Identification of Genes Required for Glucan Exopolysaccharide Production in Lactobacillus johnsonii Suggests a Novel Biosynthesis Mechanism.

Lactobacillus johnsonii FI9785 makes two capsular exopolysaccharides-a heteropolysaccharide (EPS2) encoded by the eps operon and a branched glucan homopolysaccharide (EPS1). The homopolysaccharide is synthesized in the absence of sucrose, and there are no typical glucansucrase genes in the genome. Quantitative proteomics was used to compare the wild type to a mutant where EPS production was reduced to attempt to identify proteins associated with EPS1 biosynthesis. A putative bactoprenol glycosyltransferase, FI9785_242 (242), was less abundant in the Δeps_cluster mutant strain than in the wild type. Nuclear magnetic resonance (NMR) analysis of isolated EPS showed that deletion of the FI9785_242 gene (242) prevented the accumulation of EPS1, without affecting EPS2 synthesis, while plasmid complementation restored EPS1 production. The deletion of 242 also produced a slow-growth phenotype, which could be rescued by complementation. 242 shows amino acid homology to bactoprenol glycosyltransferase GtrB, involved in O-antigen glycosylation, while in silico analysis of the neighboring gene 241 suggested that it encodes a putative flippase with homology to the GtrA superfamily. Deletion of 241 also prevented production of EPS1 and again caused a slow-growth phenotype, while plasmid complementation reinstated EPS1 synthesis. Both genes are highly conserved in L. johnsonii strains isolated from different environments. These results suggest that there may be a novel mechanism for homopolysaccharide synthesis in the Gram-positive L. johnsoniiIMPORTANCE Exopolysaccharides are key components of the surfaces of their bacterial producers, contributing to protection, microbial and host interactions, and even virulence. They also have significant applications in industry, and understanding their biosynthetic mechanisms may allow improved production of novel and valuable polymers. Four categories of bacterial exopolysaccharide biosynthesis have been described in detail, but novel enzymes and glycosylation mechanisms are still being described. Our findings that a putative bactoprenol glycosyltransferase and flippase are essential to homopolysaccharide biosynthesis in Lactobacillus johnsonii FI9785 indicate that there may be an alternative mechanism of glucan biosynthesis to the glucansucrase pathway. Disturbance of this synthesis leads to a slow-growth phenotype. Further elucidation of this biosynthesis may give insight into exopolysaccharide production and its impact on the bacterial cell.

Production of multiple bacteriocins, including the novel bacteriocin gassericin M, by Lactobacillus gasseri LM19, a strain isolated from human milk.

Bacteriocins are antimicrobial peptides produced by bacteria, and their production is regarded as a desirable probiotic trait. We found that Lactobacillus gasseri LM19, a strain isolated from human milk, produces several bacteriocins, including a novel bacteriocin, gassericin M. These bacteriocins were purified from culture and synthesised to investigate their activity and potential synergy. L. gasseri LM19 was tested in a complex environment mimicking human colon conditions; it not only survived, but expressed the seven bacteriocin genes and produced short-chain fatty acids. Metagenomic analysis of these in vitro colon cultures showed that co-inoculation of L. gasseri LM19 with Clostridium perfringens gave 16S ribosomal RNA metagenomic profiles with more similarity to controls than to vessels inoculated with C. perfringens alone. These results indicate that L. gasseri LM19 could be an interesting candidate for maintaining homeostasis in the gut environment.

Serine-rich repeat protein adhesins from Lactobacillus reuteri display strain specific glycosylation profiles.

Lactobacillus reuteri is a gut symbiont inhabiting the gastrointestinal tract of numerous vertebrates. The surface-exposed serine-rich repeat protein (SRRP) is a major adhesin in Gram-positive bacteria. Using lectin and sugar nucleotide profiling of wild-type or L. reuteri isogenic mutants, MALDI-ToF-MS, LC-MS and GC-MS analyses of SRRPs, we showed that L. reuteri strains 100-23C (from rodent) and ATCC 53608 (from pig) can perform protein O-glycosylation and modify SRRP100-23 and SRRP53608 with Hex-Glc-GlcNAc and di-GlcNAc moieties, respectively. Furthermore, in vivo glycoengineering in E. coli led to glycosylation of SRRP53608 variants with α-GlcNAc and GlcNAcß(1→6)GlcNAcα moieties. The glycosyltransferases involved in the modification of these adhesins were identified within the SecA2/Y2 accessory secretion system and their sugar nucleotide preference determined by saturation transfer difference NMR spectroscopy and differential scanning fluorimetry. Together, these findings provide novel insights into the cellular O-protein glycosylation pathways of gut commensal bacteria and potential routes for glycoengineering applications.

Acute Consumption of Flavan-3-ol-Enriched Dark Chocolate Affects Human Endogenous Metabolism.

Flavan-3-ols and methylxanthines have potential beneficial effects on human health including reducing cardiovascular risk. We performed a randomized controlled crossover intervention trial to assess the acute effects of consumption of flavan-3-ol-enriched dark chocolate, compared with standard dark chocolate and white chocolate, on the human metabolome. We assessed the metabolome in urine and blood plasma samples collected before and at 2 and 6 h after consumption of chocolates in 42 healthy volunteers using a nontargeted metabolomics approach. Plasma samples were assessed and showed differentiation between time points with no further separation among the three chocolate treatments. Multivariate statistics applied to urine samples could readily separate the postprandial time points and distinguish between the treatments. Most of the markers responsible for the multivariate discrimination between the chocolates were of dietary origin. Interestingly, small but significant level changes were also observed for a subset of endogenous metabolites. 1H NMR revealed that flavan-3-ol-enriched dark chocolate and standard dark chocolate reduced urinary levels of creatinine, lactate, some amino acids, and related degradation products and increased the levels of pyruvate and 4-hydroxyphenylacetate, a phenolic compound of bacterial origin. This study demonstrates that an acute chocolate intervention can significantly affect human metabolism.

Structure and biosynthesis of two exopolysaccharides produced by Lactobacillus johnsonii FI9785.

Exopolysaccharides were isolated and purified from Lactobacillus johnsonii FI9785, which has previously been shown to act as a competitive exclusion agent to control Clostridium perfringens in poultry. Structural analysis by NMR spectroscopy revealed that L. johnsonii FI9785 can produce two types of exopolysaccharide: EPS-1 is a branched dextran with the unusual feature that every backbone residue is substituted with a 2-linked glucose unit, and EPS-2 was shown to have a repeating unit with the following structure: -6)-α-Glcp-(1-3)-ß-Glcp-(1-5)-ß-Galf-(1-6)-α-Glcp-(1-4)-ß-Galp-(1-4)-ß-Glcp-(1-. Sites on both polysaccharides were partially occupied by substituent groups: 1-phosphoglycerol and O-acetyl groups in EPS-1 and a single O-acetyl group in EPS-2. Analysis of a deletion mutant (ΔepsE) lacking the putative priming glycosyltransferase gene located within a predicted eps gene cluster revealed that the mutant could produce EPS-1 but not EPS-2, indicating that epsE is essential for the biosynthesis of EPS-2. Atomic force microscopy confirmed the localization of galactose residues on the exterior of wild type cells and their absence in the ΔepsE mutant. EPS2 was found to adopt a random coil structural conformation. Deletion of the entire 14-kb eps cluster resulted in an acapsular mutant phenotype that was not able to produce either EPS-2 or EPS-1. Alterations in the cell surface properties of the EPS-specific mutants were demonstrated by differences in binding of an anti-wild type L. johnsonii antibody. These findings provide insights into the biosynthesis and structures of novel exopolysaccharides produced by L. johnsonii FI9785, which are likely to play an important role in biofilm formation, protection against harsh environment of the gut, and colonization of the host.

A third mode of surface-associated growth: immobilization of Salmonella enterica serovar Typhimurium modulates the RpoS-directed transcriptional programme.

Although the growth of bacteria has been studied for more than a century, it is only in recent decades that surface-associated growth has received attention. In addition to the well-characterized biofilm and swarming lifestyles, bacteria can also develop as micro-colonies supported by structured environments in both food products and the GI tract. This immobilized mode of growth has not been widely studied. To develop our understanding of the effects of immobilization upon a food-borne bacterial pathogen, we used the IFR Gel Cassette model. The transcriptional programme and metabolomic profile of Salmonella enterica serovar Typhimurium ST4/74 were compared during planktonic and immobilized growth, and a number of immobilization-specific characteristics were identified. Immobilized S.Typhimurium did not express motility and chemotaxis genes, and electron microscopy revealed the absence of flagella. The expression of RpoS-dependent genes and the level of RpoS protein were increased in immobilized bacteria, compared with planktonic growth. Immobilized growth prevented the induction of SPI1, SPI4 and SPI5 gene expression, likely mediated by the FliZ transcriptional regulator. Using an epithelial cell-based assay, we showed that immobilized S.Typhimurium was significantly less invasive than planktonic bacteria, and we suggest that S.Typhimurium grown in immobilized environments are less virulent than planktonic bacteria. Our findings identify immobilization as a third type of surface-associated growth that is distinct from the biofilm and swarming lifestyles of Salmonella.

Prediction of variability in CYP3A4 induction using a combined 1H NMR metabonomics and targeted UPLC-MS approach.

The activity of Cytochrome P450 3A4 (CYP3A4) enzyme is associated with many adverse or poor therapeutic responses to drugs. We used (1)H NMR-based metabonomics to identify a metabolic signature associated with variation in induced CYP3A4 activity. A total of 301 female twins, aged 45--84, participated in this study. Each volunteer was administered a potent inducer of CYP3A4 (St. John's Wort) for 14 days and the activity of CYP3A4 was quantified through the metabolism of the exogenously administered probe drug quinine sulfate (300 mg). Pre- and postintervention fasting urine samples were used to obtain metabolite profiles, using (1)H NMR spectroscopy, and were analyzed using UPLC--MS to obtain a marker for CYP3A4 induction, via the ratio of 3-hydroxyquinine to quinine (3OH-Q:Q). Multiple linear regression was used to build a predictive model for 3OH-Q:Q values based on the preintervention metabolite profiles. A combination of seven metabolites and seven covariates showed a strong (r = 0.62) relationship with log(3OH-Q:Q). This regression model demonstrated significant (p < 0.00001) predictive ability when applied to an independent validation set. Our results highlight the promise of metabonomics for predicting CYP3A4-mediated drug response.

Metabolomics of fecal extracts detects altered metabolic activity of gut microbiota in ulcerative colitis and irritable bowel syndrome.

(1)H NMR spectroscopy of aqueous fecal extracts has been used to investigate differences in metabolic activity of gut microbiota in patients with ulcerative colitis (UC) (n = 13), irritable bowel syndrome (IBS) (n = 10), and healthy controls (C) (n = 22). Up to four samples per individual were collected over 2 years giving a total of 124 samples. Multivariate discriminant analysis, based on NMR data from all three groups, was able to predict UC and C group membership with good sensitivity and specificity; classification of IBS samples was less successful and could not be used for diagnosis. Trends were detected toward increased taurine and cadaverine levels in UC with increased bile acid and decreased branched chain fatty acids in IBS relative to controls; changes in short chain fatty acids and amino acids were not significant. Previous PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of the same fecal material had shown alterations of the gut microbiota when comparing UC and IBS groups with controls. Hierarchical cluster analysis showed that DGGE profiles from the same individual were stable over time, but NMR spectra were more variable; canonical correlation analysis of NMR and DGGE data partly separated the three groups and revealed a correlation between the gut microbiota profile and metabolite composition.

Microbial Diversity and Metabolite Profile of Fermenting Millet in the Production of Hausa koko, a Ghanaian Fermented Cereal Porridge.

Hausa koko is an indigenous porridge processed from millet in Ghana. The process involves fermentation stages, giving the characteristic organoleptic properties of the product that is produced largely at a small-scale household level and sold as a street food. Like many other indigenous foods, quality control is problematic and depends on the skills of the processor. In order to improve the quality of the product and standardize the process for large-scale production, we need a deeper understanding of the microbial processes. The aim of this study is to investigate the microbial community involved in the production of this traditional millet porridge and the metabolites produced during processing. High-throughput amplicon sequencing was used to identify the bacterial (16S rRNA V4 hypervariable region) and fungal [Intergenic Transcribed Spacer (ITS)] communities associated with the fermentation, while nuclear magnetic resonance (NMR) was used for metabolite profiling. The bacterial community diversity was reduced during the fermentation processes with an increase and predominance of lactobacilli. Other dominant bacteria in the fermentation included Pediococcus, Weissella, Lactococcus, Streptococcus, Leuconostoc, and Acetobacter. The species Limosilactobacillus fermentum and Ligilactobacillus salivarius accounted for some of the diversities within and between fermentation time points and processors. The fungal community was dominated by the genus Saccharomyces. Other genera such as Pichia, Candida, Kluyveromyces, Nakaseomyces, Torulaspora, and Cyberlindnera were also classified. The species Saccharomyces cerevisiae, Stachybotrys sansevieriae, Malassezia restricta, Cyberlindnera fabianii, and Kluyveromyces marxianus accounted for some of the diversities within some fermentation time points. The species S. sansevieria and M. restricta may have been reported for the first time in cereal fermentation. This is the most diverse microbial community reported in Hausa koko. In this study, we could identify and quantify 33 key different metabolites produced by the interactions of the microbial communities with the millet, composed of organic compounds, sugars, amino acids and intermediary compounds, and other key fermentation compounds. An increase in the concentration of organic acids in parallel with the reduction of sugars occurred during the fermentation process while an initial increase of amino acids followed by a decrease in later fermentation steps was observed.

Bifidobacterium breve UCC2003 Exopolysaccharide Modulates the Early Life Microbiota by Acting as a Potential Dietary Substrate.

BACKGROUND: Bifidobacterium represents an important early life microbiota member. Specific bifidobacterial components, exopolysaccharides (EPS), positively modulate host responses, with purified EPS also suggested to impact microbe-microbe interactions by acting as a nutrient substrate. Thus, we determined the longitudinal effects of bifidobacterial EPS on microbial communities and metabolite profiles using an infant model colon system. METHODS: Differential gene expression and growth characteristics were determined for each strain; Bifidobacterium breve UCC2003 and corresponding isogenic EPS-deletion mutant (B. breve UCC2003del). Model colon vessels were inoculated with B. breve and microbiome dynamics monitored using 16S rRNA sequencing and metabolomics (NMR). RESULTS: Transcriptomics of EPS mutant vs. B. breve UCC2003 highlighted discrete differential gene expression (e.g., eps biosynthetic cluster), though overall growth dynamics between strains were unaffected. The EPS-positive vessel had significant shifts in microbiome and metabolite profiles until study end (405 h); with increases of Tyzzerella and Faecalibacterium, and short-chain fatty acids, with further correlations between taxa and metabolites which were not observed within the EPS-negative vessel. CONCLUSIONS: These data indicate that B. breve UCC2003 EPS is potentially metabolized by infant microbiota members, leading to differential microbial metabolism and altered metabolite by-products. Overall, these findings may allow development of EPS-specific strategies to promote infant health.

Shall I compare thee to a GM potato?

A fundamental issue in the safety assessment of genetically modified crops is the question of whether unintentional changes have occurred in the crop plant as a consequence of the genetic modification. This question was addressed recently by using a powerful metabolite fingerprinting and metabolite profiling method to assess whether genetically modified potatoes are substantially similar to their corresponding conventional cultivars.

Architectural plasticity in young Eucalyptus marginata on restored bauxite mines and adjacent natural forest in south-western Australia.

The aboveground architecture of Eucalyptus marginata (Jarrah) was investigated in chronosequences of young trees (2.5, 5 and 10 m height) growing in a seasonally dry climate in a natural forest environment with intact soils, and on adjacent restored bauxite mine sites on soils with highly modified A and B horizons above an intact C horizon. Compared to forest trees, trees on restored sites were much younger and faster growing, with straighter, more clearly defined main stems and deeper, narrower crowns containing a greater number of branches that were longer, thinner and more vertically angled. Trees on restored sites also had a higher fraction of biomass in leaves than forest trees, as indicated by 20-25% thicker leaves, 30-70% greater leaf area, 10-30% greater leaf area to sapwood area ratios and 5-30% lesser branch Huber values. Differences in crown architecture and biomass distribution were consistent with putatively greater soil-water, nutrient and light availability on restored sites. Our results demonstrate that under the same climatic conditions, E. marginata displays a high degree of plasticity of aboveground architecture in response to the net effects of resource availability and soil environment. These differences in architecture are likely to have functional consequences in relation to tree hydraulics and growth that, on larger scales, is likely to affect the water and carbon balances of restored forest ecosystems. This study highlights substrate as a significant determinant of tree architecture in water-limited environments. It further suggests that the architecture of young trees on restored sites may need to change again if they are to survive likely longer-term changes in resource availability.

Preparation of gentiobiose-derived oligosaccharides by glucansucrase E81 and determination of prebiotic and immune-modulatory functions.

Gentiobiose-derived oligosaccharides were synthesized by the acceptor reaction of glucansucrase E81 obtained from Lactobacillus reuteri E81 with sucrose and gentiobiose as donor-acceptor sugars, respectively. The reaction products were monitored by TLC analysis and gentiobiose-derived oligosaccharides up to DP 8 were formed during the acceptor reaction as determined by ESI-MS/MS analysis. The glycosylation of the gentiobiose with α-(1 → 6) linkages and α-(1 → 3) linkages was shown by 1H and 13C NMR analysis confirming the structure of these gentiobiose-derived oligosaccharides. The in vitro prebiotic function of the oligosaccharides was determined in which probiotic strains were stimulated whereas no growth was observed in pathogen strains. Gentiobiose-derived oligosaccharides showed immune-modulatory functions in vitro and triggered the production of IL-4, IL12 and TNF-α cytokines in HT29 cells in a dose dependent manner. This study showed the production and functional characterisation of gentiobiose-derived oligosaccharides establishing a promising avenue for future applications.

Structural analysis of the α-d-glucan produced by the sourdough isolate Lactobacillus brevis E25.

Cereal-associated Lactic Acid Bacteria (LAB) are well known for homopolymeric exopolysaccharide (EPS) production. Herein, the structure of an EPS isolated from sourdough isolate Lactobacillus brevis E25 was determined. A modified BHI medium was used for production of EPS-E25 in order to eliminate potential contaminants. Analysis of sugar monomers in EPS revealed that glucose was the only sugar present. Structural characterisation of EPS by NMR and methylation analysis revealed that E25 produced a highly branched α-glucan with (α1→3) and (α1→6) glycosidic linkages, and was similar in structure to a previously reported EPS from Lactobacillus reuteri 180. The 1H and 13C NMR data were contrasted with newly recorded data for known polysaccharides (alternan, commercial dextran) which also contain α-(1,3,6)Glc branch points. It was found in both E25 EPS and alternan that NMR parameters could be used to distinguish glucose residues that had the same substitution pattern but occupied different positions in the structure.

16-O-methylcafestol is present in ground roast Arabica coffees: Implications for authenticity testing.

High-field and low-field proton NMR spectroscopy were used to analyse lipophilic extracts from ground roast coffees. Using a sample preparation method that produced concentrated extracts, a small marker peak at 3.16 ppm was observed in 30 Arabica coffees of assured origin. This signal has previously been believed absent from Arabicas, and has been used as a marker for detecting adulteration with robusta. Via 2D 600 MHz NMR and LC-MS, 16-O-methylcafestol and 16-O-methylkahweol were detected for the first time in Arabica roast coffee and shown to be responsible for the marker peak. Using low-field NMR, robusta in Arabica could be detected at levels of the order of 1-2% w/w. A surveillance study of retail purchased "100% Arabica" coffees found that 6 out of 60 samples displayed the 3.16 ppm marker signal to a degree commensurate with adulteration at levels of 3-30% w/w.

Optimising conditions for bioethanol production from rice husk and rice straw: effects of pre-treatment on liquor composition and fermentation inhibitors.

BACKGROUND: Rice straw and husk are globally significant sources of cellulose-rich biomass and there is great interest in converting them to bioethanol. However, rice husk is reportedly much more recalcitrant than rice straw and produces larger quantities of fermentation inhibitors. The aim of this study was to explore the underlying differences between rice straw and rice husk with reference to the composition of the pre-treatment liquors and their impacts on saccharification and fermentation. This has been carried out by developing quantitative NMR screening methods. RESULTS: Air-dried rice husk and rice straw from the same cultivar were used as substrates. Carbohydrate compositions were similar, whereas lignin contents differed significantly (husk: 35.3% w/w of raw material; straw 22.1% w/w of raw material). Substrates were hydrothermally pre-treated with high-pressure microwave processing across a wide range of severities. 25 compounds were identified from the liquors of both pre-treated rice husk and rice straw. However, the quantities of compounds differed between the two substrates. Fermentation inhibitors such as 5-HMF and 2-FA were highest in husk liquors, and formic acid was higher in straw liquors. At a pre-treatment severity of 3.65, twice as much ethanol was produced from rice straw (14.22% dry weight of substrate) compared with the yield from rice husk (7.55% dry weight of substrate). Above severities of 5, fermentation was inhibited in both straw and husk. In addition to inhibitors, high levels of cellulase-inhibiting xylo-oligomers and xylose were found and at much higher concentrations in rice husk liquor. At low severities, organic acids and related intracellular metabolites were released into the liquor. CONCLUSIONS: Rice husk recalcitrance to saccharification is probably due to the much higher levels of lignin and, from other studies, likely high levels of silica. Therefore, if highly polluting chemical pre-treatments and multi-step biorefining processes are to be avoided, rice husk may need to be improved through selective breeding strategies, although more careful control of pre-treatment may be sufficient to reduce the levels of fermentation inhibitors, e.g. through steam explosion-induced volatilisation. For rice straw, pre-treating at severities of between 3.65 and 4.25 would give a glucose yield of between 37.5 and 40% (w/DW, dry weight of the substrate) close to the theoretical yield of 44.1% w/DW, and an insignificant yield of total inhibitors.

Yeast diversity in relation to the production of fuels and chemicals.

In addition to ethanol, yeasts have the potential to produce many other industrially-relevant chemicals from numerous different carbon sources. However there remains a paucity of information about overall capability across the yeast family tree. Here, 11 diverse species of yeasts with genetic backgrounds representative of different branches of the family tree were investigated. They were compared for their abilities to grow on a range of sugar carbon sources, to produce potential platform chemicals from such substrates and to ferment hydrothermally pretreated rice straw under simultaneous saccharification and fermentation conditions. The yeasts differed considerably in their metabolic capabilities and production of ethanol. A number could produce significant amounts of ethyl acetate, arabinitol, glycerol and acetate in addition to ethanol, including from hitherto unreported carbon sources. They also demonstrated widely differing efficiencies in the fermentation of sugars derived from pre-treated rice straw biomass and differential sensitivities to fermentation inhibitors. A new catabolic property of Rhodotorula mucilaginosa (NCYC 65) was discovered in which sugar substrate is cleaved but the products are not metabolised. We propose that engineering this and some of the other properties discovered in this study and transferring such properties to conventional industrial yeast strains could greatly expand their biotechnological utility.

Low-field (1)H NMR spectroscopy for distinguishing between arabica and robusta ground roast coffees.

This work reports a new screening protocol for addressing issues of coffee authenticity using low-field (60MHz) bench-top (1)H NMR spectroscopy. Using a simple chloroform-based extraction, useful spectra were obtained from the lipophilic fraction of ground roast coffees. It was found that 16-O-methylcafestol (16-OMC, a recognized marker compound for robusta beans) gives rise to an isolated peak in the 60MHz spectrum, which can be used as an indicator of the presence of robusta beans in the sample. A total of 81 extracts from authenticated coffees and mixtures were analysed, from which the detection limit of robusta in arabica was estimated to be between 10% and 20% w/w. Using the established protocol, a surveillance exercise was conducted of 27 retail samples of ground roast coffees which were labelled as "100% arabica". None were found to contain undeclared robusta content above the estimated detection limit.

Membrane-enclosed multienzyme (MEME) synthesis of 2,7-anhydro-sialic acid derivatives.

Naturally occurring 2,7-anhydro-alpha-N-acetylneuraminic acid (2,7-anhydro-Neu5Ac) is a transglycosylation product of bacterial intramolecular trans-sialidases (IT-sialidases). A facile one-pot two-enzyme approach has been established for the synthesis of 2,7-anhydro-sialic acid derivatives including those containing different sialic acid forms such as Neu5Ac and N-glycolylneuraminic acid (Neu5Gc). The approach is based on the use of Ruminoccocus gnavus IT-sialidase for the release of 2,7-anhydro-sialic acid from glycoproteins, and the conversion of free sialic acid by a sialic acid aldolase. This synthetic method, which is based on a membrane-enclosed enzymatic synthesis, can be performed on a preparative scale. Using fetuin as a substrate, high-yield and cost-effective production of 2,7-anhydro-Neu5Ac was obtained to high-purity. This method was also applied to the synthesis of 2,7-anhydro-Neu5Gc. The membrane-enclosed multienzyme (MEME) strategy reported here provides an efficient approach to produce a variety of sialic acid derivatives.

EpsA is an essential gene in exopolysaccharide production in Lactobacillus johnsonii FI9785.

Lactobacillus johnsonii FI9785 has an eps gene cluster which is required for the biosynthesis of homopolymeric exopolysaccharides (EPS)-1 and heteropolymeric EPS-2 as a capsular layer. The first gene of the cluster, epsA, is the putative transcriptional regulator. In this study we showed the crucial role of epsA in EPS biosynthesis by demonstrating that deletion of epsA resulted in complete loss of both EPS-1 and EPS-2 on the cell surface. Plasmid complementation of the epsA gene fully restored EPS production, as confirmed by transmission electron microscopy and nuclear magnetic resonance (NMR) analysis. Furthermore, this complementation resulted in a twofold increase in the expression levels of this gene, which almost doubled amounts of EPS production in comparison with the wild-type strain. Analysis of EPS by NMR showed an increased ratio of the heteropolysaccharide to homopolysaccharide in the complemented strain and allowed identification of the acetylated residue in EPS-2 as the (1,4)-linked ßGlcp unit, with the acetyl group located at O-6. These findings indicate that epsA is a positive regulator of EPS production and that EPS production can be manipulated by altering its expression.