Correlation of activities of the enzymes alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase with exopolysaccharide biosynthesis by Streptococcus thermophilus LY03

The effects of different carbohydrates or mixtures of carbohydrates as substrates on bacterial growth and exopolysaccharide (EPS) production were studied for the yoghurt starter culture Streptococcus thermophilus LY03. This strain produces two heteropolysaccharides with the same monomeric composition (galactose and glucose in the ratio 4:1) but with different molecular masses. Lactose and glucose were fermented by S. thermophilus LY03 only when they were used as sole energy and carbohydrate sources. Fructose was also fermented when it was applied in combination with lactose or glucose. Both the amount of EPS produced and the carbohydrate source consumption rates were clearly influenced by the type of energy and carbohydrate source used, while the EPS monomeric composition remained constant (galactose-glucose, 4:1) under all circumstances. A combination of lactose and glucose resulted in the largest amounts of EPS. Measurements of the activities of enzymes involved in EPS biosynthesis, and of those involved in sugar nucleotide biosynthesis and the Embden-Meyerhof-Parnas pathway, demonstrated that the levels of activity of alpha-phosphoglucomutase, UDP-galactose 4-epimerase, and UDP-glucose pyrophosphorylase are highly correlated with the amount of EPS produced. Furthermore, a weaker relationship or no relationship between the amounts of EPS and the enzymes involved in either the rhamnose nucleotide synthetic branch of the EPS biosynthesis or the pathway leading to glycolysis was observed for S. thermophilus LY03.     

UDP-N-Acetylglucosamine 4-Epimerase Activity Indicates the Presence of N-Acetylgalactosamine in Exopolysaccharides of Streptococcus thermophilus Strains

The monomer composition of the exopolysaccharides (EPS) produced by Streptococcus thermophilus LY03 and S. thermophilus Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and N-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-N-acetylglucosamine 4-epimerase, converting UDP-N-acetylglucosamine into UDP-N-acetylgalactosamine, is responsible for the presence of N-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-N-acetylglucosamine 4-epimerase was higher in both S. thermophilus strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during S. thermophilus Sfi20 fermentations. A correlation between activities of the enzymes α-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for S. thermophilus LY03, although S. thermophilus Sfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain.     

Exopolysaccharide (EPS) biosynthesis by Lactobacillus sakei 0-1: production kinetics, enzyme activities and EPS yields

AIMS: To determine optimal exopolysaccharide (EPS) production conditions of the mesophilic lactic acid bacterium strain Lactobacillus sakei 0-1 and to detect possible links between EPS yields and the activity of relevant enzymes. METHODS AND RESULTS: Fermentation experiments at different temperatures using either glucose or lactose were carried out. EPS production took place during the exponential growth phase. Low temperatures, applying glucose as carbohydrate source, resulted in the best bacterial growth, the highest amounts of EPS and the highest specific EPS production. Activities of 10 important enzymes involved in the EPS biosynthesis and the energy formation of Lact. sakei 0-1 were measured. The obtained results revealed that there is a clear link for some enzymes with EPS biosynthesis. It was also demonstrated clearly that the presence of rhamnose in the EPS building blocks is due to high activities of the enzymes involved in the rhamnose synthetic branch. CONCLUSION: EPS production in Lact. sakei 0-1 is growth-associated and displays primary metabolite kinetics. Glucose as carbohydrate source and low temperatures enhance the EPS production. The enzymes involved in the biosynthesis of the activated sugar nucleotides play a major role in determining the monomeric composition of the synthesized EPS. SIGNIFICANCE AND IMPACT OF THE STUDY: The proposed results contribute to a better understanding of the physiological factors influencing EPS production and the key enzymes involved in EPS biosynthesis by Lact. sakei.     

Requirement for Phosphoglucomutase in Exopolysaccharide Biosynthesis in Glucose- and Lactose-Utilizing Streptococcus thermophilus

To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.     

Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal⁺) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal⁺ strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal⁺ strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal⁺ strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.     

UDP-N-acetylglucosamine 4-epimerase activity indicates the presence of N-acetylgalactosamine in exopolysaccharides of Streptococcus thermophilus strains

The monomer composition of the exopolysaccharides (EPS) produced by Streptococcus thermophilus LY03 and S. thermophilus Sfi20 were evaluated by high-pressure liquid chromatography with amperometric detection and nuclear magnetic resonance spectroscopy. Both strains produced the same EPS composed of galactose, glucose, and N-acetylgalactosamine. Further, it was demonstrated that the activity of the precursor-producing enzyme UDP-N-acetylglucosamine 4-epimerase, converting UDP-N-acetylglucosamine into UDP-N-acetylgalactosamine, is responsible for the presence of N-acetylgalactosamine in the EPS repeating units of both strains. The activity of UDP-N-acetylglucosamine 4-epimerase was higher in both S. thermophilus strains than in a non-EPS-producing control strain. However, the level of this activity was not correlated with EPS yields, a result independent of the carbohydrate source applied in the fermentation process. On the other hand, both the amounts of EPS and the carbohydrate consumption rates were influenced by the type of carbohydrate source used during S. thermophilus Sfi20 fermentations. A correlation between activities of the enzymes alpha-phosphoglucomutase, UDP-glucose pyrophosphorylase, and UDP-galactose 4-epimerase and EPS yields was seen. These experiments confirm earlier observed results for S. thermophilus LY03, although S. thermophilus Sfi20 preferentially consumed glucose for EPS production instead of lactose in contrast to the former strain.     

Exopolysaccharide biosynthesis by Lactobacillus helveticus ATCC 15807

Exopolysaccharide (EPS) production and the activities of the enzymes involved in sugar nucleotide biosynthesis in Lactobacillus helveticus ATCC 15807 under controlled pH conditions were investigated. Batch fermentations using lactose as energy source showed higher EPS synthesis by L. helveticus ATCC 15807 at pH 4.5 with respect to pH 6.2, the enzyme -phosphoglucomutase (-PGM) being correlated with both total and specific EPS production. When glucose was used as carbon source instead of lactose, the lower EPS synthesis obtained was linked to a decrease in -PGM and galactose 1-phosphate-uridyltransferase (GalT) activities, the reduction of the latter being more pronounced. Higher EPS production by L. helveticus ATCC 15807 at the acidic constant pH of 4.5 requires that both -PGM and GalT activities are high. These enzymes are needed to synthesize UDP-glucose and UDP-galactose for supplying the corresponding monomers for EPS biosynthesis. Although differences are observed in EPS production by this strain regarding the energy source (lactose or glucose), the monomeric composition of the polymers produced is independent of the carbohydrate used. The obtained results contribute to a better understanding of the physiological factors that affect EPS biosynthesis by lactobacilli, which could help in the correct handling of the fermentation parameters within the fermented dairy industry.     

Enhanced exopolysaccharide production by metabolic engineering of Streptococcus thermophilus

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal(+)) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal(+) strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal(+) strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal(+) strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.     

Comparative Transcriptome Analysis Reveals That Lactose Acts as an Inducer and Provides Proper Carbon Sources for Enhancing Exopolysaccharide Yield in the Deep-Sea Bacterium Zunongwangia profunda SM-A87

Many marine bacteria secrete exopolysaccharides (EPSs) that have important ecological and physiological functions. Numerous nutritional and environmental factors influence bacterial EPS production. However, the regulatory mechanisms of EPS production are poorly understood. The deep-sea Bacteroidetes bacterium Zunongwangia profunda SM-A87 can produce high quantities of EPS, and its EPS production is enhanced significantly by lactose. Here, we studied the reasons behind the significant advantage that lactose has over other carbon sources in EPS production in SM-A87. RNA-seq technologies were used to study lactose-regulated genes in SM-A87. The expression level of genes within the EPS gene cluster was up-regulated when lactose was added. Supplement of lactose also influenced the expression of genes located outside the EPS gene cluster that are also involved in EPS biosynthesis. The major glycosyl components of SM-A87 EPS are mannose, glucose and galactose. Genomic metabolic pathway analyses showed that the EPS precursor GDP-mannose can be synthesized from glucose, while the precursor UDP-glucose must be synthesized from galactose. Lactose can provide glucose and galactose simultaneously and prevent glucose inhibition. Lactose can also greatly stimulate the growth of SM-A87. Taken together, lactose acts not only as an inducer but also as a carbohydrate source for EPS production. This research broadens our knowledge of the regulation of EPS production in marine bacteria.     

Metabolically Improved Exopolysaccharide Production by Streptococcus thermophilus and Its Influence on the Rheological Properties of Fermented Milk

Altered levels of enzymes in the central carbon metabolism in Streptococcus thermophilus increased the exopolysaccharide (EPS) production 3.3 times over that of the parent strain. The influence of enhanced EPS production on the rheological properties of fermented milk is described for engineered strains of S. thermophilus which produce different levels of EPSs.     

Requirement for phosphoglucomutase in exopolysaccharide biosynthesis in glucose- and lactose-utilizing Streptococcus thermophilus

To study the influence of phosphoglucomutase (PGM) activity on exopolysaccharide (EPS) synthesis in glucose- and lactose-growing Streptococcus thermophilus, a knockout PGM mutant and a strain with elevated PGM activity were constructed. The pgmA gene, encoding PGM in S. thermophilus LY03, was identified and cloned. The gene was functional in Escherichia coli and was shown to be expressed from its own promoter. The pgmA-deficient mutant was unable to grow on glucose, while the mutation did not affect growth on lactose. Overexpression of pgmA had no significant effect on EPS production in glucose-growing cells. Neither deletion nor overexpression of pgmA changed the growth or EPS production on lactose. Thus, the EPS precursors in lactose-utilizing S. thermophilus are most probably formed from the galactose moiety of lactose via the Leloir pathway, which circumvents the need for a functional PGM.     

Mutational analysis of exopolysaccharide biosynthesis by Lactobacillus sakei 0-1

Lactobacillus sakei strain 0-1 produces an exopolysaccharide (EPS) consisting of glucose and rhamnose in a ratio of 3:2. As part of a biochemical and molecular analysis of the EPS biosynthetic pathway in L. sakei strain 0-1, we have isolated a random set of EPS-negative mutants. Following treatment of cells with the mutagen ethylmethane sulfonic acid, a total of 10 mutants were identified that lacked the clear ropy appearance of wild-type colonies on agar plates. Their characterization revealed that eight mutants had completely lost the ability to synthesize the normal EPS. Six of these mutants lacked activities of enzymes involved in the biosynthesis of dTDP-rhamnose, required for EPS production. Only mutant strains 12 and 20 were directly affected in EPS synthesis. Strain 12 synthesized EPS with a different sugar composition, however. Interestingly, strain 12 showed temperature-dependent EPS synthesis, with the highest amounts synthesized at 12°C, and low amounts at the optimal temperature for growth (30°C). Two mutants were in fact EPS-positive, producing the normal EPS, but displayed a different cell morphology (elongated cells), indicating a modification in cell wall synthesis.     

Indication that the Nitrogen Source Influences Both Amount and Size of Exopolysaccharides Produced by Streptococcus thermophilus LY03 and Modelling of the Bacterial Growth and Exopolysaccharide Production in a Complex Medium

Streptococcus thermophilus LY03 is a yogurt strain producing the same exopolysaccharide material in both milk and MRS broth. Actually, two types of exopolysaccharides are produced simultaneously. The two exopolysaccharides are identical in monomer composition (galactose and glucose in a 4:1 ratio) but differ in molecular size. Gel permeation chromatography revealed a high-molecular-mass exopolysaccharide (1.8 × 10⁶) and a low-molecular-mass exopolysaccharide (4.1 × 10⁵). Both exopolysaccharides can be isolated from the fermentation broth separately. The proportion in which they are produced is strongly dependent on the carbon/nitrogen ratio of the fermentation broth. A shift from a high-molecular-mass exopolysaccharide to a low-molecular-mass exopolysaccharide was observed with increasing initial complex nitrogen concentrations. All necessary biokinetic parameters to study the kinetics of S. thermophilus LY03 fermentations were obtained from a mathematical model which describes both S. thermophilus LY03 growth and exopolysaccharide production and degradation. The model is valid with various initial complex nitrogen concentrations and can be applied to simulate exopolysaccharide production in a milk medium.     

Citrate Metabolism by Enterococcus faecalis FAIR-E 229

Citrate metabolism by Enterococcus faecalis FAIR-E 229 was studied in various growth media containing citrate either in the presence of glucose or lactose or as the sole carbon source. In skim milk (130 mM lactose, 8 mM citrate), cometabolism of citrate and lactose was observed from the first stages of the growth phase. Lactose was stoichiometrically converted into lactate, while citrate was converted into acetate, formate, and ethanol. When de Man-Rogosa-Sharpe (MRS) broth containing lactose (28 mM) instead of glucose was used, E. faecalis FAIR-E 229 catabolized only the carbohydrate. Lactate was the major end product, and small amounts of ethanol were also detected. Increasing concentrations of citrate (10, 40, 70, and 100 mM) added to MRS broth enhanced both the maximum growth rate of E. faecalis FAIR-E 229 and glucose catabolism, although citrate itself was not catabolized. Glucose was converted stoichiometrically into lactate, while small amounts of ethanol were produced as well. Finally, when increasing initial concentrations of citrate (10, 40, 70, and 100 mM) were used as the sole carbon sources in MRS broth without glucose, the main end products were acetate and formate. Small amounts of lactate, ethanol, and acetoin were also detected. This work strongly supports the suggestion that enterococcal strains have the metabolic potential to metabolize citrate and therefore to actively contribute to the flavor development of fermented dairy products.     

嗜热链球菌IMAU20246胞外多糖基因簇及其表达分析北大核心

【目的】嗜热链球菌IMAU20246是一株具有良好发酵特性且高产胞外多糖(exopolysaccharides,EPS)的菌株,但其EPS基因簇及合成途径尚不清晰。因此可通过全基因组测序及生物信息学分析菌株基因组序列,探究EPS合成及调控机制。【方法】本实验对嗜热链球菌IMAU20246进行全基因组测序并进行生物信息学分析,解析EPS生物合成相关基因簇及EPS合成途径,同时采用实时荧光定量PCR技术(quantitative real-time PCR,qRT-PCR)对其不同时间点EPS基因簇的表达进行定量分析。【结果】嗜热链球菌IMAU20246基因组中有一个18.1 kb的EPS生物合成基因簇,编码15个与EPS生物合成相关的基因。嗜热链球菌IMAU20246通过转运葡萄糖、甘露糖、果糖、半乳糖、乳糖、海藻糖、纤维二糖及蔗糖合成UDP-葡萄糖、dTDP-葡萄糖、dTDP-鼠李糖、UDP-半乳糖、UDP-呋喃半乳糖、UDP-N-乙酰葡萄糖胺和UDP-N-乙酰半乳糖胺等7种糖核苷酸。qRT-PCR的结果表明,EPS基因簇中的基因在细胞生长阶段均能表达,特别是糖基转移酶基因epsE、epsF、epsH和epsJ在培养6 h时表达量最高,此时EPS产量达到最高。【结论】本研究从基因组解析了嗜热链球菌IMAU20246 EPS基因簇及其合成途径,为菌株的进一步开发提供了理论依据。     

Exopolysaccharides Produced by Intestinal Bifidobacterium Strains Act as Fermentable Substrates for Human Intestinal Bacteria

Eleven exopolysaccharides (EPS) isolated from different human intestinal Bifidobacterium strains were tested in fecal slurry batch cultures and compared with glucose and the prebiotic inulin for their abilities to act as fermentable substrates for intestinal bacteria. During incubation, the increases in levels of short-chain fatty acids (SCFA) were considerably more pronounced in cultures with EPS, glucose, and inulin than in controls without carbohydrates added, indicating that the substrates assayed were fermented by intestinal bacteria. Shifts in molar proportions of SCFA during incubation with EPS and inulin caused a decrease in the acetic acid-to-propionic acid ratio, a possible indicator of the hypolipidemic effect of prebiotics, with the lowest values for this parameter being obtained for EPS from the species Bifidobacterium longum and from Bifidobacterium pseudocatenulatum strain C52. This behavior was contrary to that found with glucose, a carbohydrate not considered to be a prebiotic and for which a clear increase of this ratio was obtained during incubation. Quantitative real-time PCR showed that EPS exerted a moderate bifidogenic effect, which was comparable to that of inulin for some polymers but which was lower than that found for glucose. PCR-denaturing gradient gel electrophoresis of 16S rRNA gene fragments using universal primers was employed to analyze microbial groups other than bifidobacteria. Changes in banding patterns during incubation with EPS indicated microbial rearrangements of Bacteroides and Escherichia coli relatives. Moreover, the use of EPS from B. pseudocatenulatum in fecal cultures from some individuals accounted for the prevalence of Desulfovibrio and Faecalibacterium prausnitzii, whereas incubation with EPS from B. longum supported populations close to Anaerostipes, Prevotella, and/or Oscillospira. Thus, EPS synthesized by intestinal bifidobacteria could act as fermentable substrates for microorganisms in the human gut environment, modifying interactions among intestinal populations.     

Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose Biosynthesis

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.     

Influence of Nutritional Factors on the Nature, Yield, and Composition of Exopolysaccharides Produced by Gluconacetobacter xylinus I-2281

The influence of substrate composition on the yield, nature, and composition of exopolysaccharides (EPS) produced by the food-grade strain Gluconacetobacter xylinus I-2281 was investigated during controlled cultivations on mixed substrates containing acetate and either glucose, sucrose, or fructose. Enzymatic activity analysis and acid hydrolysis revealed that two EPS, gluconacetan and levan, were produced by G. xylinus. In contrast to other acetic acid strains, no exocellulose formation has been measured. Considerable differences in metabolite yields have been observed with regard to the carbohydrate source. It was shown that glucose was inadequate for EPS production since most of this substrate (0.84 C-mol/C-mol) was oxidized into gluconic acid, 2-ketogluconic acid, and 5-ketogluconic acid. In contrast, sucrose and fructose supported a 0.35 C-mol/C-mol gluconacetan yield. In addition, growing G. xylinus on sucrose produced a 0.07 C-mol/C-mol levan yield. The composition of EPS remained unchanged during the course of the fermentations. Levan sucrase activity was found to be mainly membrane associated. In addition to levan production, an analysis of levan sucrase's activity also explained the formation of glucose oxides during fermentation on sucrose through the release of glucose. The biosynthetic pathway of gluconacetan synthesis has also been explored. Although the activity of key enzymes showed large differences to be a function of the carbon source, the ratio of their activities remained similar from one carbon source to another and corresponded to the ratio of precursor needs as deduced from the gluconacetan composition.