123

5
Chitin and Chitosan in Fungi

Prof. Dr. Martin G. Peter

University of Potsdam, Institute of Organic Chemistry and Structure Analysis,
and Interdisciplinary Research Center for Biopolymers, Karl-Liebknecht-Str. 25,
D-14476 Golm, Germany; Tel.: ‡ 49-331-977-5401; Fax: ‡ 49-331-977-5300;
E-mail: peter@serv.chem.uni-potsdam.de

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

2 Chemical Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

2.1 Chitin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
2.2 Chitosan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
2.3 Polyphenolic Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

3 Occurrence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126

4 Physiological Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127

5 Chemical Analysis and Detection . . . . . . . . . . . . . . . . . . . . . . . . . . 127

6 Biosynthesis of Chitin and Chitosan . . . . . . . . . . . . . . . . . . . . . . . . 128

6.1 Chitin Synthases (CS ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
6.1.1 Enzymology and Subcellular Localization of CS . . . . . . . . . . . . . . . . . 128
6.1.2 Genetics of CS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
6.1.3 Regulation of CS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
6.1.4 Inhibition of CS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.2 Glucan Transferase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.3 Chitin Deacetylase (CDA ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.3.1 Enzymology of CDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6.3.2 CDA Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
6.3.3 Regulation of CDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134

7 Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
7.1 Chitinase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
124 5 Chitin and Chitosan in Fungi

7.1.1 Enzymology of Fungal Chitinases . . . . . . . . . . . . . . . . . . . . . . . . . 134

7.1.2 Chitinase Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
7.1.3 Regulation of Chitinases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 136
7.2 Chitosanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137
7.2.1 Structure and Mechanism of Chitosanases . . . . . . . . . . . . . . . . . . . . 138
7.2.2 Enzymology of Chitosanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
7.2.3 Chitosanases in Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
7.2.4 Chitosanase Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.2.5 Regulation of Chitosanases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
7.3 Exo-b-d-glucosaminidases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

8 Biotechnological Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

8.1 Screening for Chitosan Producer Strains . . . . . . . . . . . . . . . . . . . . . 141
8.2 Isolation of Chitin and Chitosan from Fungal Biomass . . . . . . . . . . . . . 142
8.3 Production of CDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142

9 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
9.1 Adsorption of Coloring Matters . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
9.2 Metal Ion Adsorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
9.3 Healthcare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143

10 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

11 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

12 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147

AA amino acid residues

CDA chitin deacetylase
CHS chitin synthase gene
CS chitin synthase
CSN chitosanase gene
DP degree of polymerization
ER endoplasmic reticulum
FA mole fraction of N-acetylglucosamine residues
FMOC 9-fluorenylmethoxycarbonyl
FT-IR Fourier transformation infrared spectroscopy
GlcN 2-amino-2-deoxy-d-glucopyranose, b-(1-4)-linked in chitin/chitosan
GlcNAc 2-acetamido-2-deoxy-d-glucopyranose, b-(1-4)-linked in chitin/chitosan
GlcNase glucosaminidase
HPLC high-performance liquid chromatography
M molecular mass (daltons)
MALDI TOF MS matrix-assisted laser desorption ionization time-of-flight mass spec-
trometry
Mv viscosity average molecular mass
 2 Chemical Structure 125

Mw mass average of molecular mass

ORF open reading frame
PITC phenylisothiocyanate
UDP-GlcNAc uridine diphospho-N-acetylglucosamine
WGA wheat germ agglutinin

1 2.1
Introduction Chitin

The aminoglucan chitin (poly-GlcNAc) is The molecular mass of chitin in fungi is not
widely distributed in nature, whereas the known. However, it was estimated that
related polysaccharide chitosan (poly-GlcN ) bakers' yeast synthesizes rather uniform
occurs in certain fungi only. Fungal chitin chains containing 120 ± 170 GlcNAc mono-
shows some special features, in particular mer units which corresponds to ca. 24,000 ±
with respect to chemical structure and 34,500 Daltons (for a reference, see Valdi-
biosynthesis. Thus, it appears appropriate vieso et al., 1999). This is roughly one order
to look at fungal chitin and chitosan in their of magnitude lower than the estimated
own rights and to treat them in a separate molecular mass of chitosan isolated from
chapter. Mucoraceae (see below).
The basic principles of chitin and chitosan In Saccharomyces cerevisiae, terminal re-
are treated elsewhere in this volume, and ducing ends of chitin chains are attached
readers who are not familiar with these through b-(1,4)- or b-(1,2)-linkages to the
aminoglucans should consult also Chapter nonreducing end of b-(1,3)-glucan branches
15, this volume (Chitin and Chitosan from which are linked to b-(1,6)-glucan. Attach-
Animal Sources). ment of chitin to glucan is catalyzed by chitin
synthase 3 (Hartland et al., 1994). A man-
noprotein is attached to b-(1,6)-glucan
through a glycosylphosphatidylinositol an-
chor containing five a-linked mannosyl
2 residues (Kollar et al., 1995, 1997). A mutant
Chemical Structure of S. cerevisiae with a reduced b-(1,3)-glucan
content shows increased cross-linking of
The chitin of fungi possesses principally the mannoproteins to chitin trough b-(1,6)-glu-
same structure as the chitin occurring in can (Kapteyn et al., 1997).
other organisms (see Chapter 15, this vol- Likewise, the cell wall of Aspergillus fumi-
ume). However, a major difference results gatus is highly complex, containing in the
from the fact that fungal chitin is associated alkali-insoluble fraction a linear b-(1,3/1,4)-
with other polysaccharides which do not glucan, galactomannan, chitin, and b-(1,3)-
occur in the exoskeleton of arthropods. glucan, whereas b-(1,6)-glucan is absent.
Furthermore, the occurrence of chitosan is The b-(1,3)-glucan shows 4% of b-(1,6)-
apparently restricted to fungi. branching. As in S. cerevisiae, chitin is b-
(1,4)-linked to b-(1,3)-glucan (Fontaine et al.,
2000). The content of GlcNAc in cell walls of
126 5 Chitin and Chitosan in Fungi

A. fumigatus in only 50% of that in Aspergillus charide±protein network by oxidative cross-

nidulans (Guest and Momany, 2000). linking or impregnation with a hydrophobic
Staining with fluorescent lectins reveals polymer. According to chemical logic, the
distinct distributions of mannoproteins, polyphenols should be covalently bound to
glucans and chitin in the cell wall of Candida other biopolymers, though this has not been
albicans (Ruiz-Herrera et al., 1994). The cell proven unequivocally. Precursors of fungal
wall of Fusarium oxysporum is composed of pigments are L-tyrosine and its hydroxyla-
an outer layer of glycoproteins covering an tion product, 3,4-dihydroxy-phenylalanine
inner layer of chitin and glucan (Schoffel- (DOPA ). Black and brown pigments, which
meer et al., 1999). Cell walls of the geophilic are sometimes incorrectly named melanins,
Dermatophytes Microsporum fulvum and are often also derived from 1,8-dihydroxy-
Epidermophyton stockdaleae contain 4.0 ± naphthalene or from simple catechols (Pro-
6.5% of glucomannan and 44.2 ± 71.0% of ta, 1992). Several fungi use g-glutaminyl-4-
a glucan±chitin complex (Guarro et al., hydroxybenzene as a precursor (Pierce and
1993). Rast, 1995). Wood- inhabiting fungi, e.g.,
Inonotus hispidus, produce a phenolic bio-
2.2 polymer that is derived from the stryrylpyr-
Chitosan one hispidin (for a general review of fungal
pigments, see Gill and Steglich, 1987).
Chitosans isolated from Mucorales typically
show Mv in the range 4 î 105 to 1.2 î
106 Daltons and FA values between 0.2 and 3
0.09. Amino acid analysis of chitosan pre- Occurrence
pared from Aspergillus niger reveals covalent-
ly bound arginine, serine, and proline (Le- Chitin is widely distributed in fungi, occur-
stan et al., 1993). The chitosan±glucan com- ring in Basidiomycetes, Ascomycetes, and
plexes from mycelia of A. niger, Humicola Phycomycetes, where it is a component of
lutea, and Fusarium moniliforme contain the cell walls and structural membranes of
0.05 ± 0.06% of amino acids, mostly as lysine mycelia, stalks, and spores. The amounts
and histidine ( Velichkov and Sotirov, 1990). vary between traces and up to 45% of the
Bacillus pumilus chitosanase is more effec- organic fraction, the rest being mostly
tive than Streptomyces griseus chitinase in proteins, glucans and mannans (Roberts,
digestion of the cell wall of Fusarium oxy- 1992). However, not all fungi contain chitin,
sporum. Besides GlcN-GlcNAc as the main and the polymer may be absent in one
product, maltose is also observed as a species that is closely related to another.
degradation product, which indicates the Variations in the amounts of chitin may
presence of a-(1,4)-linked glucan (Fukamizo depend on physiological parameters in nat-
et al., 1996). ural environments as well as on the fermen-
tation conditions in biotechnological proc-
2.3 essing or in cultures of fungi.
Polyphenolic Pigments Chitin is the major component in primary
septa between mother and daughter cells of
Cell walls or sporophore capsules of fungi S. cerevisiae, and also one of the main
contain often polyphenolic pigments which components of the hyaline outer wall of
presumably function to enforce the polysac- spores of four arbuscular mycorrhizal Glo-
 5 Chemical Analysis and Detection 127

mus species (Sbrana et al., 1995). Hyphal and chitosan, as revealed by mutants bearing
walls of the Oomycete Pythium ultimum a defect in the complex machinery of chitin
contain cellulose and chitin, whereas only biosynthesis, intracellular trafficking of chi-
cellulose is present in another Oomycete, tin synthases, or deposition of the polysac-
Phythophthora parasitica. Both polysaccha- charide in cell walls, although the morphol-
rides are present in cell walls of the Asco- ogy of a mutant may be indistinguishable
mycetes Ophiostoma ulmi and Colletotrichum from that of the wild-type. Thus, chsD
lindemuthianum, whereas the Ascomycete disruptants of Aspergillus nidulans show
Fusarium oxysporum and the Basidiomycete excessive swelling and lysis of conidia in
Rhizoctonia solani contain only chitin (Cherif hypoosmotic media (Specht et al., 1996) and
et al., 1993). The zoopathogenic fungi Cryp- S. cerevisiae chs5D null mutants fail to
tococcus neoformans, Pityrosporum canis and undergo cell fusion in mating (Santos et al.,
Rhizopus oryzae contain chitin, but not b- 1997) (for details, see Section 6.1).
(1,3)-glucan (Nicholas et al., 1994).
The chitin of the cell wall of the white-rot
fungus Rigidoporus lignosus is degraded by 5
enzymes excreted as a defense response by Chemical Analysis and Detection
the host cell, and therefore is not detectable
during the process of infection (Nicole and Determination of fungal chitin in biological
Benhamou, 1991). The fungal sheaths of samples is important for estimating fungal
another white-rot fungus, Phellinus noxius, biomass, e.g., in infected plant tissues. A
do not contain chitin (Nicole et al., 1995). comprehensive review of the most common
The mycelia, and the caps and stalks of methods was given by Muzzarelli (1997) (see
fruiting bodies of four edible mushrooms, also Chapter 15, this volume). Frequently,
Lentinus edodes, Lycophyllum shimeji, Pleuro- GlcN is quantified by colorimetric methods
tussajor-caju, and Volvariella volvacea contain in hydrolyzates of alkali-resistant fractions to
chitin as a minor component (Cheung, determine the amounts of chitin and chito-
1996). san (Plassard, 1997). GlcN was also deter-
Chitosan occurs naturally in the Mucor- mined in acid hydrolyzates by high-perfor-
ales, in particular Mucor, Absidia, and Rhi- mance liquid chromatography (HPLC ) of 9-
zopus species. There is apparently only one fluorenylmethoxycarbonyl (FMOC )- or phe-
report on the presence of chitosan in a nylisothiocyanate (PITC )-GlcN (Ekblad and
Basidiomycete, Lentinus edodes (Shiitake Naesholm, 1996; Osswald et al., 1995), or by
mushroom) (Crestini and Giovannozzi-Ser- gas chromatography-mass spectrometry
manni, 1996; Crestini et al., 1996). (GC-MS ) techniques (Penman et al., 2000).
Slime molds (Myxomycetes) and bacteria Localization of chitin in cell walls or spores
(Schizomycetes) are devoid of chitin. of fungi is achieved by using dyes that
intercalate with polysaccharides. Calcofluor
white shows enhanced fluorescence when
4 binding to b-(1,4)-glucans, such as chitin,
Physiological Function chitosan and cellulose, whereas b-(1,3)-glu-
cans are selectively stained with aniline blue
Chitin serves as a fibrous strengthening (Nicholas et al., 1994). Various wheat germ
element responsible for cell wall rigidity. agglutinin ( WGA ) labeling techniques in
However, there are other functions of chitin combination with fluorochromes or gold
128 5 Chitin and Chitosan in Fungi

labeling are also described for the detection 2.4.1.16) belongs to family 2 glycosyltrans-
of chitin in fungi (Sbrana et al., 1995; Hu ferases which catalyze glycosyltransfer with
and Rijkenberg, 1998; Ekramoddoullah inversion of the anomeric configuration
et al., 2000). (Coutinho and Henrissat, 1999). Further
Fourier transform (FT ) Raman spectros- classification is based on sequence similar-
copy of cell walls of fungi was applied to ities or identities, and to date five CS classes
discriminate between different mixed spe- have been assigned (cf. Table 1).
cies in culture media (Edwards et al., 1995). In general, b-glycosyltransferases, includ-
ing cellulose and chitin synthases, have a
highly conserved common motif `D, D,
6 D35Q(R,Q )XRW'. The second residue (R
Biosynthesis of Chitin and Chitosan or Q ) in the Q(R,Q )XRW sequence is
probably involved in determining the degree
Chitin is biosynthesized in all chitinous of polymerization (DP ) of the glucan chain
fungi, including the relatively few investi- (Saxena and Brown, 1997). Alignment with
gated examples of Mucorales which contain the deduced protein sequences of most
chitosan. Chitin synthases as well as chitin known chitin synthase genes (CHS ) reveals
deacetylases are reviewed in this section. five to seven conserved domains (Xoconos-
tle-Cazares et al., 1996).
6.1 CS is detected in membrane fractions and
Chitin Synthases (CS ) in chitosomes, the latter constituting small
secretory vesicles (ca. 100S ) which function
In contrast to the situation in arthropods, as conveyors of CS to the cell surface (for a
many details of chitin biosynthesis are review, see Ruiz-Herrera and Martinez-Es-
known in fungi. Most of the current knowl- pinoza, 1999). Chitosome-membrane traf-
edge is based on studies on baker's yeast, S. ficking of CS was also observed in Neuro-
cerevisiae. The earlier literature was dis- spora crassa (Leal-Morales et al., 1994b).
cussed comprehensively by Cabib (1987) Solubilization of CS is achieved with deter-
and, since then, various aspects of chitin gents, such as digitonin, yielding a catalyti-
synthesis in fungi have been reviewed cally active 16S protein complex of molecular
(Bulawa, 1993; Martinez and Gozalbo, mass ca. 500 kDa. CS isolated from a micro-
1994; Ruiz-Herrera and Xoconostle-Cazares, somal fraction of Absidia glauca is a 30-kDa
1995; Bruyere et al., 1996; Cabib et al., 1996; zymogenic polypeptide (Machida and Saito,
Gooday, 1996; Merz et al., 1999a; Ruiz- 1993). A catalytically active CS was isolated
Herrera and Martinez-Espinoza, 1999; Val- as a 60-kDa polypeptide from 100S chito-
divieso et al., 1999; Karnezis et al., 2000). somes of Mucor rouxii (Merz et al., 1999b).
In S. cerevisiae, CSI is more abundant than
6.1.1 CSII, and both are localized in low- density
Enzymology and Subcellular Localization of CS chitosomes (d ˆ 1.15 g mL 1) as well as in
The biosynthesis of chitin takes place vecto- high-density membrane fractions (d ˆ
rially in a membrane-bound protein com- 1.21 g mL 1) (Leal-Morales et al., 1994a).
plex. Chain elongation occurs by sequential Likewise, the transcripts from the Ustilago
transfer of GlcNAc from UDP-GlcNAc to the maydis gene, UmCHS1, appear to be present
nonreducing end of the growing polymer. at a higher level than those from UmCHS2,
CS (chitin-(UDP-GlcNAc)-transferase, EC and both transcripts appear to be more
 6 Biosynthesis of Chitin and Chitosan 129

Tab. 1 Chitin synthase genes from fungi (alphabetical listing by name of organism)

Organism Gene Comments Reference

Agaricus bisporus CHS1 CS class III; 2727 bp (ORF); 909 AA; Sreenivasaprasad et al.
(2000)
Ampelomyces quisqualis AqCHSA CS class I; 2786 bp; 910 AA Weiss et al. (1996)
Aspergillus fumigatus CHSD CS-like; low but significant similarity to Mellado et al. (1996)
other CS
Aspergillus nidulans CHSA 1013 AA Yanai et al. (1994)
CHSB 916 AA
A. nidulans CHSD CS class V and CS class Specht et al. (1996)
CHSE IV; high sequence identity to ScCHS3 and
CaCHS3
Beauveria brongniartii BbCHS1 Fragment; CS class II; 95.8% similarity Nam et al. (1997)
with CHS2 of Metarhizium anisopliae
Candida albicans CaCHS1A 775 AA Sudoh et al. (1995)
Fonsecaea pedrosoi FpCHS1 600 bp and 366 bp; CS Karuppayil et al.
FpCHS2 class I and II; homology (1996)
FpCHS3 to S. cerevisiae CS
Metarhizium MaCHS1 CS class I Nam et al. (1998)
anisopliae MaCHS2 CS class I
MaCHS3 CS class III
Mucor circinelloides McCHS1 CS class TI; expressed during exponen- Lopez-Matas et al.
tially growing hyphal stage (2000)
Neurospora crassa CHS2 Similar to CHS from other fungi Din and Yarden (1994)
Paracoccidioides brasi- CHS2 CS class II; 1043 AA Nino-Vega et al. (1998)
liensis
Penicillium chrysogenum PcCHS1 CS class I Namgung et al.
PcCHS2 CS class II (1996)
PcCHS3 CS class II
PcCHS4 CS class III
P. chrysogenum CHS4 CS class III; 915 AA (ORF); close rela- Park et al. (2000)
tionship between P. chrysogenum and
Aspergillus CHS
Phialophora verrucosa PvCHS1 CS class I and II; 614 bp Peng et al. (1995)
PvCHS2 CS class III; 366 bp;
PvCHS3 88.2% similarity and 78.4% identity; with
the S. cerevisiae enzyme
Pyricularia oryzae Fragment 340 bp; 86% homologous to A. fumigatus Hwang et al. (1997)
CHSE
Rhizopus oligosporus CHS3 CS class IV; sequence similarity to CHS3 Motoyama et al. (1998)
of S. cerevisiae; 46.7% identity with class IV
CS of N. crassa
Saccharomyces cerevisiae CHS4 696 AA Trilla et al. (1997)
S. cerevisiae CHS5 671 AA Santos et al. (1997)
S. cerevisiae CHS6 See text Ziman et al. (1998)
S. cerevisiae CHS7 See text Trilla et al. (1999)
Saprolegnia monoica CHS2 Oomycetes and chitinous fungi have con- Mort-Bontemps et al.
served CS (1997)
Tuber borchii ± CS class II; ca. 600 bp Lanfranco et al. (1995)
130 5 Chitin and Chitosan in Fungi

Tab. 1 (cont.)

Organism Gene Comments Reference

T. magnatum TmCHS4 1230 AA; 62% homology to class IV CHS Garnero et al. (2000)
of N. crassa
Ustilago maydis UmCHS1 See text Xoconostle-Cazares
UmCHS2 et al. (1996)
U. maydis UmCHS5 Predicted CHS class IV; high similarity Xoconostle-Cazares
CHS3 from S. cerevisiae and C. albicans, et al. (1997)
CHS4 from N. crassa, CHSE from A.
nidulans
Wangiella dermatitidis WdCHS4 High homology with CS class IV (Chs3p) Wang et al. (1999)
of S. cerevisiae

abundant in the mycelial form (Xoconostle- 6.1.2

Cazares et al., 1996). Genetics of CS
In N. crassa, CSII is compartmentalized in The genetics of fungal CS are investigated very
chitosomes which are abundant in the intensively, often using CHS from S. cerevisiae
vicinity of the hyphal tip. Immunological as hybridization probes, yielding useful in-
studies have revealed that several peptides of formation for taxonomy and phylogenetic
microsomal membrane fractions react with relations, as well as the basis for the under-
a polyclonal antibody to CSII (Sietsma et al., standing of CS functions and their regulation
1996). (for reviews, see Bulawa, 1993; Ruiz-Herrera
Though not proven conclusively in vivo, it and Xoconostle-Cazares, 1995; Valdivieso et al.,
appears that CSI and CSII are activated by 1999). The variety of CHS is illustrated with
proteolysis, occurring in a zymogenic form a few recent examples in Table 1. A compre-
which contains a cytosolic amino-terminal hensive listing of sequences is available, e.g.,
region. The CS from Saprolegnia monoica is in the CAZy database (Coutinho and Hen-
stimulated by digitonin and remains zymo- rissat, 1999; see also Campbell et al., 1997).
genic after dissociation (Leal-Morales et al., Much insight into the functions of CHS
1997). Proteolytic activation of CSIII is and their transcripts has been obtained from
observed in the presence of substrate (for studies on the effects of gene deletion or
discussion and references, see Merz et al., disruption. Eight CHS have been described
1999a; Valdivieso et al., 1999). to date in S. cerevisiae. ScCHS1 and ScCHS2
As a rule, CSI and CSII are activated by are the structural genes for CSI and CSII,
divalent metal ions as co-factors, most respectively, whereas the remaining genes
commonly Mg2‡ or Mn2‡. However, CSII are components of the CSIII complex ( Val-
of S. cerevisiae requires Co2‡ rather than divieso et al., 1999). Disruption of ScCHS2
Mg2‡ (Leal-Morales et al., 1994a). or simultaneous disruption of ScCHS2 and
In the cellulosic, nonfibrillar a-chitin- ScCHS3, but not of ScCHS1 or ScCHS3, is
producing Oomycete Saprolegnia monoica, lethal. However, a gene suppressing the
CS is found in high-density membrane lethality of disruption of ScCHS2 occurs in a
components, but not in chitosomes (Leal- S. cerevisiae strain which does not require
Morales et al., 1997). ScCHS2 for viability. A mutant containing
 6 Biosynthesis of Chitin and Chitosan 131

the suppressor and lacking ScCHS1 and 1994; Borgia et al., 1996). Chitin synthesized
ScCHS2 has normal amounts of chitin in its by the CHSD-encoded isoenzyme contrib-
cell wall. Apparently, the suppressor gene utes to the rigidity of the walls of germinat-
encodes or controls the expression of CSIII ing conidia, of the subapical region of
(Baymiller and McCullough, 1993). Chs6p is hyphae, and of conidiophore vesicles, but
required for anterograde transport of Chs3p is not necessary for normal morphology of
from the chitosome to the plasma mem- these cells. Hyphae from both, chsD and
brane (Ziman et al., 1998). The CHS7 gene chsE disruptants contain ca. 60 ± 70% of the
is specifically involved in Chs3p export from chitin present in wild-type hyphae. The
the endoplasmic reticulum (ER ) (Trilla morphology and development of chsE dis-
et al., 1999). ruptants are indistinguishable from those of
S. cerevisiae CHS show significant homol- wild-type cells (Specht et al., 1996).
ogy to insect CHS and to bacterial and An interesting feature of CHS is the
vertebrate hyaluronan synthase HAS genes presence of a N-terminal myosin motor-like
(DeAngelis et al., 1994; Ibrahim et al., 2000; sequence that has first been observed in the
see also Chapter 15 in Volume 5 and Chapter A. nidulans csmA gene which contains a
15 in this volume). large open reading frame (ORF ) encoding a
The CS of C. albicans, called CaChs1p, polypeptide of 1852 amino acids (Fujiwara
CaChs2p, and CaChs3p, are structurally and et al., 1997; see also Zhang and Gurr, 2000).
functionally analogous to the S. cerevisiae CS. Apparently, the csmA transcript has impor-
CaChs1p is involved in septum formation tant roles in polarized cell wall synthesis and
and is required for the viability of C. albicans. maintenance of cell wall integrity (Horiuchi
Inhibition of CaChsp1 with RO-09-3143 (see et al., 1999).
below) causes cell death in the cachs2D null,
but not in cachs3D null mutants (Sudoh 6.1.3
et al., 2000). Regulation of CS
In U. maydis, six CHS or fragments are On the enzymatic level, CS of Mucor rouxii is
presently known which could operate to allosterically activated by GlcNAc which
compensate an eventual loss of one activity shows cooperative binding (Horsch and
by the remaining enzymes, thus maintain- Rast, 1993; Merz et al., 1999a).
ing fungal viability. Umchs5 null mutants Septum formation in mycelial fungi and
display significant reduction in growth rate, yeasts, as well as apical growth of the hyphae
chitin content, and chitin synthase activity, of filamentous fungi requires a precisely
especially in the mycelial form, and reduced regulated, complex interplay of CS and
virulence to corn plants (Xoconostle-Cazares chitinases. In S. cerevisiae, CSI is involved
et al., 1997). in repair functions at the end of cytokinesis.
Inactivation of the N. crassa CHS2 gene CSII deposits a disk of chitin in the mother-
produces progeny which is indistinguish- bud neck, forming the primary septum at
able from those of the wild-type, though a the end of mitosis, and CSIII synthesizes a
significant reduction in CS activity and ring of chitin at the onset of bud emergence.
increased sensitivity to the phosphatidylcho- Genomic analysis reveals multigenic control
line biosynthesis inhibitor edifenphos were of chitin synthesis ( Valdivieso et al., 1999).
observed (Din and Yarden, 1994). Post-translational regulation, probably by
CHSB, but not CHSA, is essential for activation of latent zymogenic forms, ap-
hyphal growth in A. nidulans (Yanai et al., pears to be predominant for the three CS of
132 5 Chitin and Chitosan in Fungi

S. cerevisiae (Choi et al., 1994). Furthermore, 6.2

Chs2p and Chs3p are spatially and timely Glucan Transferase
regulated, involving also differential traffick-
ing (Chuang and Schekman, 1996). The The formation of branched glucan, glucan±
ScCHS4 gene which encodes a protein with glucan cross-links, and glucan±chitin cross-
no potential transmembrane domain regu- links in fungal walls involves the action of
lates the catalytic activity of CSIII, as Vmax is glucanases, glycosyltransferases, and trans-
reduced in the enzyme of chs4 null mutants. glycosylases. Glucosyltransferases from cell
In addition to the chitin defect, the chs4 walls of S. cerevisiae and C. albicans were
mutant shows a severe defect in mating partly characterized. An activated intermedi-
(Trilla et al., 1997). Chitin synthesis in S. ate is formed from a donor b-(1,3)-glucan by
cerevisiae is suppressed on the transcrip- cleaving off a disaccharide (Goldman et al.,
tional level by the KNR4 gene (Martin et al., 1995). A chitin±glucan-b-(1,4)-transferase
1999). which catalyzes formation of the linkage
Chitin synthesis in S. cerevisiae is also between the terminal reducing GlcNAc
under control of the a mating factor (Martin residue of chitin and the nonreducing Glc
et al., 1999; Santos and Snyder, 1997; for residue of b-(1,3)-glucan, as well as a
references, see also Valdivieso et al., 1999). potential use of the enzyme for assaying
antifungal agents, are described in a patent
(Kollar et al., 1996).
6.1.4
Inhibition of CS 6.3
Benzoylphenylureas and tunicamycin are Chitin Deacetylase (CDA )
not inhibitors of chitin synthesis in fungi,
whereas the nucleotide analogous nikkomy- Enzymatic deacetylation of chitin by CDA
cins and polyoxins are highly effective (for [ EC 3.5.1.41] is apparently restricted to fungi
reviews, see Cohen, 1993; Munro and Gow, and bacteria (for reviews, see Kolodziejska
1995; Palli and Retnakaran, 1999; Zhang et al., 1995; Tsigos et al., 2000). A few earlier
and Miller, 1999; Rast et al., 2000). However, reports on the occurrence of CDA in arthro-
Nikkomycin Z is not active against S. pods (Aruchami et al., 1986) have so far been
cerevisiae, as CSIII but not CSII is inhibited neither confirmed nor disproved. A review of
(Gaughran et al., 1994). CDA is given in the following section. The
A potent inhibitor of CSI of C. albicans has selective deacetylation at the nonreducing
recently been identified as 8-(6,6-dimethyl- end of (GlcNAc)n for Nod-factor biosynthesis
aminohepta-2,4-diynyl)amino-4H-benz[1,4]- as well as applications of deacetylases for the
oxazin-3-one (RO-09-3143) (Ki for CaChs1p synthesis of partially acetylated chitooligo-
0.55 nM ) which arrests cell growth in wild- saccharides, and the deacetylation of small
type yeast at MIC50 0.27 mM (Sudoh et al., substrates for the terminal metabolism of
2000). chitin are excluded, as these topics are
A number of natural products isolated discussed in Chapter 15, this volume.
from plants or microorganisms show anti-
fungal effects by inhibiting CS or functional 6.3.1
components required for CS activity. Further Enzymology of CDA
review is beyond the scope of this article, CDA deacetylates preferentially amorphous
however. chitin of medium FA (for details, see Chapter