Fux017 EPS CPS

FEMS Microbiology Reviews, fux017, 41, 2017, S168–S200
doi: 10.1093/femsre/fux017
Advance Access Publication Date: 19 June 2017
Review Article
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REVIEW ARTICLE
Polysaccharide production by lactic acid bacteria:

from genes to industrial applications
Ahmad A. Zeidan1,† , Vera Kuzina Poulsen1,† , Thomas Janzen1 ,
Patrizia Buldo2 , Patrick M. F. Derkx1 , Gunnar Øregaard1
and Ana Rute Neves1,∗
1
Discovery, Research and Development and 2 Global Application, Food Cultures and Enzymes, Chr. Hansen A/S,
2970 Hørsholm, Denmark
∗
Corresponding author: Discovery, Research and Development, Bøge Allé 10–12, 2970 Hørsholm, Denmark. Tel: +45 60 43 10 12;
E-mail: [email protected]
†
Both authors contributed equally to this work.
One sentence summary: This review describes the recent findings regarding exocellular polysaccharide production in lactic acid bacteria, and provides
an overview of their applications in food and future trends in polysaccharide research.
Editor: Oscar Kuipers
ABSTRACT
The ability to produce polysaccharides with diverse biological functions is widespread in bacteria. In lactic acid bacteria
(LAB), production of polysaccharides has long been associated with the technological, functional and health-promoting
benefits of these microorganisms. In particular, the capsular polysaccharides and exopolysaccharides have been implicated
in modulation of the rheological properties of fermented products. For this reason, screening and selection of exocellular
polysaccharide-producing LAB has been extensively carried out by academia and industry. To further exploit the ability of
LAB to produce polysaccharides, an in-depth understanding of their biochemistry, genetics, biosynthetic pathways,
regulation and structure–function relationships is mandatory. Here, we provide a critical overview of the latest advances in
the field of glycosciences in LAB. Surprisingly, the understanding of the molecular processes involved in polysaccharide
synthesis is lagging behind, and has not accompanied the increasing commercial value and application potential of these
polymers. Seizing the natural diversity of polysaccharides for exciting new applications will require a concerted effort
encompassing in-depth physiological characterization of LAB at the systems level. Combining high-throughput
experimentation with computational approaches, biochemical and structural characterization of the polysaccharides and
understanding of the structure–function–application relationships is essential to achieve this ambitious goal.
Keywords: polysaccharides; lactic acid bacteria; EPS, CPS; strain improvement; polysaccharide applications
INTRODUCTION >10 units; oligosaccharides, ≤10 units) bound together by

glycosydic linkages, and thus are characterized by high molec-
Polysaccharides or glycans are widespread in nature, and dis-
ular weight. In contrast to DNA, RNA or protein synthesis,
play diverse chemical structures, physical properties and bio-
polysaccharide synthesis is not a template-driven process, but
logical functions that are usually either structure or storage re-
instead the structures are determined primarily by the com-
lated (Ullrich 2009). These polymeric carbohydrate molecules
plement of polysaccharide-modifying enzymes present in any
consist of a large number of monosaccharides (generally,
organism. Polysaccharides can range in structure from linear
Received: 17 January 2017; Accepted: 29 March 2017

C FEMS 2017. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
S168
Zeidan et al. S169

Figure 1. Cell surface-associated polysaccharides in bacteria. Capsular polysaccharides (CPS) and exopolysaccharides (EPS) are common to Gram-negative (a) and Gram-
positive bacteria (b). Lipopolysaccharides (LPS) and lipooligosaccharides (LOS) are found only in Gram-negative bacteria, whereas Gram-positive bacteria synthesize
cell wall polysaccharides (CW-PS) such as wall teichoic acids (TA), lipotheichoic acids (LTA) and pellicles. A peptidoglycan (PG) layer is present in the cell walls of both
Gram-positive and Gram-negative bacteria. Closed circles, monosaccharides.
to highly branched, and in composition they can be classified et al. 2010; Mistou, Sutcliffe and van Sorge 2016). Whilst most
as homopolysaccharides (HoPS) when composed of only one of these polysaccharides are produced by both Gram-negative
type of monosaccharide or heteropolysaccharides (HePS) if two and Gram-positive bacteria, the latter cannot synthesize LPS and
or more types of sugars are present. Variations in the sugar LOS, but instead produce TA and LTA (Fig. 1).
building blocks, anomeric configuration, glycosidic linkage, non- Storage polysaccharides accumulate as carbon and energy
sugar decorations of the monosaccharides, conformation and reserves to cope with the starvation conditions temporarily
molecular weight result in an enormous diversity of polysac- present in the environment. Cell surface-associated polysac-
charides. This structural diversity contributes to the ensemble charides play critical roles in interactions between bacteria
of biological functions and renders an array of physicochemical and their surroundings. Accordingly, they serve a multitude of
and rheological properties that can be exploited for varied com- biological functions, such as maintenance of cell shape and
mercial applications in the industrial, food and medical sectors structural integrity, charge and cation homeostasis, protec-
(Ullrich 2009; Schmid, Sieber and Rehm 2015). Even though the tion against adverse conditions such as desiccation, toxic com-
global market is so far dominated by polysaccharides produced pounds (bile salts, hydrolyzing enzymes, e.g. lysozyme, gastric
by plants and algae (e.g. pectin, cellulose, alginate), bacteria rep- and pancreatic enzymes, metal ions, antibiotics, ethanol, etc.)
resent a relatively untapped source of an immense polysaccha- and antibacterial stresses (varying pH, osmolarity and gas atmo-
ride repertoire. sphere), predation by protozoans, evasion of the immune system
Bacteria can synthesize cytoplasmic storage polysaccharides and phage attack (Donot et al. 2012; Patel and Prajapat 2013; Ryan
(e.g. glycogen, bacterial starch; Wilkinson 1963) and cell surface- et al. 2015; Caggianiello, Kleerebezem and Spano 2016; Mahony
associated polysaccharides (peptidoglycan [PG]; lipopolysac- et al. 2016). CPS and EPS have been postulated to play important
charides [LPS]; lipooligosaccharides [LOS]; teichoic acids [TA]; roles in bacteria–host interactions, namely facilitating coloniza-
lipoteichoic acids [LTA] and other cell wall polysaccharides [CW- tion through their ability to adhere to surfaces (e.g. adhesion
PS], such as pellicles, exopolysaccharides [EPS] and capsular to eukaryotic cells and mucosa), and in microbial-mediated im-
polysaccharides [CPS]) (reviewed in Chapot-Chartier 2014; Tytgat munomodulation (Mazmanian and Kasper 2006; Ryan et al. 2015;
and Lebeer 2014; Schmid, Sieber and Rehm 2015; Mistou, Sut- Caggianiello, Kleerebezem and Spano 2016). Furthermore, EPS
cliffe and van Sorge 2016). EPS and CPS are exocellular polysac- play key roles in bacterial biofilms as summarized by Flemming
charides that at most differ in their degree of attachment to the and Wingender (2010). Hitherto, a comprehensive understand-
cell surface: CPS are tightly linked, often covalently, to the cell ing of the biological functions of the exocellular polysaccharides,
surface and form a capsule around the cell, whereas EPS are se- and especially EPS, has not been obtained.
creted into the extracellular matrix or loosely associated with Despite the vast structural diversity, bacteria produce
the cell surface via electrostatic interactions often forming a polysaccharides by using either a sequential or an en bloc mech-
slime layer (Tytgat and Lebeer 2014). Pellicles are interpolated anism via four different pathways: the Wzy-dependent path-
with the cell wall (PG) layer, as opposed to capsules that are typ- way (en bloc), the ATP-binding ABC transporter pathway (se-
ically the outermost layer of the cell envelope (Chapot-Chartier quential), the synthase-dependent pathway (sequential) and
S170 FEMS Microbiology Reviews, 2017, Vol. 41, No. Supp 1

Figure 2. Pathways for the synthesis of bacterial polysaccharides. (a) The Wzy-dependent pathway (synthesis of LPS O-antigen polysaccharide in Gram-negative
bacteria, CPS and EPS in both Gram-negative and Gram-positive bacteria) represented for Gram-positive bacteria. Main players are the Wzy polymerase and Wzx
flippase; details for this pathway are shown on Fig. 5, but a repeating unit is synthesized intracellularly, flipped and polymerized in the outer face of the cytoplasmic
membrane. (b) The ABC transporter-dependent (CPS in Gram-negative bacteria; potentially other CW-PS) represented for Gram-negative bacteria. The polysaccharide
chain, anchored on a poly-2-keto-3-deoxyoctulosonic acid linker in the cytoplasmic face of the inner membrane, is assembled by the action of GTs; the finished
polysaccharide is exported via an efflux pump complex composed of ABC transporters spanning the inner membrane and periplasmatic proteins of the OPX family
spanning the outer membrane. (c) The synthase-dependent pathway (CPS, EPS) performs the polymerization and transport by a single synthase complex, which
secretes the complete polymer strands across the membranes and cell wall. (d) Extracellular synthesis of HoPS by the use of a single GT (sucrase) protein. Blue
circle, glucose; yellow diamond, galactose; pink square, rhamnose; green triangle, fructose. CM, cytoplasmic membrane, OM, outer membrane; PG, peptidoglycan; GT,
glycosyltransferase; Und-P, undecaprenylphosphate; P, phosphate; k, poly-2-keto-3-deoxyoctulosonic acid linker.
extracellular synthesis by use of a single glycosyltransferase (su- tion to acidification, LAB can also contribute to the functionality
crase) enzyme (Fig. 2; Tytgat and Lebeer 2014; Schmid, Sieber and and organoleptic properties of the fermented products by pro-
Rehm 2015). In one bacterial species, two or more pathways can ducing antimicrobial compounds (e.g. bacteriocins), bioactive
coexist for the production of different macromolecules. A more peptides, vitamins, low calorie sugars, flavor and aroma com-
detailed description of selected pathways will be provided in the pounds, and polysaccharides (Gaspar et al. 2013). In particular,
following sections of this review. the in situ production of polysaccharides by LAB has strongly
Lactic acid bacteria (LAB) are a heterogeneous group of Gram- been associated with the diverse technological, functional and
positive bacteria known for their key roles in the manufacture health-promoting properties displayed by these microorgan-
of fermented foods and beverages, such as cheese, yoghurt isms (recently reviewed in Ryan et al. 2015; Torino, Font de Valdez
and other fermented milks; fermented meat; sourdough and and Mozzi 2015; Caggianiello, Kleerebezem and Spano 2016;
other breads; vegetables and alcoholic beverages, such as wine, Mende, Rohm and Jaros 2016). Among the cell surface-associated
cider and beer; as well as their ubiquitous presence in animal polysaccharides known to be produced by LAB (PG, TA, LTA, pel-
microbiomes, a phenomenon that is frequently associated with licles, EPS and CPS; Fig. 1), it is the exocellular polysaccharides
beneficial effects (reviewed in Mozzi, Raya and Vignolo 2010; EPS and CPS that are generally identified as contributing to the
Lahtinen et al. 2011). As indicated by their designation, the main functional attributes in food. In LAB literature, the term EPS
fermentation product of LAB from carbohydrate is lactate (Kan- is often used to refer to both exocellular polysaccharides, but
dler 1983). This feature has been extensively exploited to extend in this review we will use the terms EPS and CPS differentially
the shelf life of milk and other raw materials, since the low pH for the sake of clarity. These polysaccharides are known to
generated by the conversion of the carbon source into lactic acid improve the rheological properties of LAB-fermented prod-
inhibits the growth of spoilage and pathogenic bacteria. In addi- ucts by influencing viscosity, syneresis, firmness and sensory
Zeidan et al. S171
properties. The location of the polysaccharide (EPS or CPS), the in Streptococcus thermophilus, but can reside on a plasmid or
primary structural features (monosaccharide type and config- the chromosome in L. lactis and Lactobacillus sp. (Ruas-Madiedo,
uration, glycosidic linkage, non-sugar decorations, charge), the Salazar and de los Reyes-Gavilán 2009). The organization of
conformation and molecular weight, the amount of polysac- the eps gene cluster in LAB is similar to that of Gram-positive
charide and the interactions of the polysaccharide with other pathogens (Fig. 3). Generally, eps gene clusters are highly diverse
system components are all factors that can contribute to and/or and their nucleotide sequences are among the most variable se-
influence the displayed technofunctional properties. Whether quences in LAB genomes. Mobile genetic elements play a role in
other cell surface-associated polysaccharides are also involved this diversity. Indeed, insertion sequence (IS) elements flanking
in technofunctional properties such as rheology augmentation or within the operon are consistently present in the architec-

remains to be investigated. ture of eps gene clusters (De Vuyst et al. 2001; Hols et al. 2005;
Regarding monomeric composition, the exocellular polysac- Cui et al. 2016). However, the modular gene organization in eps
charides (EPS and CPS) produced by LAB can also be classified as gene clusters is conserved (De Vuyst et al. 2001; Jolly and Stingele
HoPS and HePS. HoPS are generally synthesized in the extracel- 2001; Bentley et al. 2006; Ruas-Madiedo, Salazar and de los Reyes-
lular matrix by an extracellular glycosyltransferase (GT) (Fig. 2) Gavilán 2009; Goh et al. 2011; Okura et al. 2013; Skov Sørensen
and are composed of D-glucose or D-fructose, and thereby de- et al. 2016). Genes in the eps operon can be categorized into
nominated glucans (α- or β-) and fructans. Glucansucrases and groups based on the putative or established functions of their
fructansucrases catalyze the polymerization of the glucans and products. These include modulatory genes (phosphoregulatory
fructans usually using sucrose as the donor of the corresponding module epsBCD), polysaccharide assembly machinery genes (ini-
monosaccharide and transferring this residue to the reducing tiation epsE, polymerization wzy, export/flippase wzx and at-
end of the growing HoPS (van Hijum et al. 2006). Different gen- tachment epsA), genes encoding the GT necessary for the assem-
era of LAB, including Weissella, Leuconostoc, Lactobacillus, Pedio- bly of the repeating units and genes encoding non-housekeeping
coccus and Streptococcus (namely oral streptococci), produce HoPS functions required for the synthesis of activated sugar precur-
via this pathway. Diversity and synthesis of sucrase-dependent sors and modification of the sugar residues (Fig. 3), according
HoPS in LAB and their applications in the food industry have to the working model for Streptococcus pneumoniae (Nourikyan
been the focus of several recent reviews (Ryan et al. 2015; Torino, et al. 2015).
Font de Valdez and Mozzi 2015) and will not be the main focus Generally, the eps gene clusters are 15–20 Kb in size and com-
of this review. prise less than 30 genes. In LAB, the eps genes usually have the
A few examples of LAB that produce HoPS constituted solely same orientation and are transcribed as a single mRNA (Guidolin
of galactose moieties have also been described (van Kranenburg et al. 1994; van Kranenburg et al. 1997; Lamothe et al. 2002). Genes
et al. 1999c; Mozzi et al. 2006). These polygalactans are com- located at the 5 end of the cluster are involved in the modu-
posed of several repeating units containing four or five galactose lation and assembly machinery of polysaccharide biosynthesis
residues, and are most likely the product of the Wzy-dependent and display the highest level of overall conservation. A typical
pathway, which in LAB is the pathway of choice for the synthesis eps gene cluster consists of five highly conserved genes epsA,
of heteropolymeric EPS and CPS (Ryan et al. 2015; Torino, Font de epsB, epsC, epsD and epsE, and a variable region, which includes
Valdez and Mozzi 2015). A detailed overview of their properties the polymerase wzy, the flippase wzx, one or more glucosyl-
in LAB is provided in the following sections. transferases and/or other polymer-modifying genes (Fig. 3). Lac-
In this review, we will describe and discuss the genetics and tococcus lactis harbors an additional gene, epsX, in the conserved
biochemistry of exocellular polysaccharide biosynthesis in LAB, 5 end. Moreover, in this organism, genes epsL and orfY represent
with particular emphasis on polysaccharides synthesized via a second conserved region at the 3 end (Fig. 3). To date, no puta-
the Wzy pathway. Furthermore, industrial applications asso- tive functions have been assigned to the lactococcal genes epsX
ciated with the ability to produce these polysaccharides and and epsL. Gene epsL can be disrupted or overproduced in L. lactis
strategies to screen and improve LAB for superior rheological NIZO B40 without any effect on EPS production (van Kranenburg
properties will be reviewed. et al. 1999c), suggesting that it is either non-essential or there is
an alternative gene in the genome which product has a similar
function. The 12-kb eps cluster of L. lactis NIZO B40 is flanked
COMPARATIVE GENOMICS OF by a gene encoding an IS element and orfY, which is oriented in
the opposite direction of the genes in the eps cluster. The clus-
POLYSACCHARIDE SYNTHESIS
ter consists of 14 genes, including the 5 conserved genes, the
Various gene clusters for the synthesis of exocellular polysac- more variable wzy and wzx, and non-conserved genes encod-
charides via the Wzy-dependent pathway and genes encoding ing GTs. Lactococcus lactis B891 harbors an eps cluster displaying
sucrases for the extracellular synthesis of HoPS have been found an identical architecture, but the gene organization in the eps
in the genomes of LAB. While sucrase-encoding genes have been cluster of L. lactis NIZO B35 differs considerably (van Kranen-
described in the LAB genera Weissella, Leuconostoc, Lactobacil- burg et al. 1999a). In S. thermophilus, despite the various archi-
lus, Pediococcus and Streptococcus, studies on Wzy-dependent eps tectures described, the eps cluster is generally flanked at the 5
gene clusters focus almost exclusively on three genera: Lactococ- end by deoD (purine-nucleoside phosphorylase presumably in-
cus, Lactobacillus and Streptococcus. Other polysaccharide biosyn- volved in the biosynthesis and catabolism of nucleotides) and at
thetic gene clusters, such as those for the synthesis of pellicles the 3 end by orf14.9 and/or bglB (6-phospho-beta-glucosidase).
in Lactococcus lactis and CW-PS in lactobacilli, have been reported, The product of orf14.9 has been putatively associated with cell
but the biosynthetic routes for these polysaccharides remain growth (De Vuyst et al. 2011), and whether the flanking gene at
largely elusive (Ruas-Madiedo, Salazar and de los Reyes-Gavilán the 3 end is orf14.9 or bglB is still under debate. In the pathogen
2009; Chapot-Chartier 2014; Tytgat and Lebeer 2014; Mistou, Sut- S. pneumoniae, genes dexB and aliA take this role, respectively.
cliffe and van Sorge 2016). In summary, despite differences in the genes flanking the eps
Genes encoding Wzy-dependent exocellular polysaccharide clusters in streptococci (both pathogenic and LAB), the conser-
biosynthesis proteins in LAB are typically organized in a clus- vation of eps gene clusters appears to be restricted to the genes
ter with an operon structure and are generally chromosomal encompassed within the cluster. This view possibly holds true

Figure 3. Schematic genetic organization of the eps gene clusters in different LAB based on several representative strains and S. pneumoniae strain D39. Gene functional
grouping is marked with different colors. Relative localizations of eps genes with homologous functions are indicated with connection bars. All the genes are transcribed
in one direction except for a few genes oriented in the opposite transcriptional sense, which are indicated with arrows. Genes with unknown functions or functions
not related to the EPS biosynthesis are in white. GT, glycosyltransferase; IS, transposase; NDP-sugar, nucleotide diphospho-sugar. The ‘GT’ functional module does
mainly include GTs, but it can also include other enzymes that modify the oligosaccharide repeat unit structure. For instance, Oenococcus oeni PSU-1 ORF marked with
a star is a putative galactoside O-acetyltransferase.
for lactobacilli, but a thorough comparative analysis of the eps tococcal eps genes are designated epsR, epsA, epsB, epsC and
gene clusters for this diverse group is still lacking. Indeed, in Lac- epsD (van Kranenburg et al. 1997, 1999c; Forde and Fitzgerald
tobacillus delbrueckii ssp. bulgaricus Lfi5, the eps cluster is flanked 2003; Dabour and LaPointe 2005; Knoshaug, Ahlgren and Trempy
by genes orfX and orfY (Lamothe et al. 2002). The variation in the 2007), whereas in S. thermophilus, the corresponding genes are
eps gene cluster architecture in lactobacilli is seemingly higher called epsA, epsB, epsC, epsD and epsE, respectively (Jolly and
than in L. lactis and S. thermophilus, which likely reflects the large Stingele 2001; Goh et al. 2011). In L. delbrueckii ssp. bulgaricus
diversity within the Lactobacillus genus. (thereafter denominated Lb. bulgaricus), the genes are named
The nomenclature currently used for designating the genes epsABCDE (Lamothe et al. 2002), but the order of the conserved
in eps clusters encoding the Wzy-dependent pathway differs genes is not the same as in S. thermophilus. Often the eps genes
among organisms (Fig. 3). In L. lactis, the first conserved lac- are merely designated alphabetically by order of occurrence in
Zeidan et al. S173
Table 1. Repeating units of EPS/CPS synthesized by the Wzy pathway. The number of elucidated repeating units, uniqueness of the repeating
unit, the size range in number of sugar residues of the repeating unit and occurrence of different sugar types and other constituents in the
structures are listed. Data derived from de Vuyst and de Vin (2007), Ruas-Madiedo, Salazar and de los Reyes-Gavilán (2009) and original works
published since 2009 (see Tables 2 and 3).
Occurrence of monosaccharides in
the 55 unique repeating units of 81 structures described
Genus RUa uRUb RU size Glc Gal Rha GlcNAc GalNAc Other Decoration

Lactococcus 6 4 5, 7c 3 (5)d 4 (6) 2 (4) – – – Ac (1)
PO4 (3) [1]e
Streptococcus 28 11 4 -8 8 (22) 11(28) 5 (8) 1f 2 (12) Fuc (1) Ac (1)
Rib (1) Pyr (1)
Sugg (1)
Lactobacillus 47 40 3-10 36(43) 34(41) 7 (13) 5 2 Man (6) Gly-P (4), Pyr (2)
Fuc (1) Ac (5)
ManNAc (2) PO4 (1)
a
RU, repeating units with structure elucidated.
b
uRU, unique repeating units among the elucidated.
c
Out of the six repeating units only one possesses seven residues.
d
Number of structures.
e
Number of repeating units.
f
Streptococcus macedonicus.
g
6-O-(3 ,9 -dideoxy-D-threo-D-altro-nononic acid-2 -yl)-D-glucopyranose.
Glc, glucose; Gal, galactose; Rha, rhamnose; GlcNAc, N-acetyltglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Rib, ribose; ManNAc, N-acetylmannosamine;
Ac, acetyl, PO4 , phosphate, Pyr, pyruvoyl, Gly-P, glycerol-phosphate.
a given locus. As a consequence, eps genes with the same name mology groups for polysaccharide polymerases (wzy) and 4 for
tag encode proteins with different functions: epsB encodes the priming GTs (wchA, wciI, wcjG, wcjH) were found. The predictions
cytoplasmic domain of a tyrosine-protein kinase in L. lactis, a for initial sugars, and subsequent repeating unit polymerization
phosphotyrosine protein phosphatase in S. thermophilus and the linkage, correlate well with the polymerase homology groups:
membrane-bound domain of the tyrosine-protein kinase in Lb. 32 polymerase homology groups associated with WchA, 5 with
bulgaricus (Fig. 3). Even for the same species, the genes can be WciI, 4 with WcjG and 1 with WcjH. These associations are
named differently: in L. lactis NIZO strain B40, epsI and epsK code mostly exclusive, with only five polymerase homology groups
for the Wzy polymerase and the Wzx flippase (polysaccharide associated with two initial transferases, which indicates a high
export), while in L. lactis SMQ-461, the genes with corresponding specificity of the initial transferases (Bentley et al. 2006). In ad-
functions are named epsH and epsM, respectively (van Kranen- dition, 13 groups of flippases and a great diversity of GTs were
burg et al. 1997; Dabour and LaPointe 2005). found in the polysaccharide gene clusters of S. pneumoniae. The
A standardized nomenclature for Wzy-dependent polysac- presence of multiple non-homologous or highly divergent forms
charide synthesis genes in pathogenic bacteria has been pro- of GTs, together with often different G+C content of the re-
posed previously (Reeves et al. 1996). In S. pneumoniae, capsule gion in which these are encoded, indicates that the genes have
biosynthesis genes utilize the prefix cps followed by the serotype been acquired from different sources. With the growing num-
number and gene designation, which follows an alphabetical or- ber of genome sequences of LAB, bioinformatic analyses similar
der based on the gene location in the cps operon, a nomencla- to those conducted in S. pneumoniae can now be applied to cat-
ture which is closely related to that of S. thermophilus. Herein, we egorize the functions encoded by the genes in eps clusters and
adopt the nomenclature commonly applied in S. thermophilus, as predict biosynthetic mechanisms in LAB.
it is arguably the best-characterized LAB in terms of exocellular Although most of the enzymes necessary for polysaccha-
polysaccharide production and the most widely used LAB in in- ride synthesis are encoded within the polysaccharide-specific
dustrial applications for its texturizing properties. For a generic loci, many of the sugar nucleotides (NDP sugars) are common to
LAB eps gene cluster, we propose to designate the five first con- other metabolic pathways, e.g. PG and TA synthesis, and are ob-
served genes epsABCDE, the polymerase wzy and the flippase tained from cellular pools without the need for unique enzymes.
wzx (Fig. 3). Of the 18 sugars and related compounds found in S. pneumo-
The genes typically encoding GT, the polymerase (wzy) and niae capsules, 7 are available from housekeeping metabolic path-
the flippase (wzx) are situated in a variable part of the eps gene ways and 9 from known dedicated pathways encoded within
cluster, and often have a low degree of similarity to already the polysaccharide gene cluster (Bentley et al. 2006). In LAB,
characterized genes, which makes the prediction of their pu- we estimate that out of the 11 sugars and their derivatives de-
tative functions difficult. A substantial effort to computation- tected so far in structures of repeating units, at least 6 (galactose,
ally group and categorize the eps gene products has been car- glucose, rhamnose, N-acetylglucosamine, mannose and ribose;
ried out for the human pathogen S. pneumoniae (Bentley et al. Table 1 and Fig. 4) result from central metabolism routes. How-
2006; Aanensen et al. 2007). Comparison of polysaccharide syn- ever, in Lactobacillus rhamnosus, the genes required to synthesize
thesis operons from 90 pneumococcal serotypes revealed that dTDP rhamnose (rmlABCD aka rbfABCD) are associated with the
central genes responsible for the synthesis and polymerization eps operon and transcribed either from their own promoter or
of the repeat unit are highly variable and often non-homologous together with the eps operon genes (Péant et al. 2005). In other
among serotypes (Bentley et al. 2006). In that study, 40 ho- LAB, like the non-EPS-producing L. lactis spp. cremoris MG1363,

Figure 4. Schematic representation of the metabolic routes for the generation of activated sugar precursors for the synthesis of exocellular polysaccharides during the
catabolism of lactose or glucose by LAB. In LAB, lactose and glucose can either be taken up via a phosphoenolpyruvate:sugar phosphotransferase system (PTS) and con-
comitantly phosphorylated to the respective sugar-phosphate (sugar-P) or via secondary carriers (SC) transporters and subsequently phosphorylated to the sugar-P via
specific kinases. For the sake of simplicity, only the lactose permease (LacS) and the main glucose PTS (PTSMan ) are represented in this scheme, but it should be noted
that by no means these are the sole routes for the transport of the sugars (Hutkins, Morris and McKay 1985, Thompson and Chassy 1985, de Vos and Vaughan 1994,
Castro et al. 2009, Solopova et al. 2012). The reactions proposed are catalyzed by the following enzymes encoded by the gene(s) in italic: PTSMan (manMNL), lactose per-
mease (lacS), phosphoglucose isomerase (pgi), 6-phosphofructo-1-kinase (pfk); fructose-1,6-bisphosphate aldolase (fba), fructose-1,6-bisphoshatase (fbp); glucokinase
(glk), β-galactosidase (lacZ), galactose mutarotase (galM), α-galactokinase (galK), galactose-1-phosphate uridylyltransferase (galT), UDP-galactose 4-epimerase (galE),
α-phosphoglucomutase (pgmA), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose 6-dehydrogenase (ugd), dTDP-glucose pyrophosphorylase (rmlA), dTDP-
glucose 4,6-dehydratase (rmlB), dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase (rmlC), dTDP-4-keto-L-rhamnose reductase (rmlD), L-glutamine:D-fructose-6-phosphate
aminotransferase (glmS), phosphoglucosamine mutase (glmM) N-acetylglucosamine-1-phosphate uridyltransferase and glucosamine-1-phosphate acetyltransferase
(glmU), UDP-N-acetylglucosamine 2-epimerase (mnaA), UDP-N-acetylglucosamine 4-epimerase (nagE), mannose 6-phosphate isomerase (manA), phosphomannomu-
tase (manB), mannose 1-phosphate guanylyltransferase (manC), GDP-mannose 4,6-dehydratase (gmd), GDP-fucose synthase (fcl). Gal, galactose; Glc, glucose; Rha, rham-
nose; Man, mannose; Fuc, fucose; GlcA, glucuronic acid; GlcNAc, N-acetyl-glucosamine; GalNAc, N-acetyl-galactosamine; ManNAc, N-acetyl-mannosamine; Glc-6-P,
glucose-6-phosphate; Fru-6-P, fructose-6-phosphate; FBP, fructose-1,6-biphosphate; α-Gal-1-P, α-galactose-1- phosphate; α-Glc-1-P, α-glucose-1- phosphate; GlcN-6-P,
glucosamine-6-phosphate; GlcN-1-P, glucosamine-1-phosphate; GlcNAc-1-P, N-acetylglucosamine-1-phosphate; Man-6-P, mannose-6-phosphate; Man-1-P, mannose-
1-phosphate;4-KDG, 4-keto-6-deoxyglucose; 4-DRha, 4-deoxyrhamnose; P, phosphate.
these genes are present elsewhere in the chromosome (Boels calized between loci LEGAS 0698 and LEGAS 0712. A putative eps
et al. 2004). The same is true for galU, galE and glmSMU genes cluster found by BLAST analysis in Lc. mesenteroides ssp. mesen-
(Fig. 4). Genes encoding proteins involved in the decoration of teroides strain BD3749 (GenBank Accession CP014610) had a sim-
the sugar residues, such as O-acetyl transferases and pyruvoyl ilar eps gene cluster structure as in the two Leuconostoc strains
transferases, have also been found in the eps gene clusters of described above.
LAB (Stingele et al. 1999; Wu et al. 2014; Fig. 3). In S. thermophilus, Oenococcus oeni, closely related to Leuconostoc, is able to syn-
genes potentially involved in the production of sugar nucleotide thesize both homo- and hetero-polysaccharides, via distinct
precursors (e.g. encoding phosphoglycerate mutase and phos- metabolic pathways (Dimopoulou et al. 2016). All 50 studied
phatase) have been found upstream of orf14.9. genomes contained at least one of the pathways: (i) a Wzy-
The eps gene clusters encoding the Wzy-dependent path- dependent synthetic pathway, allowing the production of HePS
way are present in the less characterized Leuconostoc, Oenococcus made of glucose, galactose and rhamnose, mainly in a capsu-
and Pediococcus, as determined by searches in publicly available lar form; (ii) a glucan synthase pathway (Gtf), involved in β-
genomes at NCBI or proprietary LAB genomes (Chr. Hansen Cul- glucan synthesis in a free and a cell-associated form, giving a
ture Collection). ropy phenotype to growth media; and (iii) HoPS synthesis from
Leuconostoc is known for its production of HoPS like dextran, sucrose (α-glucan or β-fructan) by glycoside-hydrolases of the
alternan and levan, but putative eps clusters for the production GH70 and GH68 families (Dimopoulou et al. 2014). For instance,
of HePS can be found in some Leuconostoc strains (Fig. 3). For in- O. oeni PSU-1 (GenBank Accession CP000411) contains a putative
stance, Leuconostoc gelidum ssp. gasicomitatum KG16-1 (GenBank HePS cluster (Wzy-dependent pathway) between loci OEOE 1507
Accession LN890331) contains a cluster of genes annotated as and OEOE 1496 (Fig. 3).
epsAHBCDEFG followed by six putative dTDP-rhamnosyl trans- An eps gene cluster can also be identified in the genome
ferase genes localized between loci LEGK 0689 and LEGK 0710. sequence of Pediococcus pentosaceus SL4 (GenBank Accession
Similarly, in Lc. gasicomitatum LMG 18811 (GenBank Accession CP006854). It is situated between locus tags T256 03080 and
FN822744), a cluster of genes annotated as epsABCDEFGHIJKX fol- T256 03130, and the order of the conserved epsBCD genes is sim-
lowed by one putative dTDP-rhamnosyl transferase gene is lo- ilar to that in L. lactis B40.
Zeidan et al. S175
In summary, the major genera of LAB used in food size, molecular weight) are essentially determined by the type
processing (Lactococcus, Streptococcus, Lactobacillus, Leuconostoc, of GT encoded in the LAB genomes, and have been extensively
Oenococcus and Pediococcus) possess eps gene clusters. Even reviewed by others (van Hijum et al. 2006; Patten and Laws 2015;
though the potential to synthesize exocellular polysaccharides Ryan et al. 2015; Torino, Font de Valdez and Mozzi 2015). Classifi-
is encoded within the genomes of many of these bacteria, pro- cation of the extracellular GTs has been briefly discussed in the
duction of polysaccharides and their functional properties need previous section and will not be further presented in this review.
to be evaluated for successful industrial applications. The biosynthesis of polysaccharides via the Wzy-dependent
The genomes of many LAB species contain a second clus- pathway is a complex intracellular process that was first stud-
ter for the production of polysaccharides, which are CW-PS an- ied for its involvement in the synthesis of the LPS O-antigen

chored to PG that frequently contain rhamnose in the repeat- polysaccharide in Gram-negative bacteria (reviewed in Islam
ing unit forming the polysaccharide (Hols et al. 2005; Thevenard and Lam 2014), and later for production of CPS and EPS in
et al. 2014; Mistou, Sutcliffe and van Sorge 2016). These CW-PS both Gram-negative and Gram-positive bacteria (Whitfield 2006;
are arguably involved in antibiotic stress response in S. ther- Tytgat and Lebeer 2014; Schmid, Sieber and Rehm 2015). The
mophilus (Thevenard et al. 2014), while in L. lactis, where CW- full biosynthetic process can be divided into two distinct steps
PS are known as the pellicle, a role as receptors for phages (Fig. 2): (i) generation of activated sugar precursors (sugar nu-
and a protective effect against host phagocytosis in murine cleotides) from central carbon metabolism in the cytoplasm
macrophages have been established (Chapot-Chartier et al. 2010; (Fig. 4) and (ii) a committed cell membrane-associated assem-
Mahony et al. 2013). CW-PS are encoded by rmlD-associated gene bly and polymerization of the polysaccharide (Fig. 5).
clusters, which vary between 14 and 28 kB in size contain- (i) Generation of activated sugar precursors: pathways for
ing between 12 and 27 genes with putative functions such as the generation of sugar nucleotides have been recurrently de-
GT, polysaccharide biosynthesis proteins, rhamnose biosynthe- scribed in LAB (Welman and Maddox 2003; Neves et al. 2005;
sis proteins (RmlABCD) and transport molecules (Mahony et al. De Vuyst and De Vin 2007). The sugar nucleotides are synthe-
2013; Mistou, Sutcliffe and van Sorge 2016). sized in multistep pathways from glycolytic intermediates, gen-
Production of extracellular HoPS in LAB has recently been ex- erally glucose-6-phosphate (Glc-6-P) or fructose-6-phosphate
tensively reviewed (Torino, Font de Valdez and Mozzi 2015; Zan- (Fru-6-P) (Fig. 4), or in certain cases from intermediates of
nini et al. 2016). In brief, these polysaccharides are synthesized sugar specific catabolic pathways, such as the Leloir path-
from an existing sugar molecule, which acts as the donor of way intermediate α-glucose-1-phosphate (α-Glc-1-P) during the
the corresponding monosaccharide, by action of an extracellular catabolism of galactose in L. lactis (Neves et al. 2006). The po-
enzyme belonging to the glycosyl hydrolase family. α-Glucans tential to produce different sugar nucleotides is intrinsically
and β-fructans are formed by glucansucrases (GH family 70) and determined by the gene content of each LAB, which ulti-
fructansucrases (GH family 68), respectively. Depending on the mately dictates the type of monomers present in the exocellular
reaction catalyzed and the specificity, glucansucrases are classi- polysaccharides. Exocellular polysaccharides produced by LAB
fied into four groups: dextransucrase, mutansucrase, alternan- via the Wzy-dependent pathway consist of repeating units usu-
sucrase and reuteransucrases, which catalyze α-(1,6), α-(1,3), α- ally composed of two or more (usually 3–8) types of monosaccha-
(1,3 and 1,6) and α-(1,4 and 1,6) glycosidic linkages, respectively. rides (Welman and Maddox 2003; Neves et al. 2005; De Vuyst and
Types of HoPS produced by LAB and their predominant linkages De Vin 2007; Ruas-Madiedo, Salazar and de los Reyes-Gavilán
were recently reviewed by Zannini et al. (2016). 2009). The most represented sugar moieties are galactose (Gal),
In the following sections, insights into the biosynthesis and glucose (Glc) and rhamnose (Rha), and to a lesser extent the N-
regulation of polysaccharide production via the Wzy-dependent acetylated sugar derivatives N-acetylglucosamine (GlcNAc) and
pathway will be presented. N-acetylgalactosamine (GalNAc) (Table 1). Other monosaccha-
rides and acetylated derivatives that have occasionally been
found in the repeating units of LAB HePS are fucose (Fuc, 2 struc-
BIOSYNTHESIS OF POLYSACCHARIDES: tures), ribose (Rib, 1 structure), glucuronic acid (GlcA, 1 struc-
FROM GENES TO STRUCTURES ture), mannose (Man, 6 structures) and N-acetylmannosamine
(ManNAc, 2 structures) (Robijn et al. 1996; Low et al. 1998; Faber
Biosynthetic pathways
et al. 2002; Li et al. 2014; Patten et al. 2014; Zhou et al. 2016). The
Hitherto, only two pathways for the biosynthesis of the exocellu- pathways to generate the respective sugar nucleotides from the
lar polysaccharides by LAB have been described in the literature: milk carbohydrate lactose and from glucose are shown in Fig. 4.
the Wzy-dependent pathway and the extracellular GT pathway The genes encoding these pathways can either be found in the
for the synthesis of glucans and fructans (Ryan et al. 2015; Torino, polysaccharide biosynthetic clusters (see previous section) or at
Font de Valdez and Mozzi 2015). The latter is a relatively sim- other locations in the genome.
ple biochemical route that involves a specific GT (glucansucrase Small quantities of uronic acids, fructose, arabinose and xy-
or fructansucrase), and an extracellular sugar donor, which is lose have also been reported in the monosaccharide composi-
sucrose for the synthesis of glucans, but can also be other tion of polysaccharides determined by complete hydrolysis of
fructose-containing oligosaccharides (e.g. raffinose) for the syn- purified HePS, but the presence of these monosaccharides has
thesis of fructans (van Hijum et al. 2006; Galle and Arendt 2014). not been confirmed in the elucidated structures of the repeating
Extracellular biosynthesis of HoPS is essentially uncoupled units. Thus, these sugars are either contaminants from other
from central metabolic processes and thereby not energetically CW-PS, cell lysis or from the growth medium as also indicated
demanding for the cell or prone to complex regulatory mecha- previously by others (De Vuyst and De Vin 2007; Ruas-Madiedo,
nisms. Indeed, cellular energy expenditure is restricted to the Salazar and de los Reyes-Gavilán 2009).
synthesis and export of the GT. In fact, the energy released by (ii) Cell membrane-associated assembly and polymeriza-
cleavage of the glycosidic bond in the substrate is used for the tion: the committed steps of the Wzy-dependent biosyn-
synthesis of new glycosidic bonds in the growing HoPS. The thetic pathway have been mainly investigated in pathogenic
structural features of the HoPS (e.g. glycosidic bond, branching, streptococci, in particular for the synthesis of capsules in

Figure 5. Proposed model for biosynthesis of polysaccharides via the Wzy-dependent pathway. This model is inspired in the findings from S. pneumoniae and adapted
from the model proposed by Nourikyan et al. (2015). The genetic locus shows the genes involved in the synthesis and export of exocellular polysaccharidess in LAB.
As an example, assembly and polymerization of the polysaccharide produced by L. lactis ssp. cremoris NIZO B40 is shown. In the eps gene clusters (generic and B40),
the genes coding for the polysaccharide assembly machinery, glycosyltransferases, the EpsBCD phosphoregulatory system and synthesis of NDP-sugars are shown in
green, orange, yellow and pink, respectively. The same color scheme is used for representing the proteins in the Wzy-dependent pathway. The reactions catalyzed by
each GT gene product are indicated in the upper-left corner gray box. These reactions occur in the cytoplasm CM, cytoplasmic membrane.
S. pneumoniae (Bentley et al. 2006; Henriques et al. 2011; Yother in LAB (Fig. 3). Some of the more recent and interesting find-
2011; Schaffner et al. 2014; Nourikyan et al. 2015; Grangeasse ings concerning the functions of these genes arise from studies
2016), but are poorly characterized in LAB, with only a few of capsule biosynthesis in the human pathogen S. pneumoniae
functional studies described so far (Minic et al. 2007; Nierop (Bentley et al. 2006; Henriques et al. 2011; Yother 2011; Eberhardt
Groot and Kleerebezem 2007; Cefalo, Broadbent and Welker et al. 2012; Schaffner et al. 2014; Nourikyan et al. 2015; Grange-
2011a; Dertli et al. 2013; Suzuki, Kobayashi and Kimoto-Nira asse 2016). Considering the relatively close genetic proximity be-
2013). Considering the importance and value of LAB exocellular tween the pathogenic streptococci and LAB, the LAB Wzy protein
polysaccharides, this is fairly surprising. Indeed, except for the functions will herein be surmised as similar to those reported for
extracellularly synthesized glucans and fructans, CPS and EPS the pneumococcal proteins. The current model for polysaccha-
are presumably the products of this pathway. Furthermore, CW- ride synthesis by the Wzy-dependent pathway starts with the
PS, such as the pellicle in L. lactis (Chapot-Chartier et al. 2010), cytoplasmic en bloc synthesis of single-repeat units by sequen-
the rhamnose-glucose polysaccharides in S. thermophilus (Hols tial addition of the activated sugar precursors (Fig. 5). The first
et al. 2005) and the recently described lactobacilli CW-PS (Vino- committed step consists of the activation of undecaprenylphos-
gradov et al. 2013, 2015, 2016) might also be synthesized via the phate (Und-P), the lipid carrier, by transfer of the first residue
Wzy-dependent pathway. However, the absence of several typ- from an activated sugar precursor via the priming GT. The sec-
ical pathway components in some CW-PS biosynthetic clusters ond step comprises the assembly of the repeating unit by se-
and the presence in others of genes with homology to ABC trans- quential addition of sugar nucleotides in reactions catalyzed by
porters (vide supra) raises the question to which pathway is used: soluble and/or membrane-bound GTs. The repeating units are
the Wzy-dependent or the ABC transporter-dependent pathway subsequently transported or flipped across the cytoplasmic
(reviewed in Mistou, Sutcliffe and van Sorge 2016). Thus, for the membrane via a flippase (Wzx, CpsJ). Polymerization is cat-
synthesis of CW-PS, more work needs to be performed in order alyzed by the Wzy polymerase (CpsH), which adds single repeat-
to firmly ascertain the biosynthetic routes. ing units via generation of new glycosidic bonds to the reduc-
Homologs of the key functions characterizing the Wzy- ing terminus of a growing polymer building up the exocellular
dependent pathway are ubiquitously present in the modular polysaccharide structure. In Gram-negative bacteria, polysac-
gene clusters for the synthesis of exocellular polysaccharides charide co-polymerase (PCP) proteins are postulated to be
Zeidan et al. S177
responsible for chain length control of the polymer (Islam to the enterobacterial WecA, respectively. These proteins belong
and Lam 2014). These proteins are, however, not present in to the bacterial sugar transferase family (Bac transf, PF02397). In
Gram-positive bacteria. The modulation proteins CpsC (Wzd or S. pneumoniae, the WbaP homolog CpsE (WchA) comprises 455
EpsC) and CpsD (Wze or EpsD), which constitute an active bac- amino acids and catalyzes the addition of Glc-1-P from UDP-
terial tyrosine (BY)-kinase, presumably act as surrogates (Yother glucose to Und-P (Kolkman, van der Zeijst and Nuijten 1997; Car-
2011; Grangeasse, Nessler and Mijakovic 2012; Nourikyan et al. tee et al. 2005). Bentley et al. (2006) observed a perfect correlation
2015; Grangeasse 2016). CpsC is a membrane-bound polypeptide between the presence/absence of cpsE and the presence/absence
that harbors two transmembrane spanning helices and a cyto- of glucose in the repeating unit of elucidated structures. These
plasmic C-terminal domain required for kinase activation. CpsD, authors proposed that CpsE performs the same function in all

a cytoplasmic protein harboring the kinase activity, possesses 65 serotypes where it is present. Streptococcus thermophilus NCFB
the Walker A and B ATP/GTP-binding motifs and a C-terminal 2393 (aka LMG18311) EpsE, a homolog of the pneumococcal CpsE,
tyrosine cluster motif. CpsC triggers CpsD kinase activity lead- was functionally characterized and shown to transfer Glc-1-P to
ing to autophosphorylation of the tyrosine cluster. Moreover, Und-P (Almiron-Roig et al. 2000) (Fig. 6). Based on NCBI BlastP
CpsC likely acts as a scaffold, keeping together the assembly searches, proteins homologous to EpsE are present in other
machinery, i.e. the priming GT (e.g. CpsE), the Wzx flippase, the S. thermophilus strains, such as Sfi39 and EU20, for which the
Wzy polymerase and CpsA. Although the mechanism by which polysaccharide structures have been elucidated (Germond et al.
the BY-kinases control exocellular polysaccharides synthesis re- 2001; Marshall et al. 2001). As for S. pneumoniae, a positive correla-
mains elusive, the current model proposed that cycling between tion between the presence of EpsE and the presence of glucose in
phosphorylated and non-phosphorylated forms of BY-kinases the repeating unit is observed for the three S. thermophilus strains
is required for proper synthesis and export of the polymer. (Fig. 6). Whether this phenomenon is more general requires fur-
Dephosphorylation is catalyzed by CpsB (Wzh or EpsB), which ther characterization.
is a metal-dependent phosphotyrosine-protein phosphatase of A different priming GT, the EpsE homolog in L. lactis NIZO
the PHP family. After completing polymerization, linkage of the B40 (EpsD), also catalyzes the transfer of Glc-1-P from UDP-Glc
polysaccharide to PG might or might not occur, but this step re- to UndP (van Kranenburg et al. 1999b). This phosphoglycosyl-
mains largely elusive. Recently, attachment of the CPS to the cell transferase is much smaller than the streptococcal EpsE, com-
wall in S. pneumoniae was shown to depend on the activity of prising only 228 amino acids. A homologous protein is present
members of the LytR-CpsA-Psr (LCP) proteins (Eberhardt et al. in NIZO B891, an L. lactis strain that produces a polysaccharide
2012), including Cps2A (EpsA, Wzg). LCP proteins also mediate containing Glc and Gal (van Kranenburg et al. 1999c). Further-
the attachment to PG of CPS in Staphylococcus aureus (Chan et al. more, homologs sharing over 90% identity and 100% coverage
2014) as well as the attachment of TAs in Bacillus subtilis (Kawai with EpsE are present in several lactococcal genomes deposited
et al. 2011). Additional modifications to the polysaccharide, in- in NCBI (Fig. 6). This smaller glucosyl-1-P transferase shares
cluding addition of other noncarbohydrate constituents such as high sequence homology with the C-terminus of the larger EpsE,
acetyl (O-acetylation), glycerol, pyruvoyl and phosphate groups, which is characterized by the presence of the bac transf domain
can also occur and the respective functions are presumably en- (PF02397) (Fig. 6).
coded in the dedicated Wzy-dependent pathway gene cluster. The S. thermophilus Sfi6 EpsE protein was shown to transfer
Indeed, in S. pneumoniae, O-acetylation of serotype 9V has been Gal-1-P from UDP-Gal to Und-P (Stingele et al. 1999). A homolog
tentatively attributed to an O-acetyl transferase encoded by the is found in strain MR-1C, for which prediction of galactosyl-1-
wcjE gene (Bentley et al. 2006), while the epsH of S. thermophilus is phosphotransferase activity can also be inferred from the eluci-
a putative O-acetyltransferase (Stingele et al. 1999). In this sec- dated structure of the repeating unit (Low et al. 1998). Further-
tion, the pneumococcal gene/protein nomenclature was used to more, BlastP searches of S. thermophilus genomes deposited in
describe the Wzy-dependent pathway proteins, but when pos- NCBI reveal additional homologs, as for example in strain LY03,
sible we will use the adopted nomenclature for LAB (epsABCDE, which contains galactose in the repeating unit of its polysac-
wzy, wzx as in Fig. 3). charide (Fig. 6). Lactococcus lactis NIZO B35 (van Kranenburg
In summary, the dedicated Wzy pathway gene clusters com- et al. 1999c), a polygalactan-producing strain harbors a homolog
prise genes involved in the synthesis and export of exocellu- of Sfi6 EpsE. The S. pneumoniae functional cognate is most likely
lar polysaccharides that can be categorized into four groups (or the WcjG galactosyl-1-P transferase described by Bentley et al.
modules) according to their functions (Figs 3–5): polysaccharide (2006). It should be noted, however, that S. thermophilus Sfi6 EpsE
assembly machinery (the priming GT, Wzx flippase, Wzy poly- and L. lactis B40 share considerable sequence homology (about
merase, EpsA), the phosphoregulatory system that controls the 50% identity) despite the different sugar specificity, which in our
polysaccharide assembly machinery (EpsB, EpsC, EpsD), the GT opinion limits firm functional assignment of putative hexose-1-
and sugar nucleotide biosynthetic pathways. Genes encoding P transferases based solely on sequence homology.
enzymes involved in monosaccharide decoration can also be Streptococcus pneumoniae possesses two additional phospho-
present in the cluster. glycosyltransferases, an alternative galactosyl-1-P transferase
The priming GTs (also known as initiating GTs) are WcjH and a hexosamine-1-P transferase WciI, postulated to
membrane-bound polyprenyl-P sugar-1-P transferases involved transfer GalNAc-1-P to the Und-P, as in the repeating unit of CPS
in the transfer of a phosphorylated monosaccharide from a of strain TIGR4 (Bentley et al. 2006). A potential homolog of the
sugar nucleotide to Und-P, thereby catalyzing the formation of latter is present in the genome sequence of S. thermophilus strain
an energy-rich phosphate bond. The membrane-bound Und-P- TH982 (Treu et al. 2014); however, no information regarding ex-
P-sugar is the substrate for the sequential action of GTs that ocellular polysaccharides synthesis is available for this strain.
build up the repeating unit in the cytoplasmic side of the mem- As for WcjH, no homologs were found in LAB by BlastP of NCBI
brane (Fig. 5). Priming GTs can either catalyze the addition of a deposited sequences.
hexose-1-phosphate (Glc-1-P or Gal-1-P) or of a hexosamine-1- A systematic investigation on the presence of the different
phosphate (GlcNAc-1-P or GalNAc-1-P) showing high degree of priming GTs in LAB has not been performed so far. Most of the
homology to the Salmonella enterica serovar typhimurium WbaP or information available on functional characterization has been

Figure 6. Priming glycosyltransferases in LAB and their characteristics. All LAB priming GTs functionally characterized so far fall into the hexose-1-phosphate trans-
ferase category. The WciI present in S. pneumoniae serotype 4 (TIGR4) is shown as an example of a hexosamine-1-phosphate transferase, but its activity as an N-
acetylgalactosamine-1-phosphate transferase has not been demonstrated experimentally. WciI can presumably catalyze the transfer of GalNAc and/or GlcNAc (Aa-
nensen et al. 2007). Abbreviations as in Fig. 4. FucNAc, N-acetylfucosamine. Rods represent the four protein types. The shaded area indicates the bacterial transfer
domain of family PF02397.
obtained from a few selected S. thermophilus or L. lactis strains knowledge functional characterization was only performed for
in the 1990s (reviewed in Jolly and Stingele 2001; Broadbent the GTs encoded in the eps gene clusters of S. thermophilus Sfi6
et al. 2003). In lactobacilli and other LAB, the information is even and L. lactis NIZO B40 (Stingele et al. 1999; van Kranenburg et al.
scarcer. A recent study postulates that EpsE of Lb. johnsonii FI9785 1999b). The Sfi6 GT gene region comprises the epsEFGHI genes,
adds Gal-1-P to Und-P, but firm confirmation is still required with epsE encoding the galactosyl-1-P transferase (priming GT).
(Dertli et al. 2013), while Lebeer et al. (2009) showed that the EpsE EpsF encodes a galactosyltransferase that adds the branching α-
protein of Lb. rhamnosus GG is a galactosyl-1-P transferase. In- 1,6-galactose, EpsG is α-1,3-N-acetylgalactosaminyltransferase
activation of epsE genes in LAB renders strains that no longer that carries out the second reaction in the build-up of the re-
secrete polysaccharides to the extracellular matrix (van Kranen- peating unit, and by exclusion EpsI was postulated to be a β-
burg et al. 1997; Germond et al. 2001; Dabour and LaPointe 2005; 1,3-glucosyltransferase. Thus, the order of biosynthesis of the S.
Minic et al. 2007; Dertli et al. 2013; Rosini et al. 2015). These ge- thermophilus Sfi6 polysaccharide repeating unit is Gal, GalNAc,
netic studies provide strong evidence that the priming GT is es- Glc and the side chain Gal. A similar functional analysis was
sential for exocellular polysaccharide production. Considering performed for the GTs encompassed in the eps gene cluster of L.
the fast build-up of LAB genomics data, it is timely to gain fur- lactis NIZO B40: EpsD (priming GT), EpsE, EpsF and EpsG link Glc-
ther insights into the characteristics of genes encoding priming 1-P to Und-P (glucosyl-1P transferase), glucose to lipid-linked
GTs. The different types of priming GTs identified in LAB are pre- glucose (glucosyltransferase) and galactose to lipid-linked Glc-
sented in Fig. 6. Glc (galactosyltransferase), respectively (van Kranenburg et al.
GTs catalyze the formation of a glycosidic bond between a 1999b). As for the priming GTs, more studies are required to
sugar moiety from an activated sugar precursor (donor) and grasp the diversity of LAB GTs.
a specific substrate molecule, which in the case of exocel- Except for the priming GTs (EpsE), functional characteriza-
lular polysaccharide synthesis via the Wzy-dependent path- tion of the Wzy-dependent pathway assembly machinery pro-
way, is the membrane-bound Und-P-P-sugar. GTs are a very di- teins, EpsA, Wzx flippase and Wzy polymerase has not been
verse group of enzymes that can recognize a range of donor pursued. Recent studies in pathogenic Gram-positive bacteria
molecules and acceptors, and are often described as promis- showed that EpsA is a phosphotransferase belonging to the
cuous. Currently, 101 GT families are described online in the LCP family of proteins that catalyzes the attachment of CPS
Carbohydrate-Active enZymes (CAZy) database (Lombard et al. to N-acetylmuramic acid residues of PG by the formation of a
2014), but promiscuity toward different substrates shown by phosphodiester bond (Eberhardt et al. 2012; Chan et al. 2014).
some GTs makes it difficult to predict activity based merely on The exact role of EpsA in LAB remains to be elucidated, as
sequence analysis. The diversity of GT-encoding genes in LAB well as its function in LAB strains that presumably produce
is enormous (see the previous section) and to the best of our mainly EPS.
Zeidan et al. S179
The presence of Wzy flippase encoding genes in the genomes Medium composition and growth conditions were shown to
of LAB is based primarily on sequence homology to genes encod- strongly influence the levels of exocellular polysaccharide pro-
ing the few Wzy proteins that have been functionally character- duction in LAB as well as its sugar composition and molecular
ized in other organisms (Islam and Lam 2014). The Wzx flippases mass, alluding to an obvious role of environmental factors in di-
are membrane-bound proteins often characterized by the pres- rectly or indirectly modulating polysaccharide biosynthesis. Ex-
ence of 12 transmembrane segments (TMS), which are classified ocellular polysaccharide production levels were found to vary
as part of the polysaccharide transporter family. Wzx flippases with the nature and/or concentration of the carbon source in
recognize the UndP-P-repeating unit and flip it across the cyto- the medium in S. thermophilus (Petit et al. 1991; De Vuyst et al.
plasmic membrane. Whether flippases display strict substrate 1998; Degeest and De Vuyst 1999, 2000; Li et al. 2016), Lb. bulgar-

specificity for a unique repeating unit is still a matter of debate, icus (Grobben et al. 1995, 1996, 1998), Lb. helveticus (Torino et al.
but the Und-P-P-linked sugar seems to play a key role serving as 2001; Torino, Mozzi and Font de Valdez 2005), Lb. casei (Cerning
an initial point of discrimination by the flippase (Islam and Lam et al. 1994; Mozzi et al. 2001), Lb. rhamnosus (Gamar, Blondeau and
2014). Simonet 1997) and L. lactis (Looijesteijn et al. 1999, 2000; Welman
The Wzy polymerases are membrane-bound proteins puta- and Maddox 2003). In Lb. casei, the sugar distribution in HePS
tively harboring 10 to 14 TMS. The role of the Wzy polymerase is also varied depending on the carbon source (Cerning et al. 1992,
to add a single repeating unit via generation of a new glycosidic 1994), an observation which has been described in S. thermophilus
bond to the reducing terminus of a polysaccharide composed when the lactose feeding rate in fed-batch cultures was reduced
of multiple copies of the repeating unit. According to Islam and (Petit et al. 1991). The proportion of high molecular weight to
Lam (2014), Wzy polymerases should be viewed as GT enzymes, low molecular weight polysaccharides in Lb. bulgaricus NCFB
but their substrate specificity remains largely uncharacterized. 2772 was shown to be dependent on the carbohydrate source
The EpsBCD phosphoregulatory system involved in the control in the growth medium (Grobben et al. 1997). In S. thermophilus
of synthesis and export of polysaccharide in S. pneumoniae will LY03, this proportion varied with the carbon/nitrogen ratio in
be discussed in more detail below. the medium (Degeest and De Vuyst 1999), which was shown to
have a strong impact on exocellular polysaccharide production
in other LAB strains (Cerning 1990; De Vuyst et al. 1998; De Vuyst
Regulation of polysaccharide synthesis
and Degeest 1999; Harutoshi 2013). Other factors reported to
Bacteria possess various regulatory mechanisms for controlling modulate polysaccharide production include pH, temperature,
their metabolic activities, which enable them to interact with oxygen tension, amino acids, phosphate and minerals (Mozzi
the surrounding environment and secure their niche among et al. 1995; Grobben et al. 1998; De Vuyst and Degeest 1999; Looi-
other forms of life. Understanding the mechanisms regulat- jesteijn et al. 2000; De Vuyst et al. 2001; Mozzi, Savoy de Giori and
ing exocellular polysaccharide production in pathogenic bacte- Font de Valdez 2003; Aslim et al. 2005; Harutoshi 2013) as well as
ria has long attracted the attention of scientists in the quest vitamins (Grobben et al. 1998). In S. thermophilus, the overall ef-
for identifying novel antibiotic targets. Likewise, comprehen- fect of optimizing medium composition and culture conditions
sive understanding of the regulatory mechanisms underlying was a 4.2-fold increase in exocellular polysaccharide titers and
polysaccharide biosynthesis in LAB allows industrial microbi- a 9-fold increase in the molecular mass of the polymer (Li et al.
ologists to maximally exploit them in a myriad of commercial 2016).
applications. Despite the presence of numerous studies demonstrating
Exocellular polysaccharide production via the Wzy- the effect of environmental factors in modulating exocellu-
dependent pathway is an energy-intensive process involving lar polysaccharide production in LAB, little is known about
various regulatory enzymes, GTs as well as transport and the underlying regulatory mechanisms. It is to be noted that,
polymerizing enzymes, in addition to housekeeping enzymes even within the same species, strains differ significantly in
that are involved in general cellular processes and not exclusive their response to changes in the above-mentioned factors. This
to polysaccharide biosynthesis (vide supra). It is, therefore, emphasizes again the lack of generalized patterns that can help
expected that bacteria employ complex regulatory mechanisms elucidating the regulatory mechanisms for exocellular polysac-
at different cellular levels for tightly controlling the production charide production in LAB. The first four genes at the 5 end of
of those polymers. It is also expected that competition for the eps gene cluster in different LAB are highly conserved (Fig. 3)
intracellular resources essential for growth, e.g. lipid carrier, and their products have been traditionally proposed to play a
sugar nucleotides and ATP, would result in maximum exo- modulatory role in exocellular polysaccharide production, based
cellular polysaccharide production occurring at the expense primarily on sequence similarity with corresponding proteins in
of the maximum specific growth rate or biomass yield of the other bacteria (van Kranenburg et al. 1999c; De Vuyst et al. 2001;
organism. However, no clear patterns or systematic correlation Jolly and Stingele 2001; Broadbent et al. 2003; Péant et al. 2005;
with growth kinetics can be drawn based on the dynamics Wu et al. 2014). In most LAB, the product of the first gene in the
of polysaccharide production observed in different bacteria. cluster, EpsA, possesses an LCP domain, which is often present
For example, exocellular polysaccharide production in some in multiple proteins in Gram-positive bacteria (Hübscher et al.
strains of S. thermophilus and Lb. bulgaricus appears to reach 2008). Transcriptional regulation of the eps operon in LAB has
maximum levels under conditions favoring optimal growth, long been attributed to EpsA (Jolly and Stingele 2001; Broadbent
whereas production of polysaccharide by mesophilic LAB was et al. 2003). In B. subtilis, the homologous protein, LytR, was sug-
reported to have its highest levels under suboptimal growth gested to play a role in the transcriptional regulation of the ly-
conditions (Cerning 1990, 1995; De Vuyst et al. 2001; Broadbent tRABC operon encoding cell wall-modifying enzymes (Lazarevic
et al. 2003; Welman and Maddox 2003; Li et al. 2016). Taken et al. 1992), a reason behind propagating the transcriptional reg-
together, the regulatory mechanisms of the energy-demanding ulatory function to EpsA in LAB by virtue of sequence homol-
process of polysaccharide production may vary considerably ogy. The EpsA homolog in L. lactis, EpsR, was also proposed to
and are likely to be dependent on the physiological significance have a regulatory function in exocellular polysaccharide produc-
of the polymer in a given organism. tion based on sequence similarity with the regulatory proteins
Xre, PrtR and RdgA, which all contain a DNA-binding domain of any regulatory mechanism controlling the metabolic fluxes to
(van Kranenburg et al. 1997). Moreover, there is another gene those precursors on polysaccharide production. This might be
in L. lactis downstream of the eps gene cluster, orfY, the prod- evident in S. thermophilus, where a correlation between exocel-
uct of which is homologous to B. subtilis LytR (van Kranenburg lular polysaccharide production and enzymes involved in sugar
et al. 1997). However, there exists limited biochemical or genetic nucleotide biosynthesis (Fig. 4) could be clearly observed (Es-
evidence supporting the regulatory role of EpsA homologs in calante et al. 1998; Degeest and de Vuyst 2000; Levander, Svens-
LAB or elucidating the nature of the proposed regulation. Only son and Radstrom 2002; Svensson et al. 2005). In S. thermophilus
a recent study in Lb. johnsonii FI9785 showed that EpsA plays LY03, the activity levels of α-phosphoglucomutase (PgmA) as
a crucial role in exocellular polysaccharide biosynthesis since well as the Leloir pathway enzymes UDP-galactose 4-epimerase

deletion of the epsA homolog was shown to completely abol- (GalE) and glucose-1-phosphate uridylyltransferase (GalU) were
ish exocellular polysaccharide production (Dertli et al. 2016). Un- highly correlated with the amount of polysaccharide produced,
like B. subtilis LytR, which acts as a transcriptional attenuator whereas no correlation with the activity of the key glycolytic
(Lazarevic et al. 1992), data from Lb. johnsonii FI9785 suggested enzyme fructosebisphosphatase (Fbp) was observed (Degeest
that EpsA is a positive regulator of the eps operon (Dertli et al. and de Vuyst 2000). A galactose-fermenting mutant of the same
2016). The role of EpsA as a positive regulator of eps operon has strain with increased activities of the Leloir pathway enzymes
been previously postulated in other Gram-positive bacteria, e.g. also showed a higher polysaccharide yield as compared to the
S. agalactiae (Cieslewicz et al. 2001) and S. pneumoniae (Morona parent strain (Levander, Svensson and Radstrom 2002). Overex-
et al. 2000; Broadbent et al. 2003). In these organisms, the epsA ho- pression of galU in combination with pgmA in S. thermophilus
molog (cpsA) was not found essential for polysaccharide repeat- LY03 or galU alone in its galactose-fermenting mutant resulted
ing unit biosynthesis, though its deletion resulted in reduced in the production of significantly higher levels of polysaccha-
capsule formation compared to the wild type. In S. agalactiae, ride, which correlated with higher levels of GalU and PgmA
cpsA deletion was associated with significant reductions in the activities (Levander, Svensson and Radstrom 2002). The com-
transcription of eps genes (Cieslewicz et al. 2001). In addition, bined effect of pgmA and galU overexpression together with the
the purified CpsA from S. agalactiae (Hanson et al. 2012) and S. enhanced Leloir enzyme activities in the galactose-fermenting
iniae (Hanson, Lowe and Neely 2011) was shown to bind specifi- strain was found to further boost exocellular polysaccharide
cally to DNA containing the putative promoter region of the cps yields (Svensson et al. 2005). In that case, UDP-glucose levels did
operon of the respective strain in vitro using electrophoretic mo- not differ from those in the parent strain suggesting that this
bility shift assays. The specific DNA binding was attributed to the sugar nucleotide is channeled in favor of exocellular polysac-
small intracellular domain of the protein, whereas the large ex- charide biosynthesis in the engineered strain rather than ac-
tracellular domain, including the LCP domain, was not required cumulated (Svensson et al. 2005). The combined data illustrates
(Hanson, Lowe and Neely 2011; Hanson et al. 2012). Besides the an important role of Leloir pathway enzymes in S. thermophilus,
proposed regulatory function, data in S. agalactiae suggested the which generally cannot ferment galactose, in the generation
implication of CpsA in modulating cell wall, in cell division and of sugar nucleotides for exocellular polysaccharide biosynthe-
in mediating CPS attachment to the cell surface (Hanson et al. sis and raises the possibility of potential control mechanisms
2012; Rowe et al. 2015; Toniolo et al. 2015). Indeed, the primary outside the eps gene cluster. In Lb. bulgaricus and Lb. casei, cor-
role recently proposed for members of the LCP protein family, relations could also be established between the levels of vari-
including EpsA, in Gram-positive bacteria points towards an en- ous enzymes involved in sugar nucleotide biosynthesis and the
zymatic function in the attachment of CW-PS to PG, which is amount of polysaccharide produced (Grobben et al. 1996; Mozzi,
supported by crystallographic (Kawai et al. 2011; Eberhardt et al. Savoy de Giori and Font de Valdez 2003).
2012) and genetic evidence (Dengler et al. 2012; Chan et al. 2013, On the other hand, studies in L. lactis did not always con-
2014; Wang et al. 2015; Baumgart et al. 2016). Furthermore, it was form to the same pattern as above. In L. lactis ssp. cremoris NIZO
assumed that many of the reported changes in polysaccharide B40, changes in the levels of exocellular polysaccharide produc-
levels in LCP mutant strains of various species are indirect con- tion with the sugar source were indeed attributed to differences
sequences of stress response induced by cell wall defects rather in the capacity to produce sugar nucleotides, whilst the tran-
than a disrupted regulatory function of LCP proteins (Kawai scriptional levels of eps genes remained constant (Looijesteijn
et al. 2011; Dengler et al. 2012). As the LCP family comprises di- et al. 1999). However, the activities of different enzymes required
verse subgroups of proteins, functional differences cannot be for sugar nucleotide biosynthesis did not change with the levels
excluded (Hübscher et al. 2008), which calls for a more cautious of sugar nucleotides. In fructose-grown cells, Fbp activity was
interpretation of protein homology searches and emphasizes the limiting factor for exocellular polysaccharide production,
the need for detailed functional studies in LAB. New insights on as the enzyme is needed for the biosynthesis of sugar nu-
transcriptional-level regulation of eps gene expression may also cleotides when fructose is used as the sugar source (Looijesteijn
be gained from the recent work in S. pneumoniae, which shows et al. 1999). In a later study, overexpression of galU, and the sub-
that sequence polymorphisms in the cps promoter play a role in sequent increase in UDP-glucose and UDP-glucose levels, did
fine-tuning the level of encapsulation (Wen et al. 2016). not result in any apparent increase in polysaccharide produc-
As exocellular polysaccharide biosynthesis requires a con- tion (Boels et al. 2001). It was suggested that the control of exo-
stant supply of intracellular metabolites essential for cellular cellular polysaccharides biosynthesis in L. lactis by the enzymes
growth and maintenance, the modulation of polysaccharide lev- involved in sugar nucleotide generation might be dependent on
els by environmental factors may also be ascribed to transcrip- the sugar composition of the polymer (Boels et al. 2001).
tional regulators outside the eps gene cluster which orchestrate In addition to the regulation of the overall amounts of ex-
growth and carbohydrate metabolism on a global level, such as ocellular polysaccharide produced, there exist apparent mech-
CcpA or other sugar-specific regulators in LAB (Ravcheev et al. anisms for regulating its polymerization and chain length.
2013). This was postulated in several LAB strains, where changes In Gram-negative pathogens, production of preferred chain
in exocellular polysaccharide levels could be correlated with the lengths of LPS O-antigen, mostly synthesized through the Wzy-
availability of sugar nucleotides, indicating the potential impact dependent pathway, was deemed crucial for their survival in
Zeidan et al. S181
different environments (Kintz and Goldberg 2011; Osawa et al. can be gained from a recent study in B. subtilis. It was suggested
2013; Chang et al. 2015) and is believed to be controlled by the PCP that the control of EpsD autophosphorylation is mediated by the
Wzz (Morona et al. 2009; Islam and Lam 2014). In Gram-positive polysaccharides produced by the organism itself, since polysac-
bacteria, this regulation appears to be achieved through the con- charide inhibits the autophophorylation through its interaction
certed actions of EpsB, EpsC and EpsD homologs, which con- with the extracellular domain of EpsC homolog (Elsholz, Wacker
stitute a phosphorylation-dependent regulatory system (Yother and Losick 2014). The unphosphorylated EpsD was suggested as
2011; Grangeasse 2016). In that system, EpsC, a transmem- the active form of the protein that can activate different GTs
brane activation protein, is required for the autophosphoryla- through phosphorylation, making the exocellular polysaccha-
tion of the tyrosine cluster of the cytoplasmic protein EpsD (vide ride a signaling molecule controlling its own production through

supra), i.e. both proteins constitute a functional autophospho- a positive feedback loop (Elsholz, Wacker and Losick 2014).
rylating BY kinase, the activity of which is modulated via in- In summary, despite the considerable advances in the under-
teraction with the phosphotyrosine phosphatase EpsB (Morona standing of the functions involved in polysaccharide biosynthe-
et al. 2000, 2002; Bender, Cartee and Yother 2003; Soulat et al. sis via the Wzy-dependent pathway, a complete picture of the
2006; Nourikyan et al. 2015; Toniolo et al. 2015). Although most underlying mechanisms in LAB is still missing, which raises the
of the knowledge on these proteins comes from the work on need for more functional studies on the proteins involved in ex-
Gram-positive pathogens, similar roles were proposed for EpsB- ocellular polysaccharide biosynthesis in this important group of
D in LAB based on sequence homology and a small number of bacteria.
functional studies. In S. thermophilus, the requirement of EpsC
for the phosphorylation of EpsD was verified and both proteins
were found essential for exocellular polysaccharide synthesis, Amount and molecular weight of polysaccharides
whereas EpsB was not (Minic et al. 2007). Protein–protein interac-
produced by LAB
tions between EpsC and EpsD as well as EpsB and EpsD homologs
were demonstrated in S. thermophilus and L. lactis using the yeast As described in the previous section, both environmental and in-
two-hybrid system (Cefalo, Broadbent and Welker 2011a,b). The trinsic factors affect the yield and molecular weight of polysac-
phosphotyrosine phosphatase function of purified EpsB has also charides produced by LAB. Amount and molecular weight are
been verified in S. thermophilus (Cefalo, Broadbent and Welker properties that have often been correlated with the techno-
2013). functional properties of the polysaccharides in diverse appli-
Since BY kinases are known to be involved in different cations (Mende, Rohm and Jaros 2016). These macromolecular
regulatory processes (Bechet et al. 2009; Kobir et al. 2011; properties have been extensively reviewed by others, but a con-
Grangeasse, Nessler and Mijakovic 2012), the alternated phos- cise overview is presented henceforth.
phorylation and dephosphorylation of EpsD represents a poten- Polysaccharides synthesized via the Wzy-dependent path-
tial regulatory system for exocellular polysaccharide assembly. way are usually produced in amounts from 20 to 600 mg L−1 (re-
In addition, in S. pneumonaie, CpsD is proposed to play a dual viewed in Vaningelgem et al. 2004; Ruas-Madiedo, Salazar and
function in capsule assembly and cell division, where its phos- de los Reyes-Gavilán 2009; Leroy and De Vuyst 2016), with only
phorylation acts as a signaling system coordinating CPS synthe- a few strains reported to produce higher amounts, e.g. 1 g L−1
sis with chromosome segregation (Nourikyan et al. 2015). in S. thermophilus ASCC 1275 (Wu et al. 2014), or over 2.7 g L−1
Mutations in the components of the phosphoregulatory sys- in Lb. rhamnosus RW-9595M (Bergmaier, Champagne and Lacroix
tem are known to affect exocellular polysaccharide levels and/or 2003). The amount of sucrase-dependent HoPS produced by LAB
molecular weight (Yother 2011). However, the detailed mecha- is usually higher than 1 g L−1 (Ruas-Madiedo, Salazar and de los
nisms and the modulatory environmental stimuli are still not Reyes-Gavilán 2009; Torino, Font de Valdez and Mozzi 2015), with
fully understood and do not appear to follow the same pattern a few strains reaching values of about 10 g L−1 as in the case of
across different exocellular polysaccharide-producing bacteria. Lb. reuteri Lb121, which produces both α-glucan and β-fructan
For instance, the phosphorylated form of EpsD homolog in S. (Van Geel-Schutten et al. 1999). The higher titers observed for
pneumoniae was found to negatively regulate CPS production in the extracellularly synthesized HoPS reflect the lack of interde-
one strain (Morona et al. 2000) while stimulating its production in pendency with central metabolic processes. On the other hand,
another (Bender, Cartee and Yother 2003). In S. agalactiae, inacti- polysaccharide synthesis via the Wzy-dependent pathway is
vation of EpsC or EpsD homologs resulted in a marked decrease strongly intertwined with carbon and energy metabolism. Care
in capsule formation (Cieslewicz et al. 2001), whereas epsC dele- should be taken when using for comparative purposes the val-
tion in Lb. johnsonii apparently enhanced polysaccharide produc- ues reported in the literature for polysaccharide titers. Indeed,
tion (Dertli et al. 2013; Horn et al. 2013). Deletion of epsC or epsD differences in isolation procedures can impact the exocellular
homologs in S. pneumoniae resulted in the production of only polysaccharide concentration measured, thus leading to either
short-chain polymers (Bender, Cartee and Yother 2003), alluding underestimation or overestimation of the ‘true’ amounts (Ruas-
to their involvement in chain-length determination. Similarly, Madiedo and de los Reyes-Gavilan 2005, Mende, Rohm and Jaros
CpsC extracellular domain in S. agalactiae appeared necessary 2016).
for the production of high molecular weight polysaccharides (To- LAB produce both polymers of high and low molecular
niolo et al. 2015). In S. thermophilus, both EpsC and EpsD were weight. Polysaccharides synthesized via the Wzy-dependent
required for the phosphoglycosyltransferase (priming GT) func- pathway can range in molecular weight from 8 to over 5000
tion of EpsE through the phosphorylation of the Tyr200 residue kDa (Mozzi et al. 2006). A simultaneous occurrence of polysac-
of the latter (Minic et al. 2007). A positive correlation between charides of different sizes is frequent, i.e. LAB can produce si-
epsC expression levels and exocellular polysaccharide molecu- multaneously the same exocellular polysaccharide with differ-
lar weight in S. thermophilus could also be established (Li et al. ent molecular weights (Mozzi et al. 2006; Ruas-Madiedo, Salazar
2016). and de los Reyes-Gavilán 2009; Mende et al. 2013). It has been
New perspectives into the control of exocellular polysaccha- speculated that the low molecular weight polymer correlates
ride production through the EpsBCD phosphoregulatory system with CPS, whereas the high molecular weight is found free in
Table 2. Monosaccharide composition and non-monosaccharide constituents present in structures of repeating units forming EPS/CPS synthe-
sized by the Wzy pathway in LAB that have been reported after the reviews by de Vuyst and de Vin (2007), Ruas-Madiedo, Salazar and de los
Reyes-Gavilán (2009). Chemical composition represented as monosaccharide ratio is shown.
Monosaccharide ratio in the repeating unit produced by LAB
Strain RUa size Glc Gal Rha GlcNAc GalNAc Other Decoration Reference
S. thermophilus ST1 6 4 2 Säwén et al. (2010)

Lb. helveticus MB2-1 6 2 1 3 Man Li et al. (2014)

10 6 2 2 Man Li et al. (2015)
Lb. helveticus Rosyjski 5 2 2 1ManNAc Patten et al. (2014)
Lb. plantarum BC-25 10 3 1 6 Man Zhou et al. (2016)
Lb. plantarum C88 5 3 2 Ac Fontana et al. (2015)
Lb. plantarum MTCC9510 3 2 1 Man Ismail and Nampoothiri (2010)
Lb. rhamnosus KL37B 9 3 6 Górska-Fraczek
et al. (2011)
Lb. rhamnosus LOCK 0900 7 1 1 1Man/4Fuc Pyr Gorska et al. (2014)
5b 2 1 1 1ManNAc PO4
Lb. johnsonii 142 5 1 4 Górska et al. (2010)
Lb. johnsonii FI9785 6 6 Gly-P, Ac Dertli et al. (2013)
6 4 2 Ac
Lb. fermentum TDS030603 4 3 1 Gerwig et al. (2013)
Lb. crispatus L1 10 10 Man Donnarumma et al. (2014)
a
RU, repeating units with structure elucidated
b
This polysaccharide was present only in the cell mass fraction, thus it is either CPS or a CW-PS.
Glc, glucose; Gal, galactose; Rha, rhamnose; GlcNAc, N-acetyltglucosamine; GalNAc, N-acetylgalactosamine; Fuc, fucose; Rib, ribose; ManNAc, N-acetylmannosamine;
Ac, acetyl, PO4 , phosphate, Pyr, pyruvoyl, Gly-P, glycerol-phosphate.
the extracellular matrix (Mende, Rohm and Jaros 2016). Despite of 81 structures elucidated so far, 55 are unique (Table 1). Re-
the variation in molecular weight, most commonly LAB produce garding structure availability, the LAB genera are not equally rep-
one type of exocellular polysaccharides. Some studies report the resented: Lactobacillus 47 structures, Streptococcus 28 structures,
ability to produce different types of polysaccharides simultane- Lactococcus 6 structures and none for other LAB, even though the
ously, for instance different types of HoPS in Lb. reuteri 121 (Van Wzy-dependent pathway gene clusters have been found in Leu-
Geel-Schutten et al. 1999), or a mixture of HoPS and HePS in e.g. conostoc and Oenococcus (Fig. 3). The greatest variety is found in
Lb. johnsonii (Dertli et al. 2013) or O. oeni (Dimopoulou et al. 2016). lactobacilli (Table 1), a trait that is likely due to the large diversity
The molecular weight of glucan and fructan HoPS is usually be- within the genus. Exocellular polysaccharides synthesized by
tween 105 and 106 Da, but polymers with molecular weight up LAB are mostly branched; so far only seven non-branched struc-
to more than 107 Da have been reported (Ruas-Madiedo, Salazar tures have been reported, with all except one being produced
and de los Reyes-Gavilán 2009; Torino, Font de Valdez and Mozzi by different species of Lactobacillus (Table 3; De Vuyst and De
2015). Vin 2007; Ruas-Madiedo, Salazar and de los Reyes-Gavilán 2009).
Streptococcus thermophilus 8S also produces a linear polysaccha-
ride. Neutral polysaccharides are the most common, but deco-
ration with phosphate, as in a few L. lactis strains and one Lb.
Polysaccharide structures
rhamnosus, renders the polymer anionic (Table 1). Other non-
The structural diversity of exocellular polysaccharides produced carbohydrate decorations like acetylation and pyruvoylation are
by LAB is enormous. These polymers are comprised of a single postulated to be of importance for the biological role and the
type of sugar (HoPS) or two or more monosaccharides (HePS), functionality of the polymers (Tables 1 and 2; Torino, Font de
can either be unbranched or branched, and neutral or charged. Valdez and Mozzi 2015; Wang et al. 2015). Besides phosphate,
This range of structural features is based on the variation of acetyl and pyruvoyl groups, glycerol-phosphate groups are also
sugar monomers, a wide range of glycosidic linkages, the pres- found as decorations on certain polymers produced by lacto-
ence of branches and decoration with non-carbohydrate con- bacilli (Tables 1 and 2).
stituents. The monosaccharides present in LAB polysaccharides The sugars galactose and glucose, and to a lesser extent
can vary in nature, anomeric configuration (α- or β-anomer), rhamnose, are the most frequently occurring in repeating units
stereochemistry (L- or D- chiral forms) and cyclic conforma- of exocellular polysaccharides. Galactose and glucose occur both
tion (pyranose or furanose). This assortment of monosaccha- in the pyranose and furanose forms, while rhamnose is gener-
rides and the ensemble of different ways they can be linked to- ally in the pyranose form. The latter is quite frequent in L. lactis
gether generate an impressive diversity. As an example, just two and S. thermophilus, but less in lactobacilli. Indeed, to date it has
glucose residues can be joined in 30 different ways (Laine 1994). only been found in the species Lb. rhamnosus and Lb. bulgaricus.
The structural features of polysaccharides produced by LAB have In contrast, mannose, which is not present in any repeating unit
been thoroughly reviewed by de Vuyst and de Vin (2007) and of polysaccharides produced by L. lactis and S. thermophilus, is rel-
Ruas-Madiedo, Salazar and de los Reyes-Gavilán (2009). More atively common in lactobacilli (Tables 2 and 3). GalNAc and Glc-
recently, Torino, Font de Valdez and Mozzi (2015) revisited the NAc are present in polysaccharides produced by a few strains
topic, but focused mainly on extracellularly synthesized HoPS. in the three genera. Other sugars that have occasionally been
An updated overview of repeating unit structures synthesized found in repeating units are as follows: ManNAc, fucose, ribose
via the Wzy-dependent pathway is presented in Tables 1–3. Out and a non-ionic sugar.
Zeidan et al. S183
Table 3. Structures of the repeating units of exocellular polysaccharides synthesized via the Wzy-dependent pathway published since 2009,
and thereby not presented in previous reviews by de Vuyst and de Vin (2007) and Ruas-Madiedo, Salazar and de los Reyes-Gavilán (2009). The
structure of all repeating units has been elucidated using nuclear magnetic resonance spectroscopy.
Genus Strain Repeating unit structure Reference
Streptococcus S. thermophilus Säwén et al.

ST1 (2010)

Lactobacillus Lb. helveticus Li et al. (2014)
MB2-1
Li et al. (2015)
Lb. helveticus Patten et al.

Rosyjski (2014)
Lb. plantarum Zhou et al. (2016)

BC-25
Lb. plantarum C88 Fontana et al.

(2015)
Lb. plantarum Ismail and

MTCC9510 Nampoothiri
(2010)
Lb. rhamnosus Górska-Fraczek

KL37B et al. (2011)
Table 3 – continued.
Genus Strain Repeating unit structure Reference
Lb. rhamnosus Gorska et al. (2014)

LOCK 0900

Lb. johnsonii 142 Górska et al. (2010)
Lb. johnsonii Dertli et al. (2013)

FI9785
Lb. fermentum Gerwig et al. (2013)

TDS030603
Lb. crispatus L1 Donnarumma et al.

(2014)
Determination of the monomeric composition of exocellu- and also in fermented bakery products, due to their positive
lar polysaccharide produced by LAB has been attempted in contribution to textural properties. The in situ production of
several studies (Ruas-Madiedo et al. 2010; Qin et al. 2011; Suzuki, exocellular polysaccharides arguably has the potential to de-
Kobayashi and Kimoto-Nira 2013; Mende et al. 2014; Wang et al. crease the amount of added ingredients and stabilizers, such
2014a,b), but these data were not considered for the analysis pre- as dairy proteins, starch, pectin and hydrocolloids, often used
sented in Table 2 as no structure for the repeating unit of the to improve the textural properties of fermented dairy and ce-
polysaccharides was reported. real products. Ultimately, replacement of these additives leads
to a clean label and a reduced production cost. In addition, exo-
cellular polysaccharides in fermented dairy and cereal products
APPLICATIONS IN FOOD can be considered as a functional ingredient due to the postu-
Exocellular polysaccharide-producing LAB cultures are on high lated health benefits (Ryan et al. 2015; Caggianiello, Kleerebezem
demand for applications in food, mainly in fermented dairy and Spano 2016). Many studies are available on the effects
products, such as yoghurt, cheese and dairy-based desserts, that polysaccharide-producing strains or cultures have on the
Zeidan et al. S185
textural properties and stability of fermented dairy products, strains. A positive correlation with EPS neutrally charged and
and several hypotheses and conclusions have been formulated with a stiff backbone was observed, whereas anionic EPS seem
(Table 4). Yet, there is not a clear understanding of the role of to negatively contribute to syneresis (Gentès, St-Gelais and
exocellular polysaccharides in the microstructure of fermented Turgeon 2011, 2013).
dairy foods. The use of different exocellular polysaccharide- CPS has better water-binding properties than EPS (Low et al.
producing strains, and the different experimental conditions 1998; Broadbent et al. 2001). In mozzarella cheese, CPS produced
used, such as fermentation temperature, composition of the by S. thermophilus MR-1C can improve water retention and melta-
milk base, including amount and type of milk proteins, leads bility (Low et al. 1998; Broadbent et al. 2001). In half-fat cheddar
to a discrepancy in the obtained conclusions (Table 4). The dif- cheese, EPS- and/or CPS-producing strains lead to higher water

ferent polysaccharide characteristics, such as attachment to the retention, cheese yield and viscosity of the whey compared to
cell (CPS vs EPS), molecular composition, molecular weight, con- EPS-negative control strains (Dabour and LaPointe 2005; Dabour
formation and size, rigidity, degree of branching and charge, will et al. 2006). CPS are less prone to increase the viscosity of the
influence the textural properties (Table 4). This is mainly due to whey compared to ropy EPS, hence resulting in a more favorable
the different interactions occurring in the system, which influ- application in cheesemaking (Broadbent et al. 2001). Generally,
ence the texture properties of the matrix. application of EPS-producing strains to cheesemaking has been
less extensively studied compared to fermented milk.
The charge of EPS, together with the EPS production time dur-
Exopolysaccharides and capsular polysaccharides
ing fermentation, determines the distribution of the EPS in the
Mesophilic and thermophilic LAB, including the common microstructure, and hence their interference with gelation and
species used in yoghurt, Streptococcus thermophilus and Lb. bul- with the protein matrix. The majority of the exocellular polysac-
garicus, are able to produce EPS and CPS. In fermented milk, charides produced by LAB is uncharged, but a few charged poly-
EPS and CPS can contribute to enhanced viscosity, creaminess, mers have also been reported (Table 1). Negatively charged EPS
shear resistance, water-holding capacity, and to low syneresis, has been associated with higher viscoelastic properties, stiff-
with some of the EPS having higher contribution to ropiness ness and viscosity (Gentès, St-Gelais and Turgeon 2011; Kristo,
than most of CPS (Hassan et al. 1996a,b, 2003; Folkenberg et al. Miao and Corredig 2011). During acidification, milk proteins go
2005, 2006a,b; Yang et al. 2010; Kristo, Miao and Corredig 2011; from negatively charged at the initial pH of the milk, approx-
Costa et al. 2012a; Buldo et al. 2016). Due to the limited knowl- imately 6.5, to positively charged at the end of the fermenta-
edge on the structure of exocellular polysaccharides produced tion, around pH 4.6–4.3. At their isoelectric point, the proteins
by different LAB, the effect that they have on textural proper- are neutrally charged. If the EPS can interact with the protein
ties is often generalized. Among the studied strains, which pos- network, e.g. via electrostatic interactions, therefore interfer-
itively contribute to texture, the following association is pro- ing with protein coagulation, a continuous branched protein
posed: generally polysaccharide-producing strains of L. lactis network will be formed, which will lead to higher viscoelastic
ssp. cremoris are known to contribute to high viscoelastic prop- properties (Girard and Schaffer-Lequart 2007a; Ayala-Hernández
erties, including viscosity and gel stiffness, and hence moduli, et al. 2009; Corredig, Sharafbafi and Kristo 2011; Gentès, St-Gelais
ropiness and sliminess (Ruas-Madiedo, Hugenholtz and Zoon and Turgeon 2013; Hahn et al. 2014; Buldo et al. 2016). In case of
2002; Girard and Schaffer-Lequart 2007a,b; Ayala-Hernández surface inert properties between EPS and proteins (e.g. casein),
et al. 2009; Kristo, Miao and Corredig 2011; Costa et al. 2012a). such as neutral or positively low charged EPS, depletion effect
Some EPS-producing strains from Lb. rhamnosus, Lb. casei and Lb. and/or phase separation can occur, leading to formation of both
helveticus result in high viscosity products (Doleyres, Schaub and protein and EPS aggregates in the serum phase (Tuinier et al.
Lacroix 2005; Yang et al. 2010). Some of the EPS-producing Lb. bul- 1999; Tuinier, Dhont and De Kruif 2000; de Kruif and Tuinier 2001;
garicus strains seem to negatively contribute to the building of Hassan, Frank and Qvist 2002; Corredig, Sharafbafi and Kristo
the structure, and thus to lower moduli, probably due to a de- 2011; Gentès, St-Gelais and Turgeon 2011; Costa et al. 2012a;
pletion effect with casein (Girard and Schaffer-Lequart 2007a, Buldo et al. 2016). This phenomenon has been linked either to
Hess, Roberts and Ziegler 1997; Purohit et al. 2009). Generally, a decrease in viscoelastic properties, due to the interference of
EPS-producing S. thermophilus strains appear to positively con- the EPS with the protein network formation or to an increase
tribute to the viscosity of the product, but negatively to the gel in viscoelastic properties, due to the formation of a dense ca-
stiffness, and thus to the moduli (Hassan et al. 2003; Amatayakul sein network (Hassan et al. 2003; Amatayakul et al. 2006; Gentès,
et al. 2006; Purohit et al. 2009; Gentès, St-Gelais and Turgeon St-Gelais and Turgeon 2011; Buldo et al. 2016). The presence of
2011; Mende et al. 2012). The cause of higher viscosity has been EPS in the serum phase also leads to higher water-binding ca-
attributed to different factors, including higher EPS molecular pacity, which results in lower syneresis and high viscosity (Costa
weight, linearity and stiffness of the backbone, and complexity et al. 2012a; Gentès, St-Gelais and Turgeon 2013). However, one
of the side chains (Faber et al. 1998; Tuinier et al. 2001; De Vuyst has to keep in mind that the explanations are mainly based
et al. 2003; Gentès, St-Gelais and Turgeon 2011, 2013). Polysaccha- on hypotheses, since the mechanism of interaction between
rides which occupy larger volume in solution tend to produce exocellular polysaccharides and other elements in the system
high viscosity of the system. In addition, the complex interac- remains largely unknown, mainly due to limitations in identifi-
tions with other elements in the system, such as proteins and/or cation and/or visualization of the polysaccharides.
cells, strongly contributed to the viscosity of the system (Folken- The most significant contribution to textural properties is re-
berg et al. 2005; Girard and Schaffer-Lequart 2007a.b; Corredig, lated to the properties of the exocellular polysaccharides, rather
Sharafbafi and Kristo 2011; Gentès, St-Gelais and Turgeon 2011; than their amount. Several studies report a lack of positive cor-
Buldo et al. 2016). The number of side chains (branching) and the relation between the amount of exocellular polysaccharides and
radius of gyration (RG ) of polysaccharides have been positively textural properties (Marshall and Rawson 1999; Petry et al. 2003;
associated to gel firmness (Tuinier et al. 2001; Ruas-Madiedo, Doleyres, Schaub and Lacroix 2005; Gentès, St-Gelais and Tur-
Hugenholtz and Zoon 2002; Petry et al. 2003). Reduced synere- geon 2011; Hahn et al. 2014; Buldo et al. 2016). This could be due to
sis is a common property conferred by different EPS-producing the higher degree of interference of the polysaccharide with the
Table 4. Overview of polysaccharide-producing strains and their effects on physicochemical properties of fermented milk products.
Species and strain Experimental conditions Polysaccharide features Observed properties Source
L. lactis ssp. cremoris Fermented milk based on 4 Glc: 2 Rha: 1 Gal PS increases viscosity Ayala-Hernández et al.
JFR1 milk permeates, 8% solid. Mw: 2 × 106 Da Interaction with whey (2009)
Various levels of whey RG 70 nm proteins
protein, 30◦ C Anionic
L. lactis ssp. cremoris Fermented skim milk, 30◦ C Very ropy High viscoelastic properties Kristo, Miao and Corredig

JFR1 Anionic Low recovery properties (2011)
after shearing Ayala-Hernandez, Goff and
Corredig (2008)
L. lactis ssp. cremoris Fermented milk based on Ropy High viscoelastic properties Costa et al. (2012b)
DPC6532 10% reconstituted skim Neutral Depletion interactions
milk, 30◦ C 1.29 Glc: 1Gal between caseins and PS
Mw: 3 × 105 g mol−1
L. lactis ssp. cremoris Set and stirred fermented NCC2771: anionic and stiff Anionic EPS: high G´ and Girard and Schaffer-Lequart
Nizo B40 (NCC2771) milk based on 11% chain shear stress, and high (2007a)
S. thermophilus reconstituted skim milk, NCC15 and NCC1971: shear resistance (attractive
NCC1971 25◦ C and 40◦ C neutral less stiff chain interaction)
Lb. bulgaricus NCC15 Structures available Neutral: depletion
(Table 1) interactions between
casein, lower G´ and shear
stress
S. thermophilus Set skim milk yoghurt, 30◦ C Low syneresis Purohit et al. (2009)
ST-5581 –37◦ C Low moduli except for
ST-4239 LB-3984
ST-PH High hysteresis loop area
Lb. bulgaricus
LB-3984
L. lactis ssp. cremoris
JFR1
S. thermophilus Fermented milk based on 0131 (neutral, flexible, Linear and stiff chain, few Gentès, St-Gelais and
0131 2104 12% reconstituted skim branched PS) branching and high Mw: Turgeon (2011)
Lb. bulgaricus milk, 42◦ C 2104 (anionic, stiff, linear high apparent viscosity,
11842 PS) firmness and low syneresis
702074 11842 (neutral, flexible, Anionic EPS positively
291 branched PS) contribute to viscoelastic
702074 (neutral, flexible, properties, but not to
branched PS) syneresis
291 (neutral, stiff, branched
PS)
S. thermophilus Fermented milk based on Ropy CPS: CHCC 3534 (3 μm Ropy CPS: High shear stress Hassan et al. (2003)
CHCC 2136 11% reconstituted skim capsules) Low moduli
CHCC 3534 milk, 40◦ C CHCC 3541 (2.5 μm Poor structure recovery
CHCC 3541 capsules) Large pores
Non-ropy EPS: CHCC 2136
S. thermophilus Set and stirred yoghurt ASCC 285: capsular Low firmness and syneresis Amatayakul et al. (2006)
ASCC 285 based on 9% (w/w) total ASCC 1275: ropy (mainly with ropy)
ASCC 1275 solid. Ropy: Higher shear stress
Different casein to whey and hysteresis area than
protein ratio capsular
42◦ C
S. thermophilus Stirred low fat yoghurt 43◦ C Non ropy: High gel stiffness Mende et al. (2012)
DSM 8713 DSM 8713 Higher viscosity and
DGCC 7710 Ropy: DGCC 7710, ST- 143 hysteresis area only for
ST-143 ropy strains
Lb. rhamnosus Stirred low fat yoghurt Anionic Dose effect on syneresis Laneuville and Turgeon
RW-9595 M EPS added to the milk base Structure available (Table 1) and gel strength. Low (2014)
acidified with GLD, 30◦ C moduli
Zeidan et al. S187
Table 4. – continued.
Species and strain Experimental conditions Polysaccharide features Observed properties Source
Lb. bulgaricus Stirred low fat yoghurt with CPS Ropy Hess, Roberts and Ziegler
RR different solid and fat level Low syneresis, gel strength, (1997)
2483 and moduli
2772 High shear resistance
EPS associated with casein

Lb. bulgaricus Fermented milk based on Composition, Mw fractions High viscosity for the ropy Petry et al. (2003)
CNRZ 397 10% reconstituted skim available strains and Lb-18
CNRZ 1187 milk, 42◦ C CNRZ 397 (non-ropy) No correlation between the
CNRZ 416 CNRZ 1187 (ropy) amount of EPS and
Lb18 CNRZ 416 (ropy) viscosity
EPS-HYD 1545 Glucose influences the
viscosity
EPS-MS 907 Purified EPS (0.01% and Anionic High EPS concentration: Girard and Schaffer-Lequart
EPS-ST 716 0.05%) added to High molecular weight and high G´, brittle gel and poor (2007b)
EPS-GY 785 reconstituted skim milk sulfate group structure recovery
(11%) prior GDL Structure available (Table 1)
fermentation
Glc, glucose; Gal, galactose; Rha, rhamnose; GDL, glucono-δ-lactone; RG , gyration radius, G´, elastic modulus; Mw; molecular weight.
protein network (Hahn et al. 2014). However, whether the con- of the dough, such as water absorption, workability and rising
centration of polysaccharide affects texture remains controver- of the dough (Tieking and Gänzle 2005). In bakery applications,
sial (Mende, Rohm and Jaros 2016). the most studied polysaccharide-producing LAB are Lb. reuteri,
Isolation and characterization of exocellular polysaccharides Lb. pontis, Lb. panis, Lb. acidophilus, Lb. sanfranciscencis and Lb. fru-
from fermented milk is still a challenging task. Several proto- menti (Tieking et al. 2003). Dextran, reuteran and levan improve
cols are available in the literature for exocellular polysaccharide the textural properties of bread, with in situ production being
isolation. Mende, Rohm and Jaros (2016) illustrate the principal more effective than added EPS (Tieking and Gänzle 2005).
steps with the related problems for exocellular polysaccharide The rational design of a LAB culture that possesses supe-
isolation. The main concerns for the isolation and purification rior rheological properties will thus require (a priori) knowl-
steps are related to the purity and/or loss of exocellular polysac- edge regarding exocellular polysaccharide production, struc-
charides during extraction and also to degradation of the structural and macromolecular features of such polysaccharides, and
ture during the extraction process. The difference in purity leads structure–function correlations in different environmental con-
to a broad and inaccurate estimation of the exocellular polysac- ditions.
charide titer, as well as an inaccurate characterization of the
structure. The exocellular polysaccharide titer varies from 20 to
600 mg L–1 , based on strain type, milk composition and fermen- DEVELOPING STRAINS FOR FOOD AND OTHER
tation conditions (Cerning 1995; Amatayakul et al. 2006; Mende, APPLICATIONS
Rohm and Jaros 2016). Polysaccharide production seems to be
Use of exocellular polysaccharide-producing strains is relevant
closely related to the amount and type of proteins present in the
for fermented milk and cheese applications (see the ‘Appli-
media (Amatayakul et al. 2006; Buldo et al. 2016), which in some
cations in food’ section), other food applications (e.g. bakery)
strains was simply attributed to the direct effect of the protein
as well as applications outside the food sector (e.g. pharma
source on bacterial growth (Zisu and Shah 2003).
and medical applications). Despite the presence of genes en-
The effect of isolated EPS from LAB, added to milk prior to fer-
coding exocellular polysaccharide synthesis in many LAB, only
mentation, on the textural properties of several fermented milk
few strains give the desired textural properties in relevant food
products has also been studied. Generally, no beneficial effects
matrices, e.g. fermented milk. Screening tools for exocellular
on textural properties or on cost-in-use have been reported (Vla-
polysaccharide production or improved texture properties of
hopoulou, Bell and Wilbey 2001; Doleyres, Schaub and Lacroix
isolates are therefore of interest to various industries includ-
2005; Laneuville and Turgeon 2014). However, Mende et al. (2013)
ing starter culture producers. Alternatively, methods to improve
have shown that the stiffness of the gel increased with the con-
strains with regard to their ability to either produce more exocel-
centration of S. thermophilus ST-143 EPS added to milk prior to
lular polysaccharides or modified exocellular polysaccharides
chemical acidification. Based on this observation, the authors
with better viscosifying features, or methods that allow devel-
proposed that the molecular weight of the polysaccharide rather
opment of strains that give enhanced texture to fermented milk,
than the charge contributes to the stiffness. The positive contri-
are of great interest to the industry.
bution of exocellular polysaccharides to textural properties of
fermented dairy products seems to be driven by high molecular
weight and complexity of the side chain. The presence of both
Screening for exocellular
non-ropy EPS and CPS improves the textural properties without
contributing to ropiness.
polysaccharide-producing strains
In addition to dairy products, exocellular polysaccharides Screening methods for exocellular polysaccharides or pheno-
produced by LAB are known to improve the textural properties types associated with these polysaccharides are valuable tools
and the staling of bread, as well as the technological properties for academia and industry. These methods allow selection of

Figure 7. Schematic overview of exocellular polysaccharide screening methods. Traditional methods used in the past 15–30 years are shown at the top, and emerging
microtiter plate-based methods are shown at the bottom. The traditional methods often rely on visual inspection of samples, or manual measurement of either run
through of liquid samples in inverted capillaries or gravimetric mass determination of EPS. The automated methods are high throughput and quantitative. See the text
for further details. UHPLC-UV-ESI-MS: ultrahigh-performance liquid chromatography with ultra violet and electrospray ionization ion trap mass spectrometer.
polysaccharide-producing strains with appropriate rheological strings longer than 6 mm. The method was used to screen 201
properties to be used in starter cultures for applications in food LAB from Argentina (Mozzi et al. 2006). Only six thermophilic
or other industrial sectors. Generally, screening methods should and six mesophilic strains were identified to generate ropy milk.
be effective in differentiating bacterial isolates with the desired This feature did not correlate with exocellular polysaccharide
phenotype, e.g. enable screening for isolates with high exocellu- production measured as polymer dry mass, but out of six high
lar polysaccharide production or alternatively screening for iso- molecular weight (>1000 kDa) polysaccharide-producing strains
lates that generate improved texture in the medium used. The five were ropy (Mozzi et al. 2006). Moreover, the method was used
screening method should ideally allow rapid measurement or to screen 94 putative LAB isolates from Indian Dahi and sour
assessment of the desired feature, permitting high numbers of raw milk, identifying 47 mesophilic isolates with ropy pheno-
isolates to be screened, and finally the method should be inex- type (Behare, Singh and Singh 2009). A variant to the ropy-screen
pensive. When designing the method, it is also important to en- method employs an inoculation wire loop to assess string gen-
sure that the screening is done in a relevant matrix, optimally eration upon touching an LAB colony. When the string is longer
close to the application; if in situ texture development in acidi- than 5 mm, the strain is considered ropy (Dierksen, Sandine and
fied milk is desired, the procedure should be suitable to screen Trempy 1997). In a study of putative LAB isolates from traditional
for texture in acidified milk. Figure 7 shows the different screen- Iranian goat and ewe’s milk, a total of 102 isolates were screened
ing methods described hereafter. for a ropy phenotype, but none of the isolates showed ropiness
Several screening strategies have been devised to identify (Hajimohammadi Farimani et al. 2016). Although the ropy screen
exocellular polysaccharide-producing bacteria. Vedamuthu and is easy to perform, it is difficult to standardize, since the output
Neville (1986) used 1.0 mL pipettes to test the ropiness of is qualitative rather than quantitative. Furthermore, although
acidified reconstituted non-fat drymilk, assessing mucoidness the ropy phenotype observed with a particular isolate demon-
by the stringiness of the free flowing milk gel. Van den Berg strates that the strain can produce exocellular polysaccharide,
et al. (1993) used this approach to screen close to 600 LAB and it is not clear whether exocellular polysaccharide is produced in
found 30 strains displaying a ropy phenotype using EPS selec- low or high amounts (Vijayendra et al. 2008).
tion medium. Using a modified version of the medium, Lud- A screening approach employing microhaematocrit capillar-
brook, Russell and Greig (1997) isolated 11 LAB from salami, ies to measure efflux, a parameter that correlates with viscos-
ham and olives that showed levels of exocellular polysaccha- ity measurements, has also been developed (Ricciardi, Parente
rides ranging from 114 to 530 mg L−1 . LAB from traditional and Clementi 1997). LAB grown on various carbon sources were
Nigerian fermented foods were screened for ropiness by the screened for viscosity using this method and several isolates
above-mentioned method allowing identification of texturiz- of Lb. bulgaricus, Lc. dextranicum and Lactococcus sp. were iden-
ing Lc. mesenteroides, Lb. plantarum, Lb. acidophilus and Lb. brevis tified as polysaccharide-producing strains. Forty-one LAB iso-
(Sanni et al. 2002). The method of measuring ropiness with a lates from Italian sourdough fermentations were screened for
pipet is also described by Mozzi et al. (2001), defining ropy as exocellular polysaccharide production by both picking colonies
Zeidan et al. S189
from solid media and detecting ropiness using microhaemat- identification of polysaccharide-producing LAB. These tests
ocrit capillaries to screen for ropy phenotype (Zotta et al. 2008). based on the agglutination reaction that occurs between the
Ropy phenotypes were detected for Lb. plantarum isolates grown polysaccharide-producing strain and the type-specific antibody
on glucose, maltose or sucrose; Lb. paraplantarum on maltose or in the serum are reported to be highly sensitive. The limitation
sucrose; and Lc. mesenteroides and Weissella cibaria grown solely however is the availability of the specific polyclonal antibody.
on sucrose as carbon source. The use of microhaematocrit capil- To the best of our knowledge, this method has not been applied
laries as a screening method is appealing, since it is easy to per- to screen for polysaccharide-producing LAB.
form and gives an indication of whether the produced polysac- All the screening approaches mentioned above rely on exo-
charide causes changes in the food matrix. cellular polysaccharides causing a physical change in the envi-

Visual inspection of slimy or mucoid colonies on solidified ronment that can be observed by visual inspection. A more di-
media is probably the most frequently used screening method rect approach involves quantification of the exocellular polysac-
for EPS production. This approach is particularly good at iden- charide produced. In a screening study of 182 Lactobacillus iso-
tifying high-level EPS producers, since the slime around the lates cultivated in liquid broth, the mass of EPS in the super-
colony is hydrated EPS. This phenotype is often associated with natant was determined gravimetrically (van Geel-Schutten et al.
extracellular HoPS (De Vuyst and De Vin 2007). Tallgren et al. 1998). Several samples with EPS dry weight above 100 mg L−1
(1999) screened 600 soil isolates by looking for slime develop- were found when the strains were grown on sucrose. Growth on
ment around colonies grown on agar plates with sucrose and raffinose or lactose only rarely resulted in EPS production. A sim-
found 170 slimy isolates. A similar strategy was used by Smiti- ilar approach to determine polymer dry weight was reported by
nont et al. (1999), screening 104 isolates from traditional Thai Smitinont et al. (1999). When LAB are grown in milk or other high
fermented foods and finding 7 slimy colonies. There are several protein content media, protein precipitation is required for reli-
other reports using slimy colony morphology as first screening able use of the polymer dry weight method (De Vuyst et al. 1998).
step (Malik et al. 2009; Bennama et al. 2012; Ishola and Adebayo- This procedure was used to determine the monomeric compo-
Tayo 2012; Paulo et al. 2012). The disadvantage of this method sition of EPS from four different S. thermophilus (De Vuyst et al.
is that it typically detects only strains with high polysaccha- 1998).
ride production. Strains that produce polysaccharides via the Recently, a high-throughput screening method was de-
Wzy-dependent pathway are rarely giving rise to slimy colonies; scribed for the determination of EPS concentration and
this was demonstrated for the polysaccharide-producing S. ther- monosaccharide composition (Rühmann, Schmid and Sieber
mophilus LY03 (De Vuyst et al. 1998). 2015, 2016). The method consists of five steps: (i) cultiva-
Staining methods are used for detecting exocellular tion, (ii) cell removal by centrifugation and filtration, (iii) sugar
polysaccharide-producing cells, e.g. by using ruthenium red, monomer removal by gel filtration, (iv) EPS hydrolysis with triflu-
neutral red, calcofluor white, congo red or Indian ink. Ruthe- oroacetic acid and (v) derivatization with 1-phenyl-3-methyl-5-
nium red was used to screen for S. thermophilus mutants with pyrazolone (PMP) and monomer detection by UHPLC-UV-ESI-MS.
loss of ropy phenotype, which appear as red colonies on The use of PMP allows high-throughput carbohydrate determi-
milk agar media, while ropy wild-type strains appear white nation (Rühmann, Schmid and Sieber 2014). Out of 94 bacteria
(Stingele, Neeser and Mollet 1996). This red/white selection (representing 21 genera spanning Firmicutes, Actinobacteria and
was also applied to L. lactis strains. Use of ruthenium red as Proteobacteria) tested, 36 strains showed carbohydrate content
a general polysaccharide indicator is difficult. We have tested above 300 mg L−1 (Rühmann, Schmid and Sieber 2015).
approximately 90 S. thermophilus and 30 L. lactis isolates with We have recently developed a method for high-throughput
ruthenium red and we observed high strain to strain variation texture screening of fermented milk samples. Bacterial isolates
and no clear correlation between ‘white’ ruthenium red isolates are grown in 96-well microtiter plates with milk as cultivation
and their ability to texturize fermented milk (unpublished medium. After acidification of the milk, samples are aspirated
results). Neutral red dye was used to select ethidium bro- and dispensed by a Hamilton MicroLab Star liquid handling
mide mutants of ropy S. thermophilus that produced higher unit. The liquid handler has a pressure sensor located in the
amounts of exocellular polysaccharide than the wild-type headspace of each pipetting channel. Pressure data from each
strain (Escalante et al. 2002). Calcofluor white has been used sensor are collected and the resulting pressure curves used to
to select polysaccharide negative mutants of Rhizobium meliloti differentiate between strains with low, medium and high tex-
(Leigh, Signer and Walker 1985) and to detect polysaccharides in ture phenotypes (Cantor et al. 2014). We find this screening ap-
biofilms by Flavobacterium columnare (Cai, De La Fuente and Arias proach useful as it allows selection of strains with texturizing
2013) and S. pneumoniae (Domenech, Garcia and Moscoso 2012). phenotype in the desired matrix, i.e. fermented milk.
Congo red has been used in agar media for biofilm staining of Molecular screening approaches have also been used in the
B. subtilis (Bedrunka and Graumann 2016) and Staphylococcus search for new exocellular polysaccharide-producing isolates or
aureus (Darwish and Asfour 2013). Although staining assays as a means to differentiate between polysaccharide-producing
are relatively easy to perform, the visual interpretation can isolates at the gene level. The strategy typically comprises the
be difficult due to small differences in positive and negative design of degenerate primers based on LAB gene sequences fol-
signals. It has been suggested that lectins, glycoproteins that lowed by PCR reaction, cloning and sequencing of PCR products.
bind to sugar monomers of polysaccharides, can be used to This approach has been mainly applied to identify glucan and
differentiate between CPS and EPS in S. thermophilus (Robitaille fructan-producing strains (Krajl et al. 2003; Tieking and Gänzle
et al. 2006). Lectins typically have specificity for certain car- 2005; Malik et al. 2009). With the drop in prices for genome se-
bohydrate epitopes and may therefore not be used as generic quencing, it is now easier to study eps gene sequences and ar-
CPS screening tools. An alternative to lectins are polyclonal chitecture of the clusters encoding eps genes, and hence differ-
antibodies, used in Quellung reaction and agglutination tests, entiate strains at the genetic level. However, the identification
which have been the gold standard for serotyping of human of eps gene clusters is unlikely to allow drawing conclusions re-
pathogens, such as S. pneumoniae (Geno et al. 2015). In particular, garding amount and type of polysaccharides or even rheological
agglutination test can be envisioned as a screening tool for properties when applied in milk fermentations.

Figure 8. Overview of strategies used to improve LAB for polysaccharide production and/or rheology enhancement. (a) Metabolic/genetic engineering approaches: (1)
introduction of complete eps and/or cps clusters; (2) overexepression of genes galU and/or pgmA; (3) heterologous expression of galK. (b) Classical strain improvement
methods for selection of strains with improved rheological properties: transfer of plasmids harboring the eps cluster by conjugation, generation of galactose-proficient
strains by galactose selective pressure, selection with antibiotics targeting the cell wall (ampicillin and D-cycloserine), selection with phages. Dashed arrow indicates
multiple step pathways. Thick red arrows indicate overexpression of genes. Thunder symbol indicates treatment with selective pressure agent. Gal, galactose; Glc,
glucose; α-Glc-1-P, α-glucose-1-phosphate; Glc-6-P, glucose-6-phosphate; Fru-6-P, fructose-6-phospahte; HA, hyaluronic acid.
Strain improvement for polysaccharide nant strains are potential hosts for the safe production and de-
production livery of pneumococcal capsule vaccine antigens or hyaluronic
acid, a polysaccharide with many applications in medicine, cos-
The demand for exocellular polysaccharide-producing LAB in metics and specialty foods.
various applications is the catalyst for the development of new In L. lactis NIZO B40, an increase in polysaccharide produc-
strains with increased production or improved technofunctional tion was achieved by cloning the entire eps gene cluster present
properties. Strain improvement can be pursued by two major ap- in plasmid pNZ4000 into a high copy number vector (Boels et al.
proaches: (i) genetic and/or metabolic engineering and (ii) clas- 2003). A 9-fold elevated copy number led to a 3-fold increase in
sical strain improvement methods (Fig. 8). The choice of either the expression level of eps genes, resulting in circa 4-fold increase
approach is dictated mainly by the final application. The tight re- in polysaccharide production. This result further substantiated
quirements of regulatory agencies and the negative perception the view that increased levels of activated sugar precursors do
by consumers of genetically modified foods exclude the use of not influence NIZO B40 exocellular polysaccharide production
recombinant DNA technologies to improve the performance of levels, but rather that the production is limited by the activity
bacteria used in food manufacture. Thus, for any food applica- levels of the enzymes in the biosynthetic pathway. The relative
tion the approach of choice is the use of natural strain improve- carbon flux towards polysaccharide production increased 3-fold,
ment methodologies. Here we will summarize the research ef- and was accompanied by a substantial reduction of growth rate
forts to improve polysaccharide production and/or the rheolog- and a lower final optical density. Together these results indicate
ical properties in LAB. that increased exocellular polysaccharide production imposes
(i) Genetic/metabolic engineering approaches. Engineering of a significant metabolic burden, possibly due to competition for
complex pathways poses great challenges, and efforts to in- sugar nucleotides and energy, which are utilized in both polysac-
crease exocellular polysaccharide production in LAB were met charide production and growth.
with limited success (Welman and Maddox 2003; Gaspar et al. The complete eps gene cluster from S. thermophilus SFi6 was
2013). This complexity is further exacerbated by the paucity of transferred to L. lactis MG1363, a non-EPS-producing model LAB.
knowledge on key functions of the Wzy-dependent pathway, The polysaccharide obtained in L. lactis had a similar high molec-
such as the Wzx flippase, Wzy polymerase and priming GTs. The ular weight, but a different structure, namely a change in back-
potential of LAB to generate sugar nucleotides required for exo- bone composition from GalNAc to Gal and a side chain mod-
cellular polysaccharide synthesis has been exploited for the het- ification, i.e. loss of the branching Gal (Stingele et al. 1999).
erologous expression of pneumococcal CPS and hyaluronic acid The substitution of GalNAc by Gal most likely results from the
in L. lactis (reviewed in Gaspar et al. 2013). The resulting recombi- absence of an UDP-N-acetylglucosamine C4-epimerase activity
Zeidan et al. S191
in L. lactis MG1363, which thereby is incapable of generat- Based on the hypothesis that polysaccharide overpro-
ing UDP-GalNAc for incorporation in the repeating unit of the ducing mutants confer bacteriophage resistance by increas-
recombinant polysaccharide. Based on the findings, the authors ing a physical barrier against infecting phages, exocellular
speculated that the S. thermophilus Sfi6 Wzy polymerase could polysaccharide production was further increased by isolating
recognize and polymerize a repeating unit that differs in the phage-resistant mutants of strain CHCC11342 (Janzen and Chris-
backbone, as well as in the side chain from its native substrate. tiansen 2011a). CHCC11342 is a galactose-proficient, exocellu-
Galactose is assimilated via the Leloir pathway in S. ther- lar polysaccharide-producing S. thermophilus strain, isolated as
mophilus (Fig 4). The enzyme that links the Leloir pathway mutant from CHCC6008. The resulting phage hardened mu-
with glycolysis is α-phosphoglucomutase (α-PGM), which cat- tant, CHCC11977, when used to acidify milk, led to viscosity,

alyzes interconversion of Glc-6-P and Glc-1-P. Streptococcus ther- shear stress and gel firmness increased approximately by 20%
mophilus mutants lacking α-PGM activity as well as mutants with as compared to CHCC6008. This stands in contrast to the re-
overexpressed pgmA produced the same amount of exocellu- sults of the study by Deveau, Van Calsteren and Moineau (2002),
lar polysaccharide as the parent strain when grown on lactose, which showed no impact of exocellular polysaccharide produc-
indicating that the Leloir pathway is important for supplying tion on either phage sensitivity or resistance. The combination
exocellular polysaccharide precursors (Levander and Radstrom of CHCC11977 with a polysaccharide-producing Lb. bulgaricus
2001). Homologous overexpression of the galU gene in S. ther- strain resulted in increased shear stress and mouth feel in yo-
mophilus LY03 led to a 10-fold increase in GalU activity, but did ghurt application trials. In a similar way, mutants isolated af-
not have any effect on the exocellular polysaccharide yield when ter phage challenge of Lb. bulgaricus strain CHCC10019 increased
lactose was the carbon source. However, when galU was overex- shear stress of fermented milk by 25% (Janzen and Christiansen
pressed in combination with pgmA the exocellular polysaccha- 2011a).
ride yield increased from 0.17 to 0.31 g mol−1 of carbon from lac- Resistance to antibiotics was recently discovered as an at-
tose. A galactose-fermenting S. thermophilus LY03 mutant, TMB tractive way to increase rheological parameters of starter cul-
6010, with increased activity of the Leloir pathway enzymes, tures. Six out of 21 Lb. bulgaricus CHCC13995 ampicillin-resistant
and higher levels of Glc-1-P and Glc-6-P, was also found to have mutants revealed doubling of efflux time of fermented milk
a higher exocellular polysaccharide yield (0.24 g mol−1 of car- measured by a volumetric pipette, indicating increased viscosity.
bon) than the parent strain. The exocellular polysaccharide yield A positive effect on ropiness and gel stiffness of the fermented
was further improved to 0.27 g mol−1 of carbon by overexpress- product was also observed when the mutant was used in com-
ing galU in this strain, but the highest exocellular polysaccha- bination with the non-polysaccharide-producing S. thermophilus
ride yield, 0.36 g mol−1 of carbon, was obtained when pgmA was strain CHCC4895 (Johansen, Soerensen and Kibenich 2013). Re-
knocked out in the galactose-proficient strain (Levander, Svens- sistance towards the antibiotic D-cycloserine was proven suc-
son and Radstrom 2002). Overexpression of both pgmA and galU cessful to increase shear stress and gel stiffness in milk acidi-
in TMB6010 led to a 3.3-fold increased polysaccharide produc- fied with the resistant Lb. bulgaricus and S. thermophilus strains.
tion (Svensson et al. 2005). Strikingly, the viscosity in fermented A D-cycloserine-resistant mutant of Lb. bulgaricus CHCC13995,
milk did not increase significantly in response to the increased CHCC12945, triggered a shear stress increase of 10% and a gel
exocellular polysaccharide amounts. stiffness increase of 50% in fermented milk as compared to the
Exocellular polysaccharide production could also be in- wild-type strain. For S. thermophilus, the D-cycloserine-resistant
creased in S. thermophilus by introducing a galactokinase (galK) mutant CHCC13236 induced a 15% increase in shear stress and
gene into a galactose-deficient strain (Robitaille et al. 2009). The 19% in gel stiffness of the fermented milk product, as compared
recombinant strain produced 1.3 times more polysaccharide, but to the wild-type CHCC13994 (Kibenich, Soerensen and Johansen
when used in combination with Lb. bulgaricus for yoghurt man- 2012).
ufacture, no significant increase in polysaccharide production Isolation of LAB strains with improved rheological properties
was detected. by selection with antibiotics, phages or essential nutrients al-
(ii) Classical strain improvement. eps gene clusters are often lows direct application of these superior strains in food applica-
plasmid encoded in L. lactis (Vedamuthu and Neville 1986; Neve, tions. The examples presented here demonstrate the feasibility
Geis and Teuber 1988; van Kranenburg et al. 1997; van Kranen- to obtain LAB isolates with improved ability to produce polysac-
burg, Kleerebezem and de Vos 2000; Forde and Fitzgerald 2003) charides. The work done so far provides the foundations for fur-
and in some lactobacilli, e.g. Lb. casei (Kojic et al. 1992) and Lb. ther exploitation of LAB-produced polysaccharides in novel ap-
paraplantarum (Zivkovic et al. 2015). When eps gene clusters are plications.
located on plasmids, the mucoid phenotype can be transferred
by conjugation from one strain to another strain as demon-
strated in L. lactis for EPS plasmids pSRQ2202 (Vedamuthu and
CONCLUDING REMARKS
Neville 1986) and pNZ4000 (van Kranenburg and de Vos 1998),
or by protoplast transformation as shown for plasmid pVS5 (von LAB have a long tradition of safe usage in the manufacture of fer-
Wright and Tynkkynen 1987). From a regulatory point of view, mented beverages and foods. One of the beneficial effects of LAB
such conjugation events are natural, and hence the plasmid reis enhancement of rheological properties through in situ pro-
cipients are not considered genetically modified organisms. duction of polysaccharides during fermentation of the raw food
The galactose-negative phenotype, identified for the major- materials. Due to their ability to form hydrocolloids in aqueous
ity of S. thermophilus strains, is due to the lack of GalK activity. solutions, polysaccharides can serve as emulsifiers, thickeners
The isolation of spontaneous galactose-proficient mutants from and gelling agents. Other attributes in food are stabilization,
the polysaccharide-producing S. thermophilus CHCC6008 by se- suspension of particulates, control of crystallization, inhibition
lective pressure in galactose broth resulted in strain CHCC11379. of syneresis, encapsulation and film formation. Despite this
This mutant showed slightly increased exocellular polysaccha- array of versatile applications, the use of LAB polysaccharides
ride production, and improved viscosity and shear stress of a as food ingredients/additives has so far been restricted to a
fermented milk (Janzen and Christiansen 2011b). single polymer, the HoPS dextran produced by Lc. mesenteroides.
Therefore, LAB are essentially an untapped reservoir of GTs observed in the loci for polysaccharide production provides
structurally diverse polysaccharides to be used as food an opportunity to continually generate new strains producing
additives. Industrial use of microbial polysaccharides is, how- unique polysaccharides by gene shuffling. Despite the relative
ever, not restricted to the food industry, and the number of simplicity in pathway assembly, main hurdles arise from poor
(bio)technological applications in pharmaceutical and cos- functional characterization of the molecular factors involved.
metics industries, medicine, water treatment, oil industry Indeed, the substrate specificity of Wzy polymerases and Wzx
(enhanced oil recovery), agriculture and construction ma- flippases has never been studied in LAB and to the best of our
terials continuously increases (Ullrich 2009; Freitas, Alves knowledge remains a mystery even in well-characterized enter-
and Reis 2011; Srinivasan 2013; Freitas, Alves and Reis 2014; obacteria. As for GTs, only a very limited number has been bio-

Moscovici 2015; Schmid, Sieber and Rehm 2015). Exploring the chemically characterized, and thus substrate specificity and GT
natural diversity and versatile technofunctional properties of activity remain largely unknown in LAB.
polysaccharides produced by LAB holds great promise in high- LAB are clearly a great source of polysaccharides with in-
value market niches, such as pharmaceuticals, cosmetics and dustrial applications. The already existing natural diversity can
medical applications (adhesives, sutures, vaccine adjuvants). even be further expanded by implementation of synthetic biol-
Furthermore, polysaccharides are biocompatible, biodegrad- ogy approaches. However, increasing polysaccharide production
able and non-toxic natural biopolymers. Thus, the demand via metabolic engineering and/or generating tailored polysac-
for biosustainable products in modern, ecofriendly societies charides requires a better understanding of the molecular
render bacterial polysaccharides as promising replacements for mechanisms underlying polysaccharide synthesis. This can be
petroleum-derived polymers. achieved by combining structural polysaccharide data, biochem-
A major limitation to the use of bacterial, and in particular ical characterization and systems biology approaches (omics
LAB, polysaccharides for industrial applications is the cost as- experimentation and bioinformatics/modeling). Finally, we sur-
sociated with their production. On the technological side, high- mise that the availability of tailored polysaccharides is an asset
value substrates (carbon sources) are used for bacterial biomass to improve our understanding of structure–function–application
production and the downstream process costs associated with relationships.
purification and isolation are also on the high end. Adding to
this, polysaccharides are generally produced by bacteria in low
titers and yields, most likely because their synthesis is metabol-
ACKNOWLEDGEMENTS
ically costly to the cell. The authors would like to acknowledge the colleagues in the Dis-
Thus, harnessing polysaccharide diversity and functional- covery function of the Research and Development organization
ity for commercial purposes requires a multidisciplinary ap- in Chr. Hansen A/S for valuable discussions and sharing of re-
proach to boost polysaccharide production in LAB in a rational sults.
way. To this end, metabolic engineering can be applied to de-
velop suitable hosts for sustainable and cost-effective produc- Conflict of interest. None declared.
tion of polysaccharides, ultimately leading to product develop-
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FEMS Microbiology Reviews, fux017, 41, 2017, S168–S200

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Polysaccharide production by lactic acid bacteria:

INTRODUCTION >10 units; oligosaccharides, ≤10 units) bound together by

Received: 17 January 2017; Accepted: 29 March 2017

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Monosaccharide ratio in the repeating unit produced by LAB

S. thermophilus ST1 6 4 2 Säwén et al. (2010)

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Genus Strain Repeating unit structure Reference

Streptococcus S. thermophilus Säwén et al.

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Lb. helveticus Patten et al.

Lb. plantarum Zhou et al. (2016)

Lb. plantarum C88 Fontana et al.

Lb. plantarum Ismail and

Lb. rhamnosus Górska-Fraczek

Genus Strain Repeating unit structure Reference

Lb. rhamnosus Gorska et al. (2014)

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Lb. johnsonii Dertli et al. (2013)

Lb. fermentum Gerwig et al. (2013)

Lb. crispatus L1 Donnarumma et al.

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