Community Research and Development Information Service - CORDIS

Trehalose production by Propionibacterium

Efrfect of stress on trehalose production:
Information was provided on the effect of aerobiosis on growth and trehalose accumulation in Propionibacterium freudenreichii subsp. shermanii NIZO B365. Growth under aerobic conditions (10% oxygen) at pH 7.0 had a drastic impact on the maximal intracellular concentration of trehalose (540 mg/g of protein in the late exponential phase). Additionally, the effect of other environmental conditions (NaCl concentration, growth temperature, pH, carbon source) on the same parameters was examined. Trehalose accumulates in response to salinity, oxygen or low pH, up to levels of 40% of the cell dry mass.

Pathways for the synthesis of trehalose in Propionibacterium:
Two pathways for the synthesis of trehalose were identified in Propionibacterium freudenreichii subsp. shermanii NIZO B365: i) the two-step pathway involving the enzymes trehalose 6-phosphate synthase (TPS) and trehalose 6-phosphate phosphatase (TPP) and ii) the single step pathway in which trehalose is synthesised from maltose by the action of trehalose synthase (TS).

The genes encoding TPS, TPP, and TS enzymes were cloned and expressed in E. coli and the recombinant enzymes characterised in detail. TPP was highly specific for trehalose 6-phosphate and was Mg2+ dependent. The kinetic parameters were KM 0.92 mM and Vmax 133 U/mg of protein. TPS was highly specific for glucose 6-phosphate but was able to use UDP-, ADP-, GDP- or TDP-glucose as glucosyl donors although there was a clear preference for UDP-glucose. The kinetic parameters of TS obtained for maltose were KM 6.4mM and Vmax 48U/mg of protein whereas KM 58.9mM and Vmax 158U/mg were determined for trehalose. The expression of the two pathways under different environmental conditions was assessed from immunoassays. A manuscript describing our results is nearly ready for submission (see Articles).

Trehalose production in fermented milk:
A natural strain accumulating high levels of trehalose (about 20% of cell mass) was identified among 18 examined strains of Propionibacterium spp. isolated from dairy sources (obtained either from DSM or NIZO culture collections). This strain was used to evaluate the production of trehalose in skim milk supplemented with casitone and lactate, to mimic the actual conditions in dairy fermentations. Trehalose accumulated to similar levels (250mg/g of cell protein). This was an encouraging result for the practical applications of this strain, aiming at trehalose production in situ. During the mid-term review of this project it was decided that the implementation of a trehalose-containing fermented dairy drink should be cancelled since the number of total Deliverables proposed was exaggerated.

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