Abstract
Leuconostoc lactis AV1 strain isolated from a Tunisian avocado was characterized as a dextran producer. The promoter PdsrLL and the dsrLL gene encoding the DsrLL dextransucrase responsible for the dextran synthesis were transcriptionally fused to the mCherry coding gene generating the pRCR20 plasmid. Upon plasmid transfer, both AV1n and the dextran non-producing Leuconostoc mesenteroides CM70 became red due to expression of the mCherry from the PdsrLL-dsr-mrfp transcriptional fusion. Characterization of the polymers present in cultures supernatants revealed that the DsrLL encoded from pRCR20 in the recombinant bacteria was able to synthesize dextran. The production of dextran by the DsrLL in AV1n increased in response to low temperature, reaching 10-fold higher levels at 20°C than at 37°C (4.15 g/L versus 0.41 g/L). To analyze if this stress response includes activation at the transcriptional level and if it was only restricted to Leuconostoc, AV1n was transformed with plasmids carrying either the PdsrLL-mrfp fusion or the PdsrLS of Lactobacillus sakei MN1 fused to the mrfp gene, and the influence of temperature and carbon source on expression from the Dsr promoters was monitored by measurement of the mCherry levels. The overall expression analysis confirmed an induction of expression from PdsrLL upon growth at low temperature (20°C versus 30°C and 37°C) in the presence of sugars tested (sucrose, glucose, maltose, and fructose). In addition, the presence of sucrose, the substrate of Dsr, also resulted in activation of expression from PdsrLL. A different behavior was detected, when expression from PdsrLS was evaluated. Similar levels of fluorescence were observed irrespectively of the carbon source or temperature, besides a sequential decrease at 30°C and 20°C, when sucrose was present in the growth medium. In conclusion, the two types of regulation of expression of Dsr presented here revealed two different mechanisms for environmental adaptation of Leuconostoc and Lactobacillus that could be exploited for industrial applications.
Highlights
Lactic acid bacteria (LAB) have been traditionally used for food fermentations as starter or co-adjuvants
Aim to identify new dextran-producing LAB from environments not previously explored and species not previously commercially exploited, the AV1n strain isolated from Tunisian avocado was selected for its ability to generate mucose colonies in a solid MRSS rich medium supplemented with sucrose (Supplementary Figure S2) and classified as belonging to the Lc. lactis species by the sequencing of its 16s rRNA coding gene
Dextrans synthesized by LAB with a similar structure and low proportion of branching (5–10%) at the O-3 position is quite common and among others have been previously reported for Leuconostoc lactis KC117496 (Saravanan and Shetty, 2016), several Lc. mesenteroides strains (Zarour et al, 2017b), and Lb. sakei MN1 (Nácher-Vázquez et al, 2015)
Summary
Lactic acid bacteria (LAB) have been traditionally used for food fermentations as starter or co-adjuvants. The increased demand for more “healthy and well-being food” has led to an expanding usage of these microorganisms for the manufacture of functional food products. This is due to the fact that they are able to synthetize several beneficial compounds such as vitamins (e.g., riboflavin and folates), enzymes (e.g., amylases and phytases), or immunomodulatory EPS (Anastasio et al, 2010; Oleksy and Klewicka, 2016; Zarour et al, 2017a; Mosso et al, 2018). The total yield of EPS production depends on the producing strain, as well as the growth conditions: (i) medium composition (carbon and nitrogen sources) or (ii) physical factors (temperature, pH, oxygen tension and incubation period) (Patel et al, 2012)
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