Appl Microbiol Biotechnol (1998) 50: 145±152
MINI-REVIEW
A. Becker á F. Katzen á A. PuÈhler á L. Ielpi
Xanthan gum biosynthesis and application:
a biochemical /genetic perspective
Received: 18 March 1998 / Received revision: 29 April 1998 / Accepted: 30 April 1998
Abstract Xanthan gum is a complex exopolysaccharide
produced by the plant-pathogenic bacterium Xantho-
monas campestris pv. campestris. It consists of D-gluco-
syl, D-mannosyl, and D-glucuronyl acid residues in a
molar ratio of 2:2:1 and variable proportions of O-acetyl
and pyruvyl residues. Because of its physical properties,
it is widely used as a thickener or viscosi®er in both food
and non-food industries. Xanthan gum is also used as a
stabilizer for a wide variety of suspensions, emulsions,
and foams. This article outlines aspects of the bio-
chemical assembly and genetic loci involved in its bio-
synthesis, including the synthesis of the sugar nucleotide
substrates, the building and decoration of the pent-
asaccharide subunit, and the polymerization and secre-
tion of the polymer. An overview of the applications and
industrial production of xanthan is also covered.
Introduction
Xanthomonas campestris pv. campestris is a plant-
pathogenic bacterium that produces the exopolysac-
charide xanthan. Many other pathovars of X. campestris
eciently produce exopolysaccharides, including the
pathovars phaseoli, malvacearum, carotae, citrumelo,
juglandis, as well as some other members of the genus
Xanthomonas (X. fragariae, X. oryzae pv. oryzae).
Xanthan has attracted particular attention because it
has found many applications as a thickener and emul-
A. Becker á A. PuÈhler (&)
Lehrstuhl fuÈr Genetik, UniversitaÈt Bielefeld,
Postfach 100131, D-33501 Bielefeld, Germany
e-mail: puehler@genetik.uni-bielefeld.de
Tel.: +49 521-106-5607
Fax: +49 521-106-5626
F. Katzen á L. Ielpi
Instituto de Investigaciones BioquõÂ micas FundacioÂn Campomar,
Facultad de Ciencias Exactas y Naturales, UBA, and CONICET,
Patricias Argentinas 435, (1405) Buenos Aires, Argentina
Ó Springer-Verlag 1998
si®er as a result of its unique rheological properties in
industrial food and non-food sectors as well as in oil
recovery. Xanthan is an acidic polymer made up of
pentasaccharide subunits, forming a cellulose backbone
with trisaccharide side-chains composed of man-
nose(b1,4)glucuronic-acid(b1,2)mannose attached to al-
ternate glucose residues in the backbone by a1,3 linkages
(Jansson et al. 1975, Fig. 1). On approximately half of
the terminal mannose residues a pyruvic acid moiety is
joined by a ketal linkage. Acetyl groups are often present
as 6-O substituents on the internal mannose residues.
Some external mannoses contain a second 6-O-acetyl
substituent (Stankowsky et al. 1993). The levels of
pyruvate and acetyl substitution vary with bacterial
growth conditions and other parameters. Here we
summarize our current knowledge of the genetics and
biochemistry of xanthan biosynthesis as well as of the
industrial production and applications of xanthan.
Biochemistry and genetics of xanthan biosynthesis
Xanthan is built up from cytoplasmic sugar nucleotides,
acetyl-CoA, and phosphoenolpyruvate with an inner-
membrane polyisoprenol phosphate as an acceptor (Ielpi
et al. 1981, 1983, 1993). The elucidation of its bio-
synthetic pathway has been greatly aided by using
X. campestris campestris cells frozen and thawed several
times in the presence of EDTA. The stepwise assembly
of the repeating pentasaccharide and the polymerization
process were resolved by incubating these permeabilized
cells with combinations of UDP-glucose, GDP-man-
nose, and UDP-glucuronic acid. The synthesis of the
repeating unit starts with the transfer of glucosyl 1-
phosphate from UDP-glucose to polyisoprenol phos-
phate, followed by the sequential transfer of the other
sugar residues to form the complete repeating unit.
Acetyl and pyruvyl residues are added at the lipid-linked
pentasaccharide level, donated by acetyl-CoA and
phosphoenolpyruvate respectively. The examination of
146
Fig. 1 Structural unit of xanthan
gum. The structure of the xanthan
repeating unit is according to
Jansson et al. (1975). The number
of substituents is variable. Some
external mannoses contain a sec-
ond O-acetyl substituent (Stank-
owsky et al. 1993)
the polymerization process indicated that xanthan
chains grow at the reducing end (Ielpi et al. 1993), as
described for Wzy-dependent (formerly Rfc-dependent)
O-antigen synthesis (Whit®eld 1995). The export mech-
anism of the polysaccharide remains to be determined
but it might be associated to the polymerization event.
Many of the genetic methods used to study Escheri-
chia coli can be used in X. campestris campestris, in-
cluding conjugation, electroporation, chemical and
transposon mutagenesis, and site-directed mutagenesis.
The isolation of a collection of non-mucoid mutants
defective in xanthan production, developed by chemical
mutagenesis or by transposon mutagenesis (BarreÁre et al.
1986; Capage et al. 1987; Harding et al. 1987; Thorne
et al. 1987; HoÈtte et al. 1990; Harding et al. 1993) re-
vealed several genetic loci involved in xanthan biosyn-
thesis.
Regions termed xpsIII, xpsIV and xpsVI (Harding
et al. 1993) as well as a 35.3-kb gene cluster (HoÈtte et al.
1990) contain several genes required for the biosynthesis
of sugar nucleotides necessary for both xanthan and li-
popolysaccharide production. The 35.3-kb gene cluster
comprises two separate regions, one of which overlaps
the xpsIII region. The DNA sequence of a common
segment of these sequences revealed two genes, desig-
nated xanA and xanB (KoÈplin et al. 1992). While the
xanA gene encodes an enzyme with phosphoglucomu-
tase and phosphomannomutase activities, xanB codes
for a bifunctional enzyme with phosphomannose iso-
merase and GDP-mannose pyrophosphorylase activities
(Fig. 2). Thus, a portion of this region encodes two en-
zymes, one of which is required for the synthesis of all
three sugar nucleotide precursors of xanthan, and the
other for the synthesis of GDP-mannose. Genetic and
biochemical analyses showed that mutations mapping in
the xpsIV region a€ect the activity of the UDP-glucose
pyrophosphorylase enzyme, whereas xpsVI mutations
totally abolished UDP-glucose dehydrogenase levels
(Harding et al. 1993) (Fig. 2). Genes encoding these
activities were recently sequenced (Lin et al. 1995; Wei
et al. 1996). Mutants mapping in all these regions exhibit
the same phenotype: they do not produce xanthan in
vivo, but did produce the polyisoprenyl diphosphate
pentasaccharide and polymer when sugar nucleotides
were added in the in vitro system (Harding et al. 1993).
The genes required for lipid-linked intermediate as-
sembly, polymerization, and secretion have been isolated
and sequenced (Harding et al. 1987; Capage et al. 1987).
They are clustered in a 16-kb region, termed xpsI or gum
(Fig. 3). Unlike other exopolysaccharide biosynthetic
systems, this region is unlinked from those required for
the synthesis of sugar nucleotide precursors (Becker et al.
1997; Canter Cremers et al. 1990; Roberts 1996; Yam-
azaki et al. 1996). Nucleotide sequence analysis pre-
dicted the presence of 12 open reading frames (gumB to
gumM) (Fig. 3; Capage et al. 1987; Becker et al. 1995;
GenBank accession number U22511). The transcrip-
tional organization of the gum region was analysed
through gum-lacZ transcriptional fusions and primer-
extension assays (Katzen et al. 1996). These analyses
indicated that the gum region is expressed as a single
operon from a promoter located upstream of the ®rst
gene, gumB. A second promoter was identi®ed upstream
of gumK.
The biochemical functions of the gum gene products
have been assigned by analysing the in vitro formation
of lipid-linked biosynthetic intermediates and polymers
employing permeabilized cells of gum mutant strains.
Much of this work has appeared as patents, abstracts,
symposia papers, personal communications, or book
chapters with the data being generally accepted but not
always having been experimentally proven, or described
 147

Fig. 2 Proposed pathway for the

synthesis of the xanthan gum.
Gene functions based on in vivo
and in vitro biochemical analysis
(Capage et al. 1987; Vanderslice
et al. 1988; Marzocca et al. 1991;
KoÈplin et al. 1992; Harding et al.
1993; Lin et al. 1995; Wei et al.
1996; Katzen et al. 1998). Glc
glucose, Fru fructose, Man man-
nose, GlcA glucuronic acid, Ac
acetyl, Pyr pyruvyl, PPi pyro-
phosphate, GK glucokinase, PGI
phosphoglucose isomerase,
UDPG-PP UDP-glucose pyro-
phosphorylase, UDPG-deH UDP-
glucose dehydrogenase. Empty
spheres represent enzymes encoded
by genes that remain to be cloned.
Empty arrows indicate that re-
peating units are variably decorat-
ed. GumJ could not be associated
with any particular xanthan-bio-
synthetic step, although the possi-
bility of its participation in pre- or
post-polymerization processes can
not be discarded
148
Fig. 3 Genetic map of the Xanthomonas campestris campestris gum
operon showing the organization of the genes. Locations and
designations of the genes are indicated as open boxes. Black arrows
indicate the size and direction of the transcripts
in the scienti®c literature. Since the biosynthesis and
genetics of xanthan are used as a model system for other
exopolysaccharides, we decided to obtain our own set of
mutants in each of the gum genes, and analysed them
using the in vitro system (Katzen et al. 1998). Figure 2
summarizes the proposed gum gene functions. The
GumD protein catalyses the addition of glucose 1-
phosphate to the polyisoprenol phosphate carrier. This
reversible reaction is the ®rst step in the biosynthesis of
lipid-linked intermediates involved in the synthesis of
xanthan. GumM catalyses the addition of a b1,4-glu-
cose, followed by the addition of the internal a1,3-
mannose by GumH, a b1,2-glucuronic acid by GumK,
and the terminal b1,4-mannose by GumI. The GumL
protein incorporates pyruvyl residues to the external b-
mannose, while the acetyl residues are incorporated into
the internal a-mannose by GumF, and into the external
b-mannose by GumG.
In addition to the polyisoprenyl diphosphate penta-
saccharide, the lipid-linked trisaccharide is able to act as
a substrate for GumF. However, the lipid-linked acetyl
trisaccharide cannot act as an acceptor of a glucuronic
acid residue, suggesting that the acetyl residues are in-
corporated into the polymer via the lipid-linked re-
peating unit (Ielpi and Dankert, unpublished data).
Most of the gum genes could be disrupted within the
wild-type strain. However, gumB, gumC, gumE, gumM,
and gumJ genes could only be mutated by using a UDP-
glucose-defective strain since their inactivation in a wild-
type background appeared to be lethal (Katzen et al.
1996, 1998). Unexpectedly, the ®rst step in the assembly
of the lipid-linked intermediate was severely a€ected in
these double mutants. This de®ciency could be recovered
by the introduction of a plasmid carrying the coding
region for the C-terminal domain of GumD, which ap-
peared to be responsible of its glucosyl-1-phosphate
transferase activity (Katzen et al. 1998; Ferreiro and
Ielpi, unpublished data). These results suggest a possible
regulatory role for the GumD protein, or that a bal-
anced expression of one or more proteins is required for
the proper expression of the GumD activity. This may
be of particular signi®cance if GumD interacts with
another protein(s). Since gumB, gumC, and gumE strains
appear to accumulate complete xanthan subunits in
vitro and are unable to synthesize polymer, the products
of these genes may be needed for polymerization or
export of the polymer. Although the function of the
gumJ product cannot be associated with a particular
gum-biosynthetic step, a secretion role for GumJ can not
be ruled out. Alternatively it might be necessary for
preventing accumulation of a harmful product or for
recycling essential substrates.
Regulation of xanthan synthesis
To date, most of what is known about regulation of
xanthan synthesis derives from phytopathogenicity
studies. No speci®c controls of xanthan synthesis have
been reported. A X. campestris campestris clp (cap-like
protein) mutant showed reduced pathogenicity and de-
creased production of xanthan and some extracellular
enzymes (de CreÂcy-Lagard et al. 1990). Synthesis of
these and other extracellular enzymes and xanthan is
activated by the products of a cluster of at least ®ve
genes, designated rpf (regulation of pathogenicity fac-
tors; Tang et al. 1991). The sequence of one of these
genes, rpfC, showed homology to members of the two-
component class of regulatory proteins. These factors
appear to be balanced by at least one unlinked gene that
negatively regulates the synthesis of the extracellular
enzymes and xanthan (Tang et al. 1990). Although
xanthan gum is not essential for plant virulence (Katzen
et al. 1998), initial stages of its biosynthesis, seem to be
regulated as a part of a pathogenicity regulon (Coplin
and Cook 1990). Few promoters and transcription start
sites have been characterized in di€erent pathovars of
X. campestris to date (Knoop et al. 1991; Kingsley et al.
1993; Katzen et al. 1996). The identi®cation of promoter
regions involved in the ®rst stages of xanthan produc-
tion will be the basis for further studies on gum gene
regulation.
Industrial production of xanthan
X. campestris campestris can use a broad range of car-
bon and nitrogen sources. Xanthan is usually produced
by fermentation of X. campestris campestris strains with
glucose, sucrose, starch, acid or enzymic hydrolysates of
starch, molasse or corn syrup as the major carbon source
in batch cultures. As nitrogen source casein hydroly-
sates, soybean meal, yeast hydrolysates or distillers sol-
ubles are frequently used. In continuous fermentation,
there are problems of maintaining sterility and the risk

of the emergence of fast-growing mutants that do not
produce the desired product xanthan. In batch cultures
xanthan is produced to some extent in all growth phases.
The maximum production was observed in the expo-
nential growth phase (Thorne et al. 1987; Sutherland
1993). Di€erent growth phases and alterations of the
growth medium, for example by using di€erent sub-
strates and limiting nutrients, do not in¯uence the pri-
mary structure, but do a€ect the degree of decoration,
the molecular mass and the yield of xanthan. Therefore,
the xanthan product recovered from a batch culture
represents a mixture of xanthan produced in the di€er-
ent growth phases and may vary with di€erent culture
conditions (Davidson 1978; Slodki and Cadmus 1978;
Tait et al. 1986; Suh et al. 1990; Peters et al. 1993).
Depending on the production strain and the substrates
used in batch cultures, approximately 10±25 xanthan/
laccumulates in the culture supernatant after 48±72 h
(Harding et al. 1987; Thorne et al. 1987; Pollock and
Thorne 1994). In continuous cultures, high conversion
rates of substrate to polymer of 60%±70% were ob-
served (Jarman and Pace 1984). The production of
xanthan is promoted by a high ratio of carbon to ni-
trogen in the substrate. In ammonia-de®cient media a
high xanthan production was observed (Davidson 1978;
Tait et al. 1986). Souw and Demain (1979) reported that
sucrose is a better substrate for xanthan production than
glucose and pyruvate. They also found that succinate
and 2-oxoglutarate have stimulatory e€ects on xanthan
production in sucrose-based medium. Davidson (1978)
demonstrated that magnesium or phosphate limitation
results in the production of xanthan with a low pyruvate
content.
One of the major problems in the industrial produc-
tion of xanthan is the growing viscosity of the culture
during fermentation, which a€ects the availability of
oxygen and nutrients. X. campestris campestris strains
are strictly aerobic. Therefore, the oxygen-transfer rate
also a€ects the xanthan yield. Moreover, Suh et al.
(1990) reported that the molecular mass of xanthan
decreases under oxygen limitation.
Owing to the growing viscosity during fermentation,
suitable aeration and mixing of the culture have to be
guaranteed to avoid stagnant areas (Nakajima et al.
1990; Amallah et al. 1998). Therefore, mechanically ag-
itated fermentation tanks instead of air-agitated tanks
are usually used for the industrial production of xanthan.
The separation of the cells from the highly viscous
xanthan solution is a cost-intensive process in the in-
dustrial production (Homma et al. 1997; Murofushi et al.
1997). Nowadays, the favoured method for recovering
xanthan from the fermentation liquid is a precipitation
with alcohol, mostly isopropanol, after pasteurization to
destroy bacterial cells and enzymes (Cottrell and Kang
1978). Subsequently the xanthan precipitate is spray-
dried and milled to a powder. The alcohol is recovered
by distillation. Concentrates containing approximately
10% xanthan for industrial non-food applications
are obtained by tangential-¯ow ®ltration (Lee 1981).
149
Antibacterial agents are added to the concentrates to
stabilize the product. Enzymatic degradation of xanthan
is used to alter the rheological properties of the product
(Hou and Barnabe 1987; Ahlgren 1991, 1993, 1995;
Cadmus and Slodki 1991).
Strains for xanthan production were selected and
improved by conventional methods. Recently, more at-
tention has been paid to genetic techniques to develop
strains with altered xanthan production. Several e€orts
have been made to simplify the repeating unit structure
by mutation of speci®c genes involved in the synthesis of
xanthan, but the xanthan yield from these mutants was
much lower as than that from the wild-type strains.
Betlach et al. (1987) constructed a mutant that produced
xanthan lacking the glucuronic acid residues and the
pyruvate decoration. Solutions of this xanthan deriva-
tive were highly viscous. A plasmid that carried a part of
the gum gene region was introduced into X. campestris
campestris (Harding et al. 1987). The resulting strain
produced xanthan with an enhanced pyruvate content.
Mutants that produced xanthan containing no pyruvate
were isolated by Wernau (1979, 1980) and Hassler and
Doherty (1990). Another strategy to alter the structure
of xanthan is the postsynthetic enzymatic treatment of
xanthan, such as the removal of the terminal b-D-glu-
curonosyl residue from xanthan missing the mannosyl
side-chain terminus (Tait and Sutherland 1989). This
truncated xanthan missing the terminal disaccharide was
more viscous than the original xanthan (Betlach et al.
1987; Tait and Sutherland 1989).
The possibility of achieving a considerable increase of
the xanthan yield by the construction of new strains
seems to be unlikely, since (like the synthesis of many
other bacterial polysaccharides) the synthetic process for
xanthan production is very ecient because of a high
conversion rate from the carbon substrates (Linton
1990). Therefore, an improvement of the xanthan yield
and quality has mainly been achieved through improved
fermenter design and through changes in the composi-
tion of the medium. Nevertheless, attempts have been
made to extend the range of substrates eciently used
for xanthan biosynthesis. X. campestris campestris does
not use lactose eciently as a carbon source because of
the low level of b-galactosidase activity. Therefore, whey
is not a suitable carbon source for xanthan production.
Fu and Tseng (1990) introduced a plasmid carrying a b-
galactosidase-encoding gene into X. campestris camp-
estris. The resulting strain was able to produce xanthan
in whey-containing medium, but the plasmid was not
maintained stably without selective pressure. Genetic
methods are also a desirable approach to eliminate un-
wanted products from the production strains. New
strains may help to improve the recovery and puri®ca-
tion of xanthan. Xanthan-producing strains synthesize
several extracellular enzymes such as cellulases (Gough
et al. 1988, 1990), which can degrade xanthan in its
disordered form (Sutherland 1984): xanthan is often
added to products that contain cellulose or derivatives
thereof. These enzymes are destroyed by heat treatment
150

or chemical treatment that involves the use of propylene lizing, thickening, gelling and emulsifying agent, but it is
oxide or propionolactone (Empey and Pettitt 1978). The also used as an adhesive and for the inhibition of ice-
construction of mutants lacking these enzymes can crystal formation.
simplify the recovery and puri®cation process.

Applications of xanthan
Although only a few microbial exopolysaccharides are
commercially available, xanthan is already a well-es-
tablished product. To date, more than 1600 patents on
production and applications of xanthan have been reg-
istered in the USA alone. Nevertheless, other polysac-
charides from plant origin, e.g. alginate, cellulose,
starch, arabic gum and guar gum, or from bacterial
origin, e.g. alginate (Rehm and Valla 1997), pullulan
(Tsujisaka and Mitsuhashi 1993), gellan (Pollock 1993)
and acetan (Jansson et al. 1993), compete with xanthan.
Xanthan was approved for food use by the U.S. Food
and Drug Administration in 1969 and received approval
from the European Union under the E-number 415 in
1980. No acceptable daily intake value was ®xed for
xanthan and it was approved quantum satis. World wide,
10 000±20 000 tonnes of xanthan per year are commer-
cially produced. The success of xanthan as the major
commercially produced microbial polysaccharide is
based on the following important rheological properties.
Solutions of xanthan have a high viscosity at low con-
centrations and they are pseudoplastic (Rinaudo and
Milas 1978; Milas and Rinaudo 1986; Richardson and
Ross-Murphy 1987; Nolte et al. 1992). Moreover, xan-
than is insensitive to a broad range of temperature, pH
and electrolyte concentrations (Lambert and Rinaudo
1985; Hatakenaka et al. 1987; Kierulf and Sutherland
1988). It also displays a high shear stability (Chen and
Sheppard 1980). Because of these properties xanthan has
found a broad range of applications in the oil, phar-
maceutical, cosmetic, paper, paint and textile industries
(Table 1; Sandvik and Maerker 1977; Brandford and
Baird 1983; Sutherland 1993). In these applications it is
mainly used as a gelling and suspending agent, as a
¯occulant or for viscosity control. Xanthan is also used
in the food industry (Table 1; Brandford and Baird
1983; Sutherland 1993). It is mainly added as a stabi-
Perspectives
Bacterial exopolysaccharides like xanthan provide an
alternative to polysaccharides of plant origin. However
bacterial polysaccharide products are relatively expen-
sive in comparison to the bulk plant polysaccharides,
such as alginate originating from plants and starch. One
advantage of xanthan is that the quality of this bacterial
product can be ensured by the use of speci®c production
strains and by the fermentation conditions. To
strengthen the position of xanthan in the polysaccharide
market, the physiology and regulation of the production
process have to be investigated and new applications
have to be established. The commercial success of xan-
than is not only based on its rheological properties, but
also on economic factors. X. campestris campestris can
use a broad range of di€erent substrates. The substrate
conversion rate is high and recovery and conversion of
xanthan to the ®nal product are simple and therefore
relatively cheap. The elimination of unwanted byprod-
ucts by genetic modi®cations of the production strains
may simplify the recovery of xanthan from the fermen-
tation liquid. New xanthan derivatives with useful
properties seem to be dicult to achieve. Decorations or
the side-chain may be subject to modi®cations. Chemical
modi®cations or genetic methods may result in xanthan
derivatives with new rheological properties. New appli-
cations for such speci®c products can be discovered in
the growing sector of sustainable resources or in the
pharmaceutical sector. A new ®eld of applications may
be identi®ed for speci®c oligosaccharides derived from
xanthan. More research will be necessary to improve the
production of speci®cally modi®ed xanthan derivatives,
to predict and characterize their rheological properties
and to identify new applications.
Acknowledgements Work at the laboratory of L.I. was partly
supported by grants from Universidad de Buenos Aires and CO-
NICET. L.I. is a member of Carerra del Investigador CONICET,
Argentina. Work at the laboratory of A.P. was partly supported by

Table 1 Examples of in-

dustrial, food and pharmaceu-
tical applications of xanthan
(Sandvik and Maerker 1977;
Brandford and Baird 1983;
Sutherland 1993)
Industrial applications
Abrasives (viscosity control)
Ceramic glazes, polishes, thixotrophic paints
(stabilization, pseudoplasticity)
Explosives (gelling agent)
Fire-®ghting ¯uids (foam stabilizer)
Hydraulic fracturing (viscosity/cross-linking)
Laundry chemicals, agricultural chemicals
including herbicides, pesticides, fertilizers,
and fungicides (suspension)
Oil-drilling muds (viscosity control, shear
thinning)
Textile dyeing (pseudoplasticity)
Water clari®cation (¯occulant)
Food and pharmaceutical applications
Beer (foam stabilizer)
Cheese (syneresis inhibitor)
Confectionery (coating)
Ice cream (stabilizer, crystallisation control)
Icings and glazes (adhesive)
Jams, sauces (thickening agent)
Juice drinks (suspension)
Pet foods (binding agent)
Pharmaceuticals (retarded drug release)
Powdered ¯avours (encapsulation)
Salad dressing, bakery ®llings (emulsifying agent)
Sausage casings (®lm formation)
Syrups (pseudoplasticity)

the grants GRK 35/2-96 and GRK 35/3-98 from Deutsche For-
schungsgemeinschaft.
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