Efficient production of isomelezitose by a glucosyltransferase activity in Metschnikowia reukaufii cell extracts

Abstract

Summary Metschnikowia reukaufii is a widespread yeast able to grow in the plants’ floral nectaries, an environment of extreme conditions with sucrose concentrations exceeding 400 g l−1, which led us into the search for enzymatic activities involved in this sugar use/transformation. New oligosaccharides were produced by transglucosylation processes employing M. reukaufii cell extracts in overload‐sucrose reactions. These products were purified and structurally characterized by MS‐ESI and NMR techniques. The reaction mixture included new sugars showing a great variety of glycosidic bonds including α‐(1→1), α‐(1→3) and α‐(1→6) linkages. The main product synthesized was the trisaccharide isomelezitose, whose maximum concentration reached 81 g l−1, the highest amount reported for any unmodified enzyme or microbial extract. In addition, 51 g l−1 of the disaccharide trehalulose was also produced. Both sugars show potential nutraceutical and prebiotic properties. Interestingly, the sugar mixture obtained in the biosynthetic reactions also contained oligosaccharides such as esculose, a rare trisaccharide with no previous NMR structure elucidation, as well as erlose, melezitose and theanderose. All the sugars produced are naturally found in honey. These compounds are of biotechnological interest due to their potential food, cosmeceutical and pharmaceutical applications.

Molecular characterization and heterologous expression of two α-glucosidases from Metschnikowia spp, both producers of honey sugars

“…S4). Despite further analysis is required, the current results may indicate that native α-glucosidase from M. reukaufii (present in the cell extracts) was involved in the hetero-GlcOs biosynthesis described before [32]. The production of these sorts of oligosaccharides by α-glucosidases from the family GH13 was previously described (Additional file 1: Table S2).…”

“…Isomelezitose and trehalulose were the main oligosaccharides produced, and to a lesser extent, the trisaccharides erlose, melezitose, theanderose and esculose. As we reported elsewhere, the glucose unit was principally transferred to the more reactive primary hydroxyl groups [25,32]. Thus, isomelezitose and trehalulose were produced when the positions 6-OH of the sucrose β-fructofuranosyl unit and 1-OH of the free fructose were glucosylated, respectively.…”

“…Besides, Mr-αGlu generated ~ 51% (w/w) of hetero-GlcOS (256 g/L, of which 173 g/L corresponded to isomelezitose, 61 g/L to threalulose, and 22 g/L to the rest of transglucosylation products). Here, performing the reactions with an apparent pure enzyme and under the optimal conditions of pH and temperature, isomelezitose production was increased almost twofold compared to our previous work (~ 81 g/L; 120 h reaction; ~ 76% (w/w) of sucrose consumed), where a cell extract from M. reukaufii was used [32]. It is worth mentioning that, to our knowledge, heterologous α-glucosidases from M. gruessii and M. reukaufii described in this work are the mayor producers of isomelezitose, not improved by molecular bioengineering.…”

“…As mentioned before, we have recently reported a potential α-glucosidase activity in M. reukaufii cell extracts that was able to hydrolyse sucrose and produced, by transglucosylation, different honey oligosaccharides, mainly isomelezitose and trehalulose [32]. Genomic DNA from M. reukaufii strain MR1 was previously sequenced and de novo assembled, which allowed Drs.…”

“…The oligosaccharides synthesised by these enzymes are of great biotechnological relevance due to their applications. For instance, isomelezitose, trehalulose, theanderose and erlose are non-cariogenic sugars with suitable sweetness and low caloric values that could be included in food products as dextrose or sucrose substitutes [9,32]. Moreover, isomelezitose is a potential prebiotic sugar and thus, a substrate for bifidobacteria [44].…”