squalens was considerably more thermostable than its P. chrysosporium homolog with half-lives 15- to 40-
fold longer at 55°C. As previously demonstrated for PcMnP, addition of exogenous MnII or CdII
conferred additional thermal stability to rDsMnP.

2.1.2 Construction of expression system in Coprinus cinereus

By using the promoters and terminator of the Lentinus edodes genes, two chromosome-integrating
vectors pLC1 and pLC2 were constructed (Fig. 3). Both vectors contains priA or ras promoter and priA
terminater. pLC1 and pLC2 are very useful for the expression of foreign genes in various
basidiomycete fungi. P. ostreatus manganese (II) peroxidase (MnP) cDNA (mnpc) was fused between
the promoter and terminator of pLC1 and pLC2, yielding the recombinant plasmids pLC1-mnp and
pLC2-mnp (Fig. 3). These plasmids were introduced into the monokaryotic C. cinereus LT2-44 (trp1-1,
1-6) genome by co-transforming with pCc1001 carrying the C. cinereus TRP1 gene, and selecting for
Trp+ transformants. Selected transformants were cultured in the medium containino lignin with shaking
and the Intense decoloriation and degradation of lignin was observed after 16 days of cultuivation.

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2.1.3 Construction of expression system in Pleurotus ostreatus
The cDNA and the structural gene fragments of P. ostreatus (IFO 36160) MnP3, the major isozyme,
was isolated and the sequence structure of these DNAs was clarified. It was indicated that the isolated
structural gene, mnp3, contains a coding sequence of 1074 nucleotides which is interrupted by 10 introns
and encodes a protein of 358 amino acids. The amino acids known to be involved in peroxidase
functions, i.e. the distal His and Arg, and the proximal His, were conserved in MnP3. The residues
which constitute the Mn binding site of P. chrysosporium MnP isozyme were also conserved. The
promoter and an allelic sequence of the iron-sulfur protein subunit (sdh1) gene were isolated from P.
ostreatus. This gene was used as a genetic marker of P. ostreatus in the construction of expression
vector of this fungus.
An expression cassette was constructed using
the promoter and terminator sequence of P. ostreatus
sdi1 gene, followed by insertion of the coding
sequence of mnp3 genomic and cDNA clones (Fig.
4). These constructs were introduced into wild-type
P. ostreatus strain by co-transformation using a
carboxin resistance marker plasmid, pTM1. Strains
with high MnP activity were isolated from the co-
transformants. One of the recombinants obtained by
a mating between the two monokaryotic
transformants, TMG9-C1, showed a several times
higher level of the original strain. MnP activity
than the wild-type control in the early stage of liquid
culture of the mycelium (Fig. 5). This is the first
report of the homologous expression of a
recombinant gene in P. ostreatus.

2.1.4 Construction of expression system in Coriolus hirsutus

Two new chromosome-integrating vectors pHGP and pHRP breeding of C. hirsutus were constructed
(Fig. 6). The former carries C. hirsutus gpd promoter and L. edodes priA terminator and the latter does C.
hirsutus ras promoter and L. edodes priA terminator. The P. ostreatus mnpc was fused between the
promoter and terminator of pHGP and pHRP, yielding the recombinant plasmids pHGP-mnp and pHRP-
mnp (Fig. 6). These plasmids were introduced into the monokaryotic C. hirsutus arg1 genome

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 2 40 0
A B 35 0

1. 5 30 0

25 0

1 20 0

15 0

0. 5 10 0

50

0 0
0 2 4 6 8 10 12 0 2 4 6 8 10 12
days days

T MG 9 -C 1 T MG 9 -C 1
w i l d - t yp e w i l d - t yp e

Fig. 5. Time course of extracellular MnP activity (A) and mycelial dry weight (B) of TMG9-C1
(〇) and wild-typ e No.261 ( △) strains

together with the C. hirsutus ARG1-containing plasmid pUCR1. The Arg+ MnP+ transformants ChTF3-
1(PoMnP) and ChTF4-55(PoMnP) showing MnP activity approximately 3 times higher than the control
Arg+ transformant, were obtained by introduction of pHGP-mnp and pHRP-mnp, respectively. ChTF3-
1(PoMnP) and ChTF4-55(PoMnP) were cultured in the medium containing spent barley grain. The
supernatants obtained were used for the measuring the decolorization activity toward Remazol brilliant
bluen R (RBBR) and degradation activity toward pentachlorophenol. The rersults showed that both
strains decolor RBBR efficiently and degrade pentachlorophenol. By using pHGP, C. hirsutus lignin
peroxidase (LiP) cDNA (lipc) was inserted into the C. hirsutus arg1 genome, resulting in an isolation of
ChTF3-10(ChLiP) which shows LiP activity two times as high as that of the control Arg+ transformant.

2.2 Genetic Engineering for the Expression of Cellulolytic and Hemicellulolytic Enzymes
2.2.1 Construction of chimera cellulase in Trichoderma reesei
Trichoderma reesei is well known to secrete a complete system of enzymes capable of hydrolyzing
crystalline cellulose. For this fungus two cellobiohydrolases (CBH I and II) and five endo-β-1,4 -
glucanases (EG I, II, III, IV and V) have been identified and characterized. To improve the degradability
of T. reesei cellulases toward crystalline cellulose, EG III, a low-molecular-mass endoglucanase and the
only one cellulase having no cellulose binding domain (CBD) from T. reesei, was modified to chimera

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EG IIIs having cellobiohydrolase (CBH) CBD using protein engineering technique. The strategy of
construction chimera cellulase is shown in Fig. 7. These chimera EG IIIs were expressed extracellularly
by transformed Schizosaccharomyces pombe. They were purified and physicochemically and
enzymologically characterized. The CBD of the chimera EG III exhibited about the same binding
ability on crystalline cellulose as that of native CBH CBD does. The activities of chimera EG IIIs
toward amorphous and crystalline cellulose were about 2-fold that of native EG III, while those to soluble
substrate (CMC and HEC) were similar. Furthermore, chimera EG IIIs showed higher synergy with
CBH than native EG III does in bacterial microcrystalline cellulose degradation.

2.2.2 Construction of expression systems in Coprinus cinereus

The Bacillus subtilis endo (β-1,4-) D-xylanase gene (xyn) was used to construct C. cinereus strains
useful for elimination of xylan, a major component of hemicellulose, from lignocellulosic materials.
Signal peptide-encoding sequence was removed from xyn and this shortened xyn gene was fused after the
signal sequence of the aforementioned P. ostreatus mnpc. The resulting modified xyn sequence (named
xyn') was inserted between promoter and terminator of pLC1 and pLC2, yielding the recombinant
plasmids pLC1-xyn' and pLC2-xyn' These plasmids were introduced into the C. cinereus trp1 genome
with pCc1001 by co-transformation. Two Trp+ transformants showing clearly high xylan-hydrolyzing
activities were obtained; they were named CcTF1-16(BsXyn) (derived from pLC1-xyn') and CcTF2-
11(BsXyn) (derived from pLC2-xyn'). Southern-blot analysis suggested that CcTF1-16(BsXyn) and
CcTF2-11(BsXyn) carry more than 10 copies of the xyn' sequence on their chromosomes. The two
transformants prpdduced xylanase nine or seven times as high as that of the control Trp+ transformant.

2.3 Lignin Degradation in Solid State Fermentation

2.3.1 Degradation of lignin by Pleurotus ostreatus
The white-rot fungus P. ostreatus produces two types of extracellular peroxidases: manganese-
dependent peroxidase (MnP) and versatile peroxidase (VP). The effect of Mn2+ on fungal growth,

6
peroxidase activity profiles and lignin degradation by P. ostreatus was studied in liquid culture and under
solid-state fermentation (SSF) conditions on perlite, the latter resembling the natural growth conditions of
this fungus. The fungus was grown in either a defined asparagine-containing basidiomycete selective
medium (BSM) or in a rich peptone medium (PM). Biomass production, as determined by respiration
experiments in SSF and liquid cultures and fungal growth on Petri dishes, was higher in the PM than in
the BSM. Mn2+ affected biomass production only in the PM on Petri dishes. In the non-amended PM, high
levels of MnP and VP activity were detected relative to the non-amended BSM (Fig. 8). Nevertheless, a
higher rate of 14C-lignin mineralization was measured in the Mn2+-amended BSM, as determined during
the course of 47 days of fermentation. Mn2+-amendment of the PM increased mineralization rate to that
obtained in the Mn2+-amended BSM (Fig. 9). The enzyme activity profiles of MnP and VP were studied

4
/ L )
P e r o x i d a s e a c t i v it y ( U

● V P activity in non-amended media

▲ MnP activity I n non-amended media

0 ■ Mn activity I n Mn-amended
0 2 4 6 8
(50 0 µM) media

20
/ L )

16
P e r o x i d a s e a c t i v it y ( U

12

0
0 2 4 6 8

Ti me (d a y s )

Fig. 8 . E z ymatic activity of MnP and V P in liq uid cultures o f P. ostreatus in BS M (A) and PM (B)

9
C-lignin)
CO 2

6 ■ in BSM containing
14
Accumulat ed

50 µM Mn 2 +
14
(% of total

▲ in PM non-amended Mn 2 +
3

0
0 10 20 30 40 50
T ime (days)

14
Fig. 9. Mineralization o f C-lignin b y P. ostreatus grown under SSF conditions.

7
in the BSM using anion-exchange chromatography. In the non-amended BSM, only minute levels of MnP
and VP were detected. Upon Mn2+ amendment, two MnP isoenzymes (B1 and B2) appeared. Isoenzyme
B2 was purified and showed 100% identity with the MnP isoenzyme purified in our previous study from
PM-SSF (P6). P6 was found to be the dominant isoenzyme in terms of activity level and gene expression
compared to the VP isoenzymes. Based on these results, we concluded that Mn2+ plays a key role in lignin
degradation under different nutritional and growth conditions, as it is required for the production of MnP
in P. ostreatus.

2.3.2 Selective delignification of wood by white rot fungi

Solid state culture of Pleurotus ostreatus, Dichomitus squalens, Ceriporiopsis subvermispora and
other basidiomycete strains was carried on the Japanese beech wood meal supplemented with liquid
medium. D. squalence and C. subvermispora degraded lignin remarkably as shown in Fig. 10.
Degradation of lignin was much higher than that of cellulose, suggesting that lignin was selectively
degraded by these fungi. Degradation of lignin by P. ostreatus was not effective compared with other
fungi. Fungal-treated and exploded wood meal was hydrolyzed by using a cellulase preparation
(Meicellase, Meiji Seika, Co. Ltd.). Enzymatic hydrolysis by the cellulase preparation resulted in the
effective production of glucose from the treated wood (Fig. 11). These results indicated that production of
glucose from lignocellulosic materials is enhanced by the selective delignification by fungi. Steam
explosion was carried out by using a bench scale apparatus. However，this process was not effective for
the application to wood meal treated with D. squalens and C. subvermispora, whereas it caused the
enhancement of saccharification of the meal treated with P. ostreatus.

80 80
D i ch om i tu s s q u al e ns D i ch om i tu s s q u al e ns
Lignin
Dry On holocellulose
weight
40 40
On untreated meal
Holocellulose
On treated meal

0 0
Yi e l d o f gl u c o se ( % )

C er i p or i op s i s s u b v er m i s p or a C er i p or i op s i s s u b v er m i s p or a
D e cr e a se ( %)

40 40

0 0
P l e ur o t us os tr ea t u s (0 - 48 )
P l e ur o t us os tr ea t u s (0 - 48 )

40 40

0
0
0 2 4 6 8 0 2 4 6 8
G r o w t h (w e e k)
G r o w t h (w e e k)

Fig. 10. Selective degradation o f lignin b y Fig. 11 Enzymic hydrolysis of wood meal
white-rot fun gi treated b y fun gi

2.3.3 Delignification of rice straw by the molecular-genetically breeded C. cinereus

CcTF2-7(PoMnP) and CcTF2-11(BsXyn), which exhibited the highest lignin- or xylan-degrading

8
activity, respectively, were cultured together at 30℃ for one to three weeks with shaking in the water
containing 0.5% (w/v) rice straw which had pretreated by growing Elfvingia (Ganoderma) applanata at
27℃ for one week. The supernatant of the culture of 3 weeks was found to contain the unprecipitable
cellulose of which amount corresponds to 29 % of the total cellulose contained in rice straw. These results
clearly show a usefulness of the molecular-genetically breeded C. cinererus strains.

2.4 Estimation of Wood Decay Process

Rapid nondestructive screening methods are of great importance, since huge numbers of progenies
and bio-treated samples should be analyzed. Recent efforts have been devoted to quantifying the content
of wood components, such as cellulose and lignin, using a micro scale sample. Furthermore, for
chemical treatment of wood, especially for pulping, not only quantity but also quality of lignin i.e. the
structure of aromatic nuclei, strongly affects the properties of the products. The Raman spectroscopic
technique was advantageous since solid or powder samples can be measured directly. The variation of
samples was enhanced and no complicated procedures were required to obtain NIR-FT Raman (NIR-
FTR) spectra of lignocellulosic materials. Utilizing these advantages of the spectroscopy, we attempted
to develop a nondestructive, rapid, and easy-handling technique for characterizing woody materials and
for monitoring wood decay process.
The aromatic C=C vibration bands for
guaiacyl and syringyl skeletons can be assigned to
individual Raman bands, which we designated as
guaiacyl and syringyl marker bands. Using
these bands, the S/G ratio of 12 different wood
samples was calculated, indicating a good
accordance with the values obtained from the
conventional chemical analysis. FT-Raman
measurement of reduced and reoxidized DHP,
MWL, and wood meal revealed the marker band
for carbonyl groups and ethylene groups found in
lignin side chain. The carbonyl marker band
was, then, utilized for monitoring wood decay
process. The Raman marker for glycosidic bond
was also assigned. It was also found that the
ratio between the Raman band intensities of
glycosidic bond marker and C-H vibration
(polysaccharide) marker was correlated well with
the crystallinity of cellulose. Fig. 12 shows the
Raman spctra of the sound wood meal samples
from C. japonica and Q. mongolica.

Wood samples decayed by Coriolus versicolor, Dichomitus squalens, and Ceriporiopsis

subvermispora were measured using FT-Raman spectroscopy. Fig. 13 shows time course of change in
Raman intensities at 1600 and 1096 cm-1. The Raman intensities for aromatic markers and glycosidic

9
bond marker exhibited a good accordance with the
quantitative values obtained from the conventional
chemical analysis. The r2 values for the
relationship between the Raman intensities and
quantitative values obtained from chemical
analysis were >0.985 when BaSO4 (SO stretching)
was utilized as an internal standard for a
quantitative Raman measurement.

CONCLUSION

Expression systems for a ligninolytic enzyme, manganese peroxidase (MnP), of a white-rot fungus,
Dichomitus squalens and Pleurotus ostreatus was expressed in Phanerochaete chrysosporium and
Coprinus cinereus, respectively. P. ostreatus MnP was also expressed in Coriolus hirsutus. Homologous
expression system was also constructed in Pleurotus ostreatus. Higher activity of MnP or lignin-
degradation than the original fungi were abserved in the transformatnt strains. Transformants of C.
cinereus was useful for the delignification of rice straw. Chimera cellulases having fused structure of exo-
and endo-glucanase were constructed. Bacterial hemicellulase, xylanase, was expressed in C. cinereus.
Lignin degradation of solid lignocellulosic materials was enhanced by P. ostreatus by controlling the
concentration of Mn2+. D. squalense and Ceriporiopsis subvermispora caused selective delignification
of wood meal, which resulted in the improvement of digestability of cellulose to produce glucose.
NIR-FT Raman spectrometry was applied to the quick and non-destructive analysis of the chemical
structure of sound and decayed wood and its components.
The results obtained in the international corroboration research suggested that the idea to utilize
genetically improved fungi can be one of the ways to produce energy and other useful chemicals from
lignocellulosic resources presented most abundantly on the earth.

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