A functional and genetic overview of exopolysaccharides produced by Lactobacillus plantarum

Journal of Functional Foods 47 (2018) 229–240

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A functional and genetic overview of exopolysaccharides produced by Lactobacillus plantarum Yunyun Jiang, Zhennai Yang

T

⁎

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Laboratory of Food Quality and Safety, Beijing Technology and Business University, 100048 Beijing, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Exopolysaccharides Lactobacillus plantarum Function Whole genome Gene cluster

Lactobacillus plantarum strains have been widely used in food processing and preservation, and capability of their production of exopolysaccharides (EPSs) contributes significantly to the improvement of food quality and function. During the last decade, rapid development and accumulation of data in the research field of EPSs produced by L. plantarum have been observed. Availability of complete genome sequences from forty-three L. plantarum strains to date has provided good basis for deep analysis and understanding of the molecular and functional properties of the EPSs. In this review, an overview of the main insights into the fundamental molecular and genetic aspects of the EPSs produced by L. plantarum was presented. The organization and function of the EPS biosynthesis gene clusters from the forty-three L. plantarum strains were analyzed and compared, including those in both the genomes in thirty-six strains and plasmids in seven strains that were not reported earlier.

1. Introduction Exopolysaccharides (EPSs) were high-molecular-weight carbohydrate polymers secreted extracellularly by many microorganisms (Gorska et al., 2017). EPSs could be produced in two forms, concretely, capsular polysaccharide (CPS) that was closely associated with the cell surface, forming a capsule, and slime polysaccharide (SPS) that was loosely attached or even totally secreted into the environment of the cell (Lee, Caggianiello et al., 2016). According to the type of monosaccharides, EPSs were classified into two groups, homopolysaccharides and heteropolysaccharides (Hidalgo-Cantabrana et al., 2014). The latter contained 100–90,000 oligosaccharide repeating units to form linear or highly branched structure (Frengova, Simova, Beshkova, & Simov, 2000; Gorska et al., 2017; Hidalgo-Cantabrana et al., 2014; Oldak, Zielinska, Rzepkowska, & Kolozyn-Krajewska, 2017). The bonds between the monomers were 1,4-β- or 1,3-β-linkages and 1,2-α- or 1,6-αlinkages (Frengova et al., 2000; Gorska et al., 2017; HidalgoCantabrana et al., 2014; Oldak et al., 2017). EPS production has been extensively studied in Lactobacillus and other lactic acid bacteria (LAB) genera of food origin (Badel, Bernardi, & Michaud, 2011). These studies were concerned with many species: L. delbrueckii subsp. bulgaricus, L. helveticus, Streptococcus thermophilus, L.

acidophilus, Lactococcus lactis, L. rhamnosus, L. plantarum and L. casei. The fundamental function of bacterial EPSs was mainly associated with their protective nature including protection of bacterial cells from heavy metals, desiccation or other environmental effect, ability of recognition, adhesion to surfaces, and biofilm formation (Limoli, Jones & Wozniak et al., 2015). Bacterial EPSs also exhibited emulsion stabilizing capacity, shear-thinning activity, suspension ability, high viscosity, and excellent biocompatibility (Hou et al., 2010). In the food industry, these polymers were used as bio-thickeners because of their stabilizing, emulsifying or gelling properties (Hou et al., 2010). Moreover, bacterial EPSs could interfere with adhesion of pathogens, lower the cholesterol and triglyceride levels, and possess antitumor, anti-HIV, and immunomodulatory activities (Ayadi, Bayer, Marras, & Athanassiou, 2016; Drogoz et al., 2008; Gorska et al., 2017; Lee, Caggianiello et al., 2016; Zaporozhets & Besednova, 2016). L. plantarum exhibited polyfunctionality in the processing and preservation of dairy products, meat, vegetables and lactic beverage, as well as preparation of various antiseptic and other medicinal formulations, and enterohepatic fermentation (Lee, Kim, & Park, 2016; Oldak et al., 2017; Russo et al., 2017; Zivkovic et al., 2015). Especially, the EPS-producing L. plantarum strains impacted physicochemical and functional properties of healthy foods (Al-Dhaheri et al., 2017; Ayyash,

Abbreviations: EPS, exopolysaccharides; SDM, semi-defined medium; MRS, deMan Rogosa Sharpe; Gtf, glycosyl transferase; Act, acyltransferase; CPS, capsular polysaccharide; SPS, slime polysaccharide; LAB, lactic acid bacteria; TNF, tumor necrosis factor; IL, interleukin; Ntr, nitroreductase; GluT, glucosyltransferase ⁎ Corresponding author. E-mail address: [email protected] (Z. Yang). https://doi.org/10.1016/j.jff.2018.05.060 Received 8 November 2017; Received in revised form 25 May 2018; Accepted 26 May 2018 1756-4646/ © 2018 Published by Elsevier Ltd.

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Table 1 Fundamental characteristics of EPSs from L. plantarum. Strain

Source of isolation

Molecular weight (Da)

Yield (mg/L)

Td (°C) TGA and medium

EPS repeating unit or monosaccharide composition

Reference

WCFS1

Hunman saliva

2.9 × 104

31.52

BC-25

Traditional Chinese fermented pickle

1.83 × 104 1.33 × 104

324.80

– 2 × CDM 320 MRS

Kleerebezem et al. (2003) and Remus et al. (2012) Zhou et al. (2016)

RJF4

Rotten Jack fruit

–

1500

Rha: NAG: Gal: Glu: Galacturonic acid = 5.40: 3.28: 17.39: 27.98: 45.66 Man, Gal and Glu in the EPS and the molar ratio was 92.21:1.79:6.00 for the EPS with Se, whereas 91.36:2.44:6.20 for the EPS without Se α-D-Man, β-D-Glu

6

SKT109

Tibet Kefir

2.1 × 10

58.66

YML009

Kimchi

–

260

70,810

Chinese Paocai

KF5

Tibet Kefir

1.70 × 105, 2.05 × 105 –

64.17, 859 75.57, 95.58 187

DM5

Ethnic fermented March beverage from Sikkim Curd

1.11 × 10

6

225 MRS, pH 7.3 – SDM – MRS 339.45 MRS 279.59 whey – modified MRS

–

– MRS

–

YW11

Tofu in Inner Mongolia Tibet Kefir

2.68 × 105, 2.55 × 105, 2.83 × 105 1.15 × 106 1.1 × 105

90

– SDM 287.7 SDM

YW32

Tibet Kefir

1.03 × 105

MTCC 9510

C88

–

4

429.4

Traditional acid beans

5.17 × 10

EP56

Corn silage

8.5 × 105, 4.0 × 104

126.4

SF2A35B

Sour cassava

5.1 × 103

59.5

2 × CDM

3

85.7

2 × CDM

Lp90

Wine

2.8 × 10

– Cell-bound exopolysaccharides (c-EPS)-α-(1 → 6)gal_-β-(1 → 4)-gal-β-(1 → 2,3)-gal -β-(1→)- gal Man, Glu and Gal in an approximate ratio of 1:4.99:6.90 a-D-glucan (dextran), α-(1 → 6) and α-(1 → 3) linkages

-α-D-Glu, α-D-Man and β-D-Gluthe sequence of carbohydrate backbone linked by α-1,3-linkages and a possibility of β-1,3-linkage at the terminal →4)[β-Glc-(1 → 3)][ β-DGalp-(1 → 6)] α-D-Galp2Ac(1 → 2)- α-D-Glcp-(1 → 3)- β-D-Glcp-(1→ Glu and Gal 2.71:1

283.5 MRS – modified MRS – CDM

ZDY2013

Fructose and Glu in an approximate molar ratio of 3:1

Man, fructose, Gal and Glu 8.2:1:4.1:4.2 Xylose and Gal Cell-bound EPS fraction (EPS-b) Glu, Gal and Nacetylgalactosamine 3:1:1 Released EPS fraction (EPS-r) Glu, Gal and Rha 3:1:1 Glu: NAG : Gal: Galactosamine = 2: 0.1: 66.6: 31.3 Glu: NAG : Gal: Galactosamine: Rha = 3.9: 24.5: 22.2: 4.4: 25

Dilna et al. (2015) Wang, Zhao, Tian, He, et al. (2015) Seo et al. (2015) Wang et al. (2014a, 2014b) Wang et al. (2010) Das and Goyal (2014) and Das et al. (2014) Ismail and Nampoothiri (2010, 2014) Fontana et al. (2015) Wang, Zhao, Tian, Yang, et al. (2015) and Zhang et al. (2017) Wang, Zhao, Yang, et al. (2015) Zhang, Liu et al. (2016) Tallon et al. (2003)

Lee, Caggianiello et al. (2016) Lee, Caggianiello et al. (2016)

EPSs was identification of efficient EPS-producing strains and nature of the EPSs, cost of production and development of downstream process (Badel et al., 2011). Genetic engineering of microbes required further developments to convert inexpensive raw materials to EPSs (HidalgoCantabrana et al., 2014; Oldak et al., 2017). In the light of current knowledge, an emphasis should be given to produce new EPSs with potential multifarious applicability (Badel et al., 2011). During the last decade, rapid development and accumulation of data in the research field of EPSs produced by LAB, e.g. L. plantarum, were observed, and summarization of the recent research progress was imperatively necessitated. In this review, an overview of the main insights into the molecular and genetic aspects of the EPSs produced by L. plantarum was presented focusing on their fundamental molecular characteristics, bioactivities, and the relationship between structure and function. Moreover, genetic characterization and organization of EPS biosynthesis gene clusters, and prediction for the functions of EPS biosynthesis genes were reviewed, and finally suggestions on further research and applications of L. plantarum EPSs were outlined.

Abu-Jdayil, Hamed, & Shaker, 2018; Russo et al., 2016). Strains KX881772 and KX881779 from camel milk conferred better qualities and health-promoting benefits on low-fat Akawi cheese. Strain Lp90 from wine improved the technological and nutritional characters of oatbased foods. The studies on functional mechanism of L. plantarum EPS deepened as the complete genome sequencing technology developed. The complete 3.3 Mb genome of L. plantarum WCSF1 was first reported within the Lactobacillus species (Kleerebezem et al., 2003). Among the forty-three L. plantarum genome sequences available in the NCBI database, fewer gene clusters related to EPS biosynthesis have been reported (Lee, Caggianiello et al., 2016; Li et al., 2016; Remus et al., 2012). The well characterized genome from strain WCFS1 was shown to contain four clusters of genes (cps1A-I, cps2A-J, cps3A-J and cps4A-J) that were associated with surface polysaccharide production (Remus et al., 2012). The cps2 and cps4 clusters encoded all functions required for capsular polysaccharide formation, while the cps1 and cps3 clusters were predicted to lack genes encoding chain-length control functions and a priming glycosyl-transferase. Moreover, strains SF2A35B and Lp90 contained complete cps3, cps4 and strain-specific cps2 without cps1, respectively, and the main role of cps2-like cluster in the strains Lp90 and SF2A35B was found to associate with production of ropy phenotype EPS (Lee, Caggianiello et al., 2016). L. plantarum strains ZJ316, ST-III and ATCC 14917 had cps3 and cps4 without cps1 and cps2 clusters. and strain JDM1 carried only cps4 cluster. Bacterial EPSs were considered to be new sources of natural polymers, which could meet the increasing demands for many industrial applications, including food, cosmetic and medicine (Ayadi et al., 2016; Gorska et al., 2017; Lee, Caggianiello et al., 2016; Zaporozhets & Besednova, 2016). The main challenge for commercialization of new

2. Fundamental characterization of EPSs from L. plantarum EPS-producing LAB strains have been isolated from a wide range of sources including sour dough, whey, fermented vegetables, snake gourd, excreta of sheep, human baby, etc. (Badel et al., 2011). The EPSproducing strains of L. plantarum were mainly isolated from human gut (Ait Seddik, Bendali, Cudennec, & Drider, 2017; Hirano et al., 2017), Drosophila melanogaster gut (Daisley et al., 2017), Tibet Kefir (Zhang, Liu et al., 2013), wine (Lamontanara et al., 2015), fermented vegetables (Tanganurat, Quinquis, Leelawatcharamas, & Bolotin, 2009) such as 230

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Fig. 1. Structures of the repeating units of the EPSs of L. plantarum C88 and BC-25 (Zhou et al., 2016; Fontana et al., 2015).

while another cell-bound EPS from L. plantarum 70810 was a galactan (Wang et al., 2014a). Many EPSs produced by L. plantarum were heteropolysaccharides containing two to three types of monosaccharides. The EPSs produced by L. plantarum C64MRb, E112, G62 and H2 of human origin contained galactose and glucose in molar ratios of 1:9, 1:5, 1:5, 1:3, respectively (Salazar, Prieto et al., 2009). L. plantarum C88 (Fontana et al., 2015) and YW11 (Wang, Zhao, Tian, Yang, et al., 2015) also produced EPSs composed of galactose, but in different molar ratios of 1:2 and 1:2.71, respectively. L. plantarum RJF4 and MTCC 9510 produced EPSs containing mannose and glucose, while the EPS from L. plantarum ZDY2013 EPS contained xylose and galactose (Dilna et al., 2015; Ismail & Nampoothiri, 2014; Zhang, Liu et al., 2016). Fructose and glucose in an approximate molar ratio of 3:1 were present in the EPS from L. plantarum SKT109 (Wang, Zhao, Tian, He, et al., 2015). Both L. plantarum KF5 (Wang et al., 2010) and BC-25 (Zhou, Zeng, Han, & Liu, 2015) produced EPSs composed of mannose, glucose and galactose. Two EPS fractions were isolated from L. plantarum EP56 (Tallon et al., 2003), the cell-bound EPS containing fraction (EPS-b) composed of glucose, galactose and N-acetylgalactosamine (3:1:1), and the released EPS fraction (EPS-r) containing glucose, galactose and rhamnose (3:1:1). Particularly, L. plantarum YW32 (Wang, Zhao, Tian, Yang, et al., 2015) produced an EPS containing four types of monosaccharides such as mannose, fructose, galactose and glucose in a ratio of 8.2:1:4.1:4.2, and another EPS produced by L. plantarum NTU 102 contained six types of monosaccharides such as fructose, arabinose, galactose, glucose, mannose and maltose (Lin & Pan, 2015). The glucosidic linkages between the monosaccharides in the EPSs produced by L. plantarum differ considerably. The α-D-glucan produced by L. plantarum DM5 possessed α-(1 → 6) and α-(1 → 3) linkages (Das et al., 2014). The galactan from L. plantarum 70810 consisted of a repeating unit of α-(1 → 6)-gal-β-(1 → 4)-gal-β-(1 → 2,3)-gal-β-(1→)- gal (Das et al., 2014; Wang et al., 2014a). Other heteropolysaccharides produced by L. plantarum had rather complex structures. For example, L. plantarum C88 (Fontana et al., 2015) produced an EPS constituted of a repeating unit of → 4) [β-Glc-(1 → 3)] [β-Galp-(1 → 6)] α-Galp2Ac(1 → 2)-α-Glcp-(1 → 3)-β-Glcp-(1 → . The EPS from L. plantarum BC-25 (Zhou et al., 2016) contained a highly branched repeating unit consisted of (1 → 2)-Man, (1 → 2,6)-Glc, (2 → 6)-Man, and (2 → 6)-Gal (Fig. 1). The thermostability of an EPS played a key role in its industrial applications. The property of tolerance to higher temperatures made

sour cassava (Bron et al., 2016), traditional Chinese fermented pickle (Xi et al., 2013) and Kimchi (Korean traditional fermented food) (Kwak, Cho, Noh, & Om, 2014). The features of EPS production by L. plantarum strains and the molecular characteristics of the polysaccharides were strain dependent and varied with the culture conditions and medium compositions (Kim, Seo, Hwang, Lee, & Park, 2008; Salazar, Prieto et al., 2009). The amount of EPS produced by L. plantarum strains ranged from 31 to 859 mg/L in deMan Rogosa Sharpe (MRS) medium or semi-defined medium (SDM), but L. plantarum RJF4 produced as much as 1500 mg/L in MRS medium (Table 1). The average molecular weights of the EPSs from twelve L. plantarum stains varied with the strains, ranging from 104 to 106 Da (Table 1), similar to those of other LAB strains reported (Kim et al., 2008; Salazar, Prieto et al., 2009). Strains of YW32 (Wang, Zhao, Yang, Zhao, & Yang, 2015), YW11 (Wang, Zhao, Tian, Yang, & Yang, 2015), MTCC 9510 (Ismail & Nampoothiri, 2010, 2014) and 70810 (Wang et al., 2014a, 2014b) had molecular weights around 105 Da, while strains of C88 (Fontana, Li, Yang, & Widmalm, 2015), DM5 (Das & Goyal, 2014) and SKT109 (Wang, Zhao, Tian, He, et al., 2015) had higher values (about 106 Da), but strains ZDY2013 (Zhang, Liu, Tao, & Wei, 2016) and BC-25 (Zhou et al., 2016) had lower values (about 104 Da). Strain C88 also produced a high-molecular-mass capsular polysaccharide (1.15 × 106 Da) when grown in a SDM (Fontana et al., 2015; Zhang, Zhang et al., 2013). Strain 70,810 produced two EPSs with similar molecular weights of about 2 × 105 Da (Wang et al., 2014a, 2014b). Strain EP56 produced two EPSs with different molecular weight of 8.5 × 105 and 4.0 × 104 Da (Tallon, Bressollier, & Urdaci, 2003). Glucose and galactose were the most frequently reported monosaccharides in the EPSs produced by lactobacilli including L. plantarum strains (Mozzi et al., 2006; Salazar, Ruas-Madiedo et al., 2009). This might be associated with the growth media used for the EPS production. Most EPS-producing L. plantarum strains reported were cultivated in MRS or SDM medium (Table 1), in which glucose was the carbon source. Carbon source was reported to have great effect on both the quality and quantity of EPSs synthesized by microorganisms (Bramhachari et al., 2007). Glucose, galactose and mannose in the EPSs from L. plantarum also had higher proportion than other monosaccharides such as fructose, xylose, rhamnose and N-acetylgalactosamine. The EPS from L. plantarum DM5 was a homopolysaccharide containing only glucose (Das, Baruah, & Goyal, 2014), 231

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dismutase, catalase and total antioxidant capacity in mice serum. The EPS from L. plantarum YW11 also had gut microbiota regulating activities, which decreased the abundance of Flexispira by 37.5 folds, and increased Blautia (36.5 folds) and Butyricicoccus (9.5 folds). Correspondingly, the fecal content of nitrogen oxides decreased to 9.87%, and the content of short-chain fatty acids increased by 2.23 folds. Hence, EPSs from the eight stains of L. plantarum were potential antitumor adjuvants. Chemical modification could be an effective way to improve the bioactivities of the EPSs from LAB (Zhang, Liu et al., 2016). Sulphonation (sulphonation degree of 0.26 ± 0.03) of the purified EPS produced by S. thermophilus GST-6 improved its antioxidant activities (DPPH, superoxide and hydroxyl radicals scavenging activities) and resistance to bacterial pathogens (against Eschericia coli, Salmonella typhimurium and Staphylococcus aureus) (Zhang, Cao et al., 2016). Modification of the EPS from L. plantarum 70810 by acetylation, phosphorylation and carboxymethylation improved its antioxidant and antitumor activities (Wang, Li et al., 2015). Sulphonation of the EPS from L. plantarum ZDY203 increased its effectiveness in counteracting the cytotoxicity induced by B. cereus enterotoxins on Caco-2 cells (Zhang, Liu et al., 2016). EPSs from L. plantarum were shown to be potentially effective for treatment of diabetes (Dilna et al., 2015; Sasikumar et al., 2017; Zhang, Liu et al., 2013). The EPS from L. plantarum BR2 was of antidiabetic potential by inhibiting α-amylase activity by 10% at 100 mg/mL of the EPS, inhibiting β-glucosidase activity by 67% at 300 mg/mL, and reducing the in vitro cholesterol level by a margin of 45% at 0.1% of the EPS. The EPS from L. plantarum RJF4 displayed cholesterol lowering activity by 42.24%, and α-amylase inhibition by 40%. The EPS from KF5 also had cholesterol lowering activity, and it might be effective in the treatment of diabetes. The EPS-producing L. plantarum strains might impact physicochemical and functional properties of healthy foods (Al-Dhaheri et al., 2017; Ayyash et al., 2018; Russo et al., 2016). The ropy-type EPSs from L. plantarum might affect favorably the rheological characteristics of the products since these polymers exhibited non-Newtonian and pseudoplastic behavior with capability of improving food texture by preventing syneresis (Ayyash et al., 2018; Das et al., 2014; Russo et al., 2016; Wang, Zhao, Tian, He, et al., 2015). The EPS-producing strains KX881772 and KX881779 from camel milk conferred better qualities and health-promoting benefits on low-fat Akawi cheese. The EPS-producing strain Lp90 from wine improved the technological and nutritional characters of oat-based foods. The EPS from L. plantarum SKT109 played an important role in the improvement of the rheology of fermented milk (Wang, Zhao, Tian, He, et al., 2015). The glucan produced by L. plantarum DM5 had shear thinning effect and it was considered potentially applicable as a food additive.

Table 2 Bioactivities of the EPSs from L. plantarum. Bioactivity Antioxidant and antitumor human liver cancer HepG-2 human gastric cancer BGC-823 human cervical cancer HeLa breast adenocarcinoma MCF-7 MiaPaCa2-pancreatic cancer colon cancer Caco-2 colon cancer HT-29 Antioxidant Nontoxic biocompatible nature Inhibitory on the biofilms formation Immunomodulatory Biosorption of Pb (II) Cholesterol-reducing Gut microbiota regulating

Strain

70810 70810 DM5 MTCC 9510 RJF4 ZDY2013, C88 70810, YW32, YW11 LP6, YML009, BR2 MTCC 9510, DM5, BR2 YW32 MTCC 9510, CGMCC No. 5222, WCFS1, C88 70810 RJF4, KF5, BR2 YW11

EPS strong enough to withstand the processing procedures in food industry (Wang, Zhao, Tian, Yang, et al., 2015; Wang, Zhao, Yang, et al., 2015). Most EPSs produced by L. plantarum were reported with good thermostability (Table 1). The EPS from L. plantarum RJF4 could tolerate temperature of 225 °C. The degradation temperature of L. plantarum KF5, YW11 and YW32 EPSs were about 280 °C. Remarkably, the EPSs from L. plantarum BC-25 and 70810 had ability to tolerate temperatures up to 320 °C and 339 °C, respectively. These highly heatstable EPSs could be exploited for application in foods processed at high temperatures, e.g. fried foods. 3. Functional properties of EPSs from L. plantarum EPSs from L. plantarum exhibited various bioactivities such as antitumor, antioxidation, immunomodulation, biocompatibility and inhibition on the formation of biofilms in pathogenic bacteria (Table 2). These health-promoting properties of the EPSs enabled exploitation of their application in different functional foods, cosmeceuticals, and pharmaceuticals. Peroxide was reported to be closely related to the malignancies and metastasis of tumor cells (Badel et al., 2011). Antioxidant EPSs often possessed antitumor activity. Eight stains of L. plantarum (70810, YW32, YW11, DM5, MTCC 9510, RJF4, ZDY2013, C88) had both antitumor and antioxidant activities (Table 2). However, the EPSs from L. plantarum strains LP6, BR2 and YML009 were found with antioxidant activity (Li, Jin, Meng, Gao, & Lu, 2013; Sasikumar, Kozhummal Vaikkath, Devendra, & Nampoothiri, 2017; Seo, Bajpai, Rather, & Park, 2015). The EPSs from L. plantarum strains 70810, YW32, YW11, LP6, YML009 were effective on colonic cancer. The EPS from L. plantarum MTCC 9510 exhibited suppression on breast adenocarcinoma cell line (MCF-7) and stimulation on proliferation of human lymphocytes. L. plantarum 70810 produced two EPSs, the cell bound EPS having in vitro antitumor activity against HepG-2 (liver cancer), BGC-823 (gastric cancer) and HT-29 (colon cancer) cells, but the released EPS having both antioxidant and antitumor activities. The EPS from L. plantarum DM5 was shown to be of nontoxic biocompatible nature (human embryonic kidney 293 and human cervical cancer HeLa cell lines), while it had antitumor activities (HeLa cell lines). The EPS from L. plantarum RJF4 was toxic to MiaPaCa2-pancreatic cancer cell line, but not toxic to normal cell line (L6 and L929 fibroblast cells). The biocompatibility of EPSs displayed non-toxic effect on tumor cell lines, suggesting their potential application in drug delivery and tissue engineering. L. plantarum YW11 EPS showed concentration-dependent in vitro antioxidant activity, being higher at 3.0 mg/mL of the EPS and lower at the low dose (0.25 mg/mL) (Zhang et al., 2017). In addition, high doses of EPS (50 mg/kg per day) effectively relieved the oxidative stress in the aging mice with increasing levels of glutathione peroxidase, superoxide

4. Relations between structure and function of EPS produced by L. plantarum Although the EPSs produced by LAB frequently contained the monosaccharide components such as galactose, glucose, rhamnose, etc., they possessed different functional properties due to their structural difference (Salazar et al., 2014). Three EPSs produced by L. helveticus MB2-1 had similar molecular weight distribution and monosaccharide composition (galactose, glucose and mannose), but they exhibited different antioxidant activities probably due to different molar ratios of their component sugars (Li, Ji et al., 2014). Many structural factors were reported likely to affect EPS functions, such as monosaccharide composition, molecular size, glucosidic linkage type, charge, presence of side chains and rigidity of the molecules (Salazar et al., 2014). L. plantarum strains were shown to produce many structurally different EPSs with a variety of functional activities, such as antitumor, antioxidant and antimicrobial activities (Freitas, Alves, & Reis, 2011). However, some EPSs produced by different L. plantarum strains had 232

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horizontal gene transfer, which often encoded virulence factors (van den Nieuwboer, van Hemert, Claassen, & de Vos, 2016). Among the forty-three L. plantarum strains that were whole-genome sequenced, all of them contained cps gene clusters in their chromosome (Fig. 2), and seven of which also contained another cps gene cluster in their plasmids (Fig. 3). The cps gene clusters in all the forty-three L. plantarum chromosomes could be divided into four types (type A-D). Type A referred to the gene clusters from only one strain, L. plantarum WCFS1, with its genome containing the most gene clusters (four) in two regions, gene clusters (cps1A-I to cps3A-J) and cps4 gene cluster (cps4A-J). Type B referred to those from twenty-one strains, which contained three gene clusters in two regions with most cps3 and cps4 gene clusters and a combination gene cluster of cps2. Type C included 6 genomes containing two gene clusters, cps4 gene cluster and others. Type D contained only the cps4 gene cluster in sixteen strains (Fig. 2). The best characterized L. plantarum WCFS1 genome contained four gene clusters in two regions. One region of 49 kb contained three gene clusters (cps1A-I to cps3A-J) separated by transposase genes and it was identified as a genomic life-style island with high variability between L. plantarum strains (Remus et al., 2012). The second region of 14 kb had cps4 gene cluster (cps4A-J), and it was conserved in all L. plantarum strains. Among other twenty-one genomes of the L. plantarum strains (type B) containing 3 gene clusters in 2 regions, 15 of them contained most cps3 and cps4 gene clusters with a combination gene cluster of cps2A-E and cps4F-I, and 6 strains (Lp90, BLS41, LP3, B21, CGMCC 1.557 and TS12) contained most cps3 and cps4 with a combination of cps2A-E and the other associative function genes (Table 4). Interestingly, the framework of the cps gene clusters from L. plantarum ZJ316, LZ206, LZ95, KP, DF and CAUH2 was basically the same, despite of their different origins; strains ZJ316 and LZ95 from infant feces, LZ206 from raw cow milk, KP and DF being the intestinal bacteria from Drosophila melanogaster, and CAUH2 from Sichuan pickled vegetables. L. plantarum TMW1.277 and TMW1.25 (type C) contained only cps2A-E and cps4A-J without cps4C or cps4F, respectively. L. plantarum JBE490 contained integrated cps1, rfb and cps4 gene clusters, and L. plantarum ZS2058 contained integrated cps4 and cps2 (the cps4F replacing cps2F) gene clusters, the same as those of strain WCFS1. Two L. plantarum strains (MF1298 and LZ227) contained cps4 and cps3 gene clusters, and strain MF1298 contained integrated cps4A-J and most cps3. The other 16 strains contained only the cps4 gene cluster (type D), seven of which contained integrated cps4A-J. Presence of cps gene clusters in the plasmids in lactobacilli was less reported. Previous study proposed sixteen gene clusters responsible for EPS-CG11 production in a 26.4-kb region of the pCG1 plasmid of L. paraplantarum BGCG11 (Nikolic et al., 2012; Zivkovic et al., 2015). Among the forty-three L. plantarum strains reported, seven of them were found in this review to possess the cps gene clusters in their plasmids with high variability between the strains, except that all of them had the cps2ABC genes (Table 5, Fig. 3). L. plantarum K25 plasmid 3 (K25p3) had the minimum cps clusters among the seven L. plantarum strains, a region of 8389 bp consisting of eight predicted ORFs (Jiang et al., 2018). L. plantarum TMW1.1623 plasmid 1 (pL11623-1) has eleven predicted ORFs responsible for the EPS production in a region of 13,744 bp. L. plantarum LZ227 plasmid 1 (LZ227p1) had thirteen predicted ORFs responsible for EPS production, which were divided into two gene clusters by a transposase gene in a region of 13,714 bp. L. plantarum C410L1 plasmid 1 (unnamed-1) had eleven predicted ORFs responsible for EPS production, which were divided into three gene clusters by two transposase genes in a region of 12,841 bp. L. plantarum16 plasmid H (Lp16H) had eighteen predicted ORFs responsible for EPS production in a region of 18,907 bp. L. plantarum ZJ316 plasmid 2 (pLP ZJ102) had fifteen predicted ORFs responsible for EPS production in a region of 17,005 bp. L. plantarum HFC8 plasmid 1 (pMK01) had twenty-three predicted ORFs responsible for EPS production, which were divided into three gene clusters by two transposase genes in the longest region of 23,786 bp among the L. plantarum strains.

similar function, probably due to their similar physicochemical characteristics (Freitas et al., 2011; Wang, Khoo et al., 2002). Some EPSs with stronger antitumor activity had relatively low molecular weight, composition of galactose and glucosans, or the main chain with (1 → 4)galactose, (1 → 3)-glucose or α-(1 → 4)-mannan (Freitas et al., 2011; Wang, Khoo et al., 2002). EPSs having negative charge (phosphate or sulfate), glucose and/or galactose in its composition, and/or low molecular weight were shown to act as mild stimulators of immune cells, whereas those non-charged polymers with higher molecular weight presented a suppressive profile (Caggianiello, Kleerebezem, & Spano, 2016; Yasuda, Serata, & Sako, 2008). L. plantarum WCFS1 EPS having more negative charge (galacturonic-acid) and smaller molecular weight was able to stimulate immune cells. Compared with the natural high molecular weight EPS, the relatively lower molecular weight EPS from the gene knockout strain of L. plantarum WCFS1 was more effective in inducing cytokines, such as TNF-α, IL-12, IL-10 and IL-6 (Remus et al., 2012). The bacterial β-glucan that was immunomodulatory had a common structure of branched (1 → 3)-β-D-glucose substituted at C2 with β-D-glucose. EPSs from LAB containing α-(1 → 3) and α-(1 → 6) linkages in the main chain and α-(1 → 3) linkages in the branched chains were found to exhibit good thickening and stabilizing effects (Zannini, Waters, Coffey, & Arendt, 2016). Primary presence of α-(1 → 6) linkages (86.5%) in the main chain of glucan with 13.5% α-(1 → 3) branched linkages in the EPS from L. plantarum DM5 was important for improvement of its water absorption capacity. The EPS from L. plantarum MTCC 9510 had a trisaccharide repeating unit of glucose and mannose with α-(1 → 3)-linkages and a possibility of β-(1 → 3)-linkages at the terminal, exhibiting good texturing properties. In addition, the EPS from L. plantarum DM5 exhibited a typical non-Newtonian pseudo-plastic behavior, which might be related to its porous structure, and this EPS was suggested to be a potentially good thickening and gelling agent in dairy and baking industries. 5. Genomic analysis of EPS biosynthesis gene cluster in L. plantarum Bacterial surface-associated polysaccharides were involved in interactions of cells with their environment, and played an important role in communication between bacteria and their host (Badel et al., 2011). However, the genetic aspects of the EPS biosynthesis from probiotic LAB were far less well described (Badel et al., 2011; Zannini et al., 2016). Improved knowledge on biosynthesis of these molecules was of great importance for understanding the strain-specific and proposed beneficial modes of probiotic action (Hidalgo-Cantabrana et al., 2014). Within the Lactobacillus genus, EPS biosynthesis genes were identified in different species such as L. helveticus, L. delbrueckii subsp. bulgaricus, L. rhamnosus, L. paraplantarum and L. plantarum (Zannini et al., 2016). The complete genome sequencing of up to forty-three L. plantarum strains to date enabled further understanding of the gene clusters related to EPS biosynthesis. The EPS biosynthesis genes were designated as cps genes of L. plantarum with surface-associated polysaccharide (Remus et al., 2012). In addition to the already reported cps gene clusters in the seven L. plantarum strains (WCFS1, ZJ316, ST-III, NC8, JDM1, P8, 16) with complete genome sequencing and two L. plantarum strains (SF2A35B and Lp90) with draft genome sequencing (Bron et al., 2016; Lamontanara et al., 2015; Li et al., 2016), the other thirty-six strains were also found in this review to possess cps gene clusters in their genomes (Table 3). The compositions of cps gene clusters varied considerably between strains within the L. plantarum species, mainly because of fitting the different circumstances by the different strains (Li et al., 2016). Despite of this diversity, some strains were shown to possess identical structure of the cps gene cluster due to their similar inhabiting environment through horizontal gene transfer (Hidalgo-Cantabrana et al., 2014). The cps gene clusters were often encoded within regions of the genome known as genomic islands or segments of the genome prone to 233

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Table 3 Information of complete whole genome sequencing of L. plantarum strains. Strain

Assembly

Size (Mb)

GC%

Protein

Gene

Plsm Number

Source of isolation

DF KP ZJ316 LZ227 DOMLa NCU116 JDM1 CLP0611 LY-78 LPL-1 MF1298 Zhang-LL RI-113 5-2 SRCM102022 SRCM100434 HFC8 WCFS1 KLDS1.0391 ST-III BLS41 GB-LP1 JBE245 Plantarum P-8 LZ95 JBE490 LP2 C410L1 LZ206 TMW1.1623 TMW 1.708 TMW 1.277 TMW 1.25 ZS2058 CAUH2 16 TS12 Dm K25 B21 LP3 BDGP2 CGMCC 1.557

GCA_001704335.1 GCA_001704315.1 GCA_000338115.2 GCA_001660025.1 GCA_000604105.1 GCA_001672035.1 GCA_000023085.1 GCA_002024845.1 GCA_001715615.1 GCA_002205775.2 GCA_001880185.1 GCA_001581895.1 GCA_001990145.1 GCA_001278015.1 GCA_002173655.1 GCA_002174195.1 GCA_001302645.1 GCA_000203855.3 – GCA_000148815.2 GCA_002116955.1 GCA_002220815.1 GCA_001596095.1 GCA_000392485.2 GCA_001484005.1 GCA_002109405.1 GCA_002109425.1 GCA_002109425.1 GCA_001659745.1 GCA_002117305.1 GCA_002117285.1 GCA_002117265.1 GCA_002117245.1 GCA_001296095.1 GCA_001617525.1 GCA_000412205.1 GCA_001908455.1 GCA_002220175.1 GCA_003020005.1.1 GCA_000931425.2 GCA_002286275.1 GCA_002290185.1 GCA_001272315.2

3.42 3.42 3.2 3.13 3.2 3.35 3.2 3.2 3.12 3.19 3.24 2.95 3.25 3.24 3.25 3.22 3.07 3.31 2.89 3.25 3.25 3.04 3.26 3.04 3.26 3.19 3.28 3.1 3.21 3.14 3.13 3.1 3.14 3.2 3.25 3.04 3.19 3.33 3.18 3.28 3.26 3.41 3.16

44.4 44.4 44.6 44.7 44.7 44.4 44.7 44.6 44.8 44.6 44.6 44.9 44.6 44.7 44.5 44.6 44.8 44.5 44.7 44.6 44.5 44.9 44.5 44.8 44.6 44.6 44.5 44.8 44.6 44.7 44.7 44.6 44.6 44.7 44.6 44.7 44.6 44.5 44.7 44.5 44.5 44.5 44.6

3204 3184 2894 2819 2914 3066 2904 2922 2824 2901 2956 2650 2987 2991 2988 2947 2766 3013 2669 2975 3004 2729 2993 2749 2982 2858 3011 2805 2843 2749 2702 2699 2738 2916 2975 2784 2808 3062 3221 3021 2911 3148 3083

3402 3384 3110 3060 3088 3264 3084 3094 3011 3090 3121 2860 3176 3148 3167 3097 3036 3124 2891 3143 3158 2930 3177 2964 3168 3126 3185 3014 3192 2933 2908 2906 2944 3077 3155 2949 3111 3253 3365 3170 3036 3379 2893

3 3 3 5 2 0 0 1 1 1 28 0 6 0 3 0 10 3 3 1 5 0 0 7 2 1 0 6 3 4 4 10 8 0 4 10 6 0 6 0 2 4 2

Intestinal bacteria from Drosophila Intestinal bacteria from Drosophila Infant feces Raw cow milk CDC Chinese pickle Commercial probiotic in China Environment Fermented chinese cabbage Fermented fish Fermented meats Fermented rice Fermented salami Fermented soybean, yunnan Food Food Human Gut Human saliva Jiaoke Kimchi Kimchi Korean fermented food Korean traditional Meju koumiss in Inner Mongolia Newborn infant fecal Nuruk, Korean traditional beverage starter Pickles Pit mud of a Chinese flavour liquor-making factory Raw cow milk Raw sausage Raw sausage Raw sausage Raw sausage Sauerkraut Sichuan pickle vegetables Steep water Stinky tofu in Malaysia Drosophila gut Tibet Kefir Vietnamese Fermented Sausage Korea Food Drosophila melanogaster gut Cell culture

6. Functional analysis of EPS biosynthesis genes in L. plantarum genomes

encode all functions required for the capsular polysaccharide formation, while the cluster 1 (cps1A-I) and cluster 3 (cps3A-J) genes were predicted to lack genes encoding a priming glycosyl-transferase. The cluster 2 and cluster 4 genes had a typical structure of a Wzy (polysaccharide polymerase)-dependent polymer gene cluster. The first three genes (cps2ABC and cps4ABC) were tyrosine kinase phosphor-regulatory (polysaccharide chain-length regulators Wzd, Wze, and Wzh). The fourth gene (cps2D, and cps4D) was predicted to encode an UDP-Nacetylglucosamine 4-epimerase catalyzing the interconversion between UDP-N-acetyl-D-glucosamine and UDP-N-acetyl-D-galactosamine. The fifth gene (cps2E and cps4E) was predicted to encode the priming glycosyltransferase catalyzing the transfer of a sugar-1-phosphate from a UDP-sugar to the undecaprenyl-phosphate, the first step in the synthesis of the repeat unit. The cps2FGJ and cps4FGI genes were in high sequence similarity to glycosyltransferase genes, which would then be predicted to be involved in the synthesis of a polysaccharide made up of quatro-saccharide repeat units. The cps2I and cps4J genes were predicted to encode flippase, and cps2H and cps4H to encode polymerase. The cluster 2 and cluster 4 genes seemed to possess similar structure and function, and combination of the first five genes (cps2ABCDE) and the next four genes (cps4FGHI) formed one cluster in most of the fortythree L. plantarum strains. The cps2-like cluster genes of L. plantarum Lp90 and SF2A35B, including the first five genes (cps2ABCDE) similar to those of strain

The functional structure of the cps gene cluster was similar to that of previously known operons for EPS production as it generally contained genes for enzymes responsible for regulation, polymerization, export, assembling repeating units and chain length determination (Remus et al., 2012). The combination analysis of cps gene function of chromosome and plasmid found that the EPSs from the seven stains of L. plantarum (TMW1.1623, LZ227, C410L1, ZJ316, HFC8, 16, K25) might possess biological functions, as indicated below in the detailed functional analysis of the cps cluster genes. The cps gene clusters in L. plantarum WCFS1 were well characterized (Remus et al., 2012). The cluster 1 genes in L. plantarum WCFS1 were in control of molecular mass and monosaccharide composition of the EPS, and deletion of cluster 1 (△cps1A-I) led to a decreasing molar mass of the EPS, and to a complete lack of rhamnose and a reducing relative amount of galactose. The cluster 2 genes affected the monosaccharide compositions, mainly managing galactose in the EPS, and deletion of it (△cps2A-J) reduced the relative abundance of galactose. Deletion of the clusters 3 (△cps3A-J) and cluster 4 (△cps4A-J) genes was found not to significantly affect the monosaccharide composition of the EPS produced. The cluster 2, 3 and 4 genes together governed the yield of the EPS. The cluster 2 (cps2A-J) and cluster 4 (cps4A-J) genes appeared to 234

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Fig. 2. Physical maps of the putative cps clusters from forty-three L. plantarum and L. plantarum Lp90 chromosomes.

Fig. 3. Physical maps of the putative cps clusters from seven L. plantarum plasmids.

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Table 4 Functions of part cps2-like cluster genes. Stain

Gene

Protein

Length (aa)

Function

SRCM100434, SRCM102022, LP2, dm, BDGP2 SRCM100434, LP2, dm, BDGP2 BLS41, LP3

rfbX

RfbX

460

Glycosyl transferase, Membrane protein involved in the export of O-antigen and teichoic acid

hypothetical gene bcsA gtf wzy gtf gtf rfbX act wecB/epsC tagB wzx epsG rfaB och1 wcaA bcsA wzy gtf wcaA tagB/spsB wecB rfaA gtf wzy gtf csaB rfbX mts

hypothetical Gtf Gtf Wzy Gtf Gtf RfbX Act Gtf Gtf Wzx Gtf RfaB OCH1 Gtf Gtf Wzy Gtf WcaA Gtf Gtf RfaA GT-A Wzy Gtf Gtf RfbX Mts

357 281 346 401 356 330 518 388 369 388 481 364 363 239 250 289 415 269 237 386 478 289 276 409 304 390 463 261

TS12

B21, CGMCC 1.557

Lp90

Glycosyl transferase A Glycosyltransferase, Stealth protein CR1, CR2, CR3 polysaccharide polymerase UDP-D-galactose:(glucosyl) lipopolysaccharide-1,6-D-galactosyltransferase Glycosyltransferase, Stealth protein CR1, CR2 Glycosyl transferase acyltransferase UDP-N-acetylglucosamine 2-epimerase (non-hydrolyzing) CDP-glycerol: polyglycerol phosphotransferase flippase Glycosyl transferase Glycosyl transferase Mannosyltransferase OCH1 or related enzyme WcaA, Gtf or PgaC Glycosyl transferase A Polymerase Glycosyl transferase alpha-L-Rha alpha-1,3-L-rhamnosyltransferase CDP-glycerol: polyglycerol phosphotransferase UDP-N-acetylglucosamine 2-epimerase (non-hydrolyzing) Glucose-1-phosphate thymidylyltransferase Glycosyltransferase family A oligosaccharide repeat unit polymerase glycosyltransferase family 2 protein Polysaccharide pyruvyl transferase Polysaccharide biosynthesis protein Putative mannosyltransferase

speculated as a polysaccharide biosynthesis protein of unknown function. Cps3F and cps3G might be wzy homologue genes, and cps3E shared distant homology with the N-terminus of a Wzc tyrosine kinase (YP_001221462), which might have the capacity of chain length regulation. Cps3I coding an O-acetyltransferase indicated that acetylation of the repeating units might take place. As in L. rhamnosus, the EPS synthesis gene cluster in L. plantarum also contained no priming glycosyltransferase gene (Nadkarni, Chen, Wilkins, & Hunter, 2014). The priming glycosyltransferase genes in L. rhamnosus were separated from the body of the polysaccharide gene cluster. The downstream of the cluster 3 in L. plantarum WCFS1 had a polysaccharide polymerase-like (lp_1231) and priming glycosyltransferase (lp_1233) gene, which could complete the polysaccharide synthesis machinery of cps3. The organization of the cluster 1 and cluster 3 genes seemed to be different from that of clusters 2 and cluster 4 in L. plantarum WCFS1. Expression of the L. plantarum WCFS1 genes was predicted to produce a polysaccharide comprising of acetylated hexasaccharide repeating unit. The cps1 genes were in a transcriptional unit of glf-cps1A-I-rmlA-glycosylhydrolase-rmlCBD. There were five predicted glycosyltransferase genes (cps1A, cps1B, cps1D, cps1G and cps1H) and an acetyltransferase gene (cps1E), particularly, cps1H as the only rhamnosyltransferase found in all the cps clusters, relating to rhamnose in the surface polysaccharide of L. plantarum WCFS1. Cps1C flippase (Wzx) was an oligosaccharide transporter. The Cps1F regulated the chain length of

WCFS1, were possibly responsible for ropy phenotype and main yield of EPS (Lee, Caggianiello et al., 2016). The cps2-like cluster genes of L. plantarum TS12, CGMCC 1.557 and B21 genomes possibly endowed them with specific traits (Table 4). The EPS biosynthesis gene cluster of L. plantarum TS12 contained a gene coding a flippase, which participated in the membrane translocation of lipopolysaccharides including those containing O-antigens, or assisted in translocating a precursor of murein to act as a lipid II flippase. The wcaA in L. plantarum TS12 coded a protein containing PgaC_IcaA domain (5–93 aa) and poly-β-1,6Nacetyl-D-glucosamine synthase. Members of this protein family include biofilm-forming enzymes that polymerize N-acetyl-D-glucosamine residues with β-(1,6) linkage. In Staphylococcus epidermis, IcaA (intercellular adhesin protein A) was one of the enzymes that acted in polysaccharide intercellular adhesin (PIA) biosynthesis with aid of subunit IcaD (Castelani, Pilon, Martins, Pozzi, & Arcaro, 2015). L. plantarum CGMCC 1.557 and B21 also possessed a bcsA gene encoding BcsA (6–212 aa) domain, a catalytic subunit of cellulose synthase and poly-β-1,6-N-acetylglucosamine synthase (Castiblanco & Sundin, 2016). The cluster 3 genes in L. plantarum WCFS1 were predicted to be involved in synthesis of a polysaccharide made up of acetylated quatrosaccharide repeating units, with three predicted glycosyltransferase genes (cps3AB and cps3J). Cps3C acted as a glf gene coding an UDPgalactopyranose mutase, an enzyme catalyzing conversion of UDP-Dgalactopyranose into UDP-D-galacto-1,4-furanose. Cps3D was Table 5 Basic information of plasmids containing cps gene cluster in L. plantarum. Stain

Plam Name

RefSeq

INSDC

Size (bp)

GC%

Protein

Gene

ZJ316 TMW 1.1623 K25 C410L1 LZ227 16 HFC8

pLP-ZJ102 pL11623-1 K25p3 unnamed1 LZ227p1 Lp16H pMK01

NC_021904.1 NZ_CP017380.1 NZ CP020096.1 NZ_CP017955.1 NZ_CP015858.1 NC_021519.1 NZ_CP012657.1

CP006248.1 CP017380.1 CP020096.1 CP017955.1 CP015858.1 CP006041.1 CP012657.1

39,116 42,745 47,145 73,978 74,177 74,078 84,759

38.7 38.2 40.1 41.5 41.3 41.5 41.0

35 43 41 69 70 72 81

45 50 54 83 83 76 93

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predicted priming glycosyltransferase genes were separated by a tnp gene encoding an IS30 family transposase, which was an integrase core domain, mediating integration of a DNA copy of the viral genome into the host chromosome (Cuecas, Kanoksilapatham, & Gonzalez, 2017). Putative transposases and insertion elements were present, indicating that possible horizontal gene transfers and homologous recombination events contributed to the ubiquity and diversity of EPSs produced by bacteria. In this gene cluster, there were other eight glycosyltransferases, including three partly Rhamnan synthesis F (RgpF), one GDP-mannose-dependent α-(1–6)-phosphatidylinositol monomannoside mannosyltransferase pimB_2 (with 58% identify of Clostridium sp. FS41 pimB_2). The RgpF family consisted of a group of proteins related to the rhamnose-glucose polysaccharide (RgpF) assembly protein from Streptococcus (Shibata, Yamashita, Ozaki, Nakano, & Koga, 2002). There was a WcaF gene coding an acetyltransferase, which was a colanic acid biosynthesis acetyltransferase with 50% identity of that from S. gallolyticus subsp. gallolyticus DSM 16831. This acetyltransferase was believed to catalyze addition of an acetyl group attached through an Olinkage to the first fucosyl residue of the colanic acid repetitive unit (E unit) (Stevenson, Andrianopoulos, Hobbs, & Reeves, 1996). This protein belonged to the transferase hexapeptide repeat family with trimeric LpxA-like domain and Hexapeptide repeat. Many bacterial transferases contained a LpxA-like domain. A variety of bacterial transferases contained a repeat structure composed of tandem repeats of a [LIV]-G-X(4) hexapeptide, which, in the tertiary structure of LpxA (UDP N-acetylglucosamine acyltransferase) (Raetz & Roderick, 1995), was shown to form a left-handed parallel β helix. A number of different transferase protein families contained this repeat, such as galactoside acetyltransferase (GAT, LacA, EC:2.3.1.18) from E. coli, a gene product of the lac operon that might assist cellular detoxification (Wang, Olsen, & Roderick, 2002). Next to the WcaF was a predicted epimerase. This portended that the EPS from L. plantarum HFC8 might be acetylated with unique physiological functions such as antitumor, antioxidation or immunoenhancement. The cps gene cluster in L. plantarum 16 plasmid H (Lp16H) contained eighteen genes (Fig. 3). The first four genes were rfbA, rfbC, rfbB, and rfbD. The next two predicted priming glycosyltransferase genes were separated by three cps2ABC genes. The tenth gene coded a rhamnosyltransferase, and the next one was an epimerase. The twelfth gene encoded a glucosyltransferase, and the next one was an acetyltransferase, following with two glycosyltransferases. The flippase and polysaccharide polymerase genes located in the upstream of the last one UDP-glucose dehydrogenase gene (ugd), which belonged to the UDPglucose/GDP-mannose dehydrogenase family, catalyzing formation of UDP-glucuronate from UDP-glucose. Hence the EPS produced by L. plantarum HFC8 might contain rhamnose, glucose and acetylated glucuronate. The cps gene cluster region in L. plantarum K25 plas3 contained the minimum genes (eight). The next cps2ABC was a tnp gene encoding an IS30 family transposase with 98% identity at the amino acid level of that from L. plantarum. The insertion of an IS3 element upstream from a gene could lead to enhanced transcription of that gene due to a “Pribnow box” (CAATTT) and a -35 region (TTGGTC) toward the end of insF, which effectively made IS3 a mobile promoter in E. coli (Mandal, Collie, Srivastava, Kauffmann, & Huc, 2016). The last four genes were rfbA, rfbC, rfbB, and rfbD. This cluster and the nearby genes contained no predicted priming glycosyltransferase, flippase and polysaccharide polymerase, and the complete polysaccharide synthesis machinery in this strain needed to be studied further. The cps gene cluster in L. plantarum ZJ316 pLP-ZJ102 contained fifteen genes (Fig. 3). The first three genes were cps2ABC and the next gene encoded an UDP-glucose 4-epimerase. The cps9E protein contained CoA_binding_3 (64–236 aa), RmlD_sub_bind (279–476 aa) and CapD-like (282–483 aa) domains. The CapD protein (mannosyl-transferase) from S. aureus was required for biosynthesis of type 1 capsular polysaccharide (Li, Ulm et al., 2014). The next one was a galactosyl

polysaccharide, and Cps1I acted as the polysaccharide polymerase. The cluster 1 contained no priming glycosyltransferase gene, but the upstream glf gene (lp_1176) of cps1A coded a UDP-galactopyranose mutase as the priming glycosyltransferase. There were four genes (rmlA–rmlD) and a glycosylhydrolase gene (lp_1187) in downstream region of the cps1 cluster. The rml gene products included glucose-1phosphate thymidylyltransferase (also known as dTDP-glucose pyrophosphorylase), dTDP-D-glucose-4,6-dehydratase, dTDP-6-deoxy-Dxylo-4-hexulose-3,5-epimerase and dTDP-6-deoxy-L-lyxo-4- hexulosereductase. These four rml genes were not generally contained in the SPS biosynthesis gene clusters of LAB, but in the cps clusters. For the EPS from L. rhamnosus LRHMDP2 and LRHMDP3, one out of four monosaccharides of the repeat unit was rhamnose, suggesting importance of this monosaccharide for EPS structure (Nadkarni et al., 2014). The transcription of L. rhamnosus rml genes could be controlled by two different promoters that had bifunctions, i.e. formation of the cell wall polysaccharides and biosynthesis of rhamnose precursor (Nadkarni et al., 2014). The rml genes might be of significance for capsule polysaccharide synthesis and regulation of monosaccharide composition in the EPS from L. plantarum. For example, both L. plantarum WCFS1 and L. plantarum JBE490 contained cluster 1 genes without the glf and rmlAD genes. 7. Functional analysis of EPS biosynthesis genes in L. plantarum plasmids The cps gene cluster region in the plasmids from the seven L. plantarum strains all contained cps2ABC genes (Fig. 3). The organization of the cps2ABC gene clusters was similar to that of the cps2A-J gene cluster in L. plantarum genomes, and their coding proteins were conserved with the lengths and sequences. The cps2ABC coding proteins possessed high identity at the amino acid level with the proteins coded by the cps gene cluster 2 from L. plantarum WCFS1 (96.44%, 95.36% and 93.95%, respectively). They were also the cps2-like cluster, possibly responsible for ropy phenotype and main yield of EPS, interacting with intestinal tract epithelium, bacterial surface traits, and stress resistance in accordance with L. plantarum Lp90 and SF2A35B (Lee, Caggianiello et al., 2016). Cps2A was predicted to encode a protein for determination of the EPS whole chain length and export. The cps2B gene product was a member of the family of the chain length determinant protein-tyrosine kinase (EpsG) with C-terminal tyrosine residues (likely to be autophosphorylation sites). The cps2C product was a capsular polysaccharide biosynthesis protein phosphotyrosine-protein phosphatase Cps2C from L. plantarum (or named CapC from L. paraplantarum (Nikolic et al., 2012)). Therefore, the products of cps2B and cps2C might be part of the phosphorylation complex. This was likely due to the fact that the cps gene clusters in plasmids connected with the cps gene cluster 2 in L. plantarum genomes. Because of Cps2B protein containing a Mrp domain (22–220 aa), this protein might function as a chromosome partitioning ATPase, and might play a role in gene expression of the chromosome (Morino, Natsui, Swartz, Krulwich, & Ito, 2008). The next highly conserved genes were four genes of rfbA, rfbC, rfbB and rfbD from L. plantarum Lp16H, pMK01 and K25p3, which encoded proteins with high identity (more than 90%) to the products of rfbABCD that located after the cluster 1 in the L. plantarum WCFS1 genome. The identity values for rfbA and rfbD were 90% and 96%, respectively, and those of rfbB and rfbC more than 99%. In the cps gene clusters, five (LZ227p1, Lp16H, pMK01, pL11623-1and pLP-ZJ102) out of the seven plasmids contained both predicted polysaccharide polymerase (Wzy) and flippase (Wzx) with similar length, but low identity (14% and 32%, respectively). The cps gene cluster in L. plantarum HFC8 pMK01 (from human gut) containing twenty-three genes was the maximal one among the seven L. plantarum plasmids (Fig. 3). The first three genes were cps2ABC and the next four genes were rfbA, rfbC, rfbB and rfbD. Then the following two 237

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one was UDP-N-acetylglucosamine (NacGluT). Hence the polysaccharide from L. plantarum C410L1 might contain UDP-N-acetylglucosamine and peptidoglycan. The cps gene cluster in L. plantarum TMW1.1623 pL11623-1 contained eleven genes. The first two genes encoded glycosyltransferases and the last three genes were cps2ABC. The third gene encoded a β-1,3glucosyltransferase (GluT) and the following one was a Wzy. The seventh gene encoded a nitroreductase (Ntr), which played an important role in activation and detoxification of drugs (Chalansonnet, Mercier, Orenga, & Gilbert, 2017). The Ntr and related oxidoreductases family utilized FMN as a cofactor and were often found to be homodimers, and such enzymes included oxygen-insensitive NAD(P)H nitroreductase (FMN-dependent nitroreductase), dihydropteridine reductase (EC:1.6.99.7) and NADH dehydrogenase (EC:1.6.99.3). A number of the proteins were described as oxidoreductases. They were primarily found in bacterial lineages though a number of eukaryotic homologues were identified: Caenorhabditis elegans P34273, Drosophila melanogaster (Fruit fly), Mus musculus (Mouse) and Homo sapiens (Human). Therefore, the EPS from L. plantarum TMW1.1623 might contain amino acid residue and nitrate, indicating possible immunoregulatory activity of the EPS. Many EPSs from LAB that were effective in immunoregulation contained active groups (Lee et al., 2011), such as phosphate groups, amino acid residue and sulfuric acid groups (Freitas et al., 2011).

transferase (GalT). The seventh gene also encoded an UDP-glucose 4epimerase, and this gene was followed by three genes for glycosyl transferase. The orf11 gene encoded hypothetical protein without known function, and the following gene encoded the Wzx (polysaccharide transporter). The hypothetical wzy encoded an analogous CDP-glycerol: poly (glycerophosphate) glycerophosphotransferase (TagF), which was responsible for the polymerisation of the main chain of the teichoic acid by sequential transfer of glycerol-phosphate units from CDP-glycerol to the linkage unit lipid (Fitzgerald & Foster, 2000). TagD protein represented glycerol-3-phosphate cytidyltransferase, also called CDP-glycerol pyrophosphorylase (Zhang, Manos, Ma, Belas, & Karaolis, 2004), which was a closely related human ethanolaminephosphate cytidylyltransferase (EC:2.7.7.14) assigned with a different function experimentally. Glycerol-3-phosphate cytidyltransferase acted in pathways of teichoic acid biosynthesis. Teichoic acids were substituted polymers, linked by phosphodiester bonds, of glycerol, ribitol, etc. The last gene might encode an acetyltransferase with a LpxA-like domain. L. plantarum ZJ316 was reported to inhibit the food-borne pathogenic gram-negative bacteria Salmonella spp., and this strain might be useful as a natural preservative candidate to improve pig growth and pork quality (Li et al., 2016). The cps gene cluster in L. plantarum LZ227p1 contained fourteen genes. The first three genes were cps2ABC and the next gene encoded a partial multidrug MFS transporter. There were six glycosyltransferases or related enzymes, and one Wzy and Wzx. The thirteenth gene was a pvg, which encoded a putative polysaccharide pyruvyltransferase. Pyruvyl-transferases were involved in peptidoglycan-associated polymer biosynthesis, such as CsaB in B. anthracis and WcaK in E. coli that were involved in the biosynthesis of colanic acid and AmsJ in Erwinia amylovora (Becker et al., 1993; Mesnage et al., 2000). This indicated that the EPS from L. plantarum LZ227 might contain amino acid residue. The cps gene cluster in L. plantarum C410L1 unnamed1 contained twelve genes. The first tarJ gene encoded aribulose-5-phosphate reductase with the zinc ion as cofactor, which catalyzed the NADPH dependent reduction of D-ribulose 5-phosphate to D-ribitol 5-phosphate. The next tarI encoded aribitol-5-phosphate cytidylyltransferase that catalyzed transfer of the cytidylyl group from CTP to D-ribitol 5-phosphate to form CDP-ribitol (Nikolic et al., 2012). This protein was also named IspD (2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase), which catalyzed formation of 4-diphosphocytidyl-2-C-methyl-D-erythritol from CTP and 2-C-methyl-D-erythritol 4-phosphate (MEP) in the deoxyxylulose pathway of isopentenyl diphosphate (IPP) biosynthesis (Nikolic et al., 2012). This mevalonate independent pathway that utilized pyruvate and glyceraldehydes 3-phosphate as starting materials for production of IPP occurred in a variety of bacteria, archaea and plant cells, but it was absent in mammals. The isoprenoid pathway was a well-known target for anti-infective drug development (Masini & Hirsch, 2014). The TarJ and TarI/IspD proteins were predicted to be involved in the pathway poly (ribitol phosphate) teichoic acid biosynthesis, which was part of cell wall biogenesis. The tarI/ispD gene encoded a protein with 100% amino acids identity of orf17 from L. paraplantarum CG11. This orf17 gene encoded a protein thought to be involved in UDP-N-acetylglucosamine and peptidoglycan biosynthesis, and thus it might be involved in the biosynthesis and degradation of surface polysaccharides. The third gene encoded transposase, belonging to IS30 family transposase. The next three genes were cps2ABC and the following one encoded VanZ protein. This protein had a conserved sequence region found in the VanZ protein and also in several phosphortransbutyrylases. VanZ conferred low-level resistance to the glycopeptide antibiotic teicoplanin. Analysis of cytoplasmic peptidoglycan precursors, accumulated in the presence of ramoplanin, showed that VanZ-mediated teicoplanin resistance did not involve in the corporation of a substituent of D-alanine into the peptidoglycan precursors (Lai et al., 2017). The eighth gene encoded transposase, belonging to IS256 family. The next five genes encoded glycosyltransferases, and the last

8. Conclusions The EPS-producing L. plantarum strains could be isolated from various sources, such as human gut, D. melanogaster gut and different fermented foods. These polysaccharides generally had molecular weights of 104–106 Da, with the average size of 105 Da. Galactose and glucose were the most frequent monosaccharide components in the EPSs from L. plantarum. Some L. plantarum strains produced homopolysaccharides, e.g. galactan from L. plantarum 70810, and glucan from L. plantarum DM5. Many other L. plantarum strains produced heteropolysaccharides with oligosaccharide repeating units containing different monosaccharides, e.g. fructose, arabinose, galactose, glucose, mannose and maltose in the EPS from L. plantarum NTU 102. The EPSs from L. plantarum were shown with health-promoting properties such as antitumor, antioxidation, immunomodulation and biocompatibility, and they were beneficial in the treatment of diabetes, inhibition of pathogens and improvement of food functions. The physiological functions of the EPSs were related to their molecular characteristics. Many bioactive EPSs were found with lower molecular weights (∼104 Da), negative charge (e.g. galacturonic-acid), and branched chains, e.g. with α-(1 → 3) linkages. Presence of α-1,3-linkages in the EPS molecules was found to be beneficial in the improvement of the texture of the dairy products. Modification of the EPSs, e.g. by acetylation, phosphorylation and carboxymethylation, could improve their bioactivities. The EPSs containing acetyl, phosphoryl groups or amino acid residues might possess specific physiological functions such as antitumor, antioxidation or immunoenhancement. On the basis of the complete genome sequences from forty-three L. plantarum strains reported to date, the organization patterns of the cps gene clusters could be divided into four types. Type A had four gene clusters, e.g. those from L. plantarum WCFS1. Type B had three gene clusters in two regions with mostly cps3 and cps4, and a combination gene cluster of cps2. Type C had two gene clusters, cps4 and others. Type D had only the cps4 gene cluster. Additionally, seven L. plantarum strains had cps2-like gene clusters in the plasmids, which contained highly conserved cps2ABC genes. Functional prediction of the cps genes indicated presence of acetyltransferase genes such as cps1E, cps3I, cps2D, and cps4D, suggesting possible physiological functions of the EPSs from L. plantarum. Furthermore, there were also genes in the plasmids of the seven L. plantarum strains, which were predicted to code acetyltransferase, phosphate, amino acid residue or nitrate related 238

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enzymes. For example, Lp16H had one UDP-glucose dehydrogenase (Ugd), and pLP-ZJ102 had a CDP-glycerol pyrophosphorylase (TagD). LZ227p1 had a Pvg involving in peptidoglycan biosynthesis. TarI in unnamed1 (L. plantarum C410L1) encoded aribitol-5-phosphate cytidylyltransferase, a well-known target for anti-infective drug development. The cps gene cluster in pL11623-1 had one Pvg and nitroreductase, which played an important role in the activation and detoxification of drugs. However, functional determination of the genes in the cps clusters of L. plantarum strains is still limited. There is no research on interaction and regulation between the cps genes in the chromosomes and plasmids in L. plantarum, though transcriptional regulators for EPS biosynthesis probably locate at the chromosome, for instance lp_2714 from L. plantarum WCFS1 encoding an EAL-domain protein (Brown, Marchesi, & Morby, 2011). In the future, more studies on regulatory mechanism and connecting genetic information with the physicochemical and functional characteristics of the EPS are required to gain deeper insight into the EPS biosynthesis in LAB and EPS functions.

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