Abstract
The connection between the gut microbiome composition and human health has long been recognized, such that the host-microbiome interplay is at present the subject of the so-called “precision medicine”. Non-digestible fructooligosaccharides (FOS) can modulate the microbial composition and therefore their consumption occupies a central place in a strategy seeking to reverse microbiome-linked diseases. We created a small library of Bacillus megaterium levansucrase variants with focus on the synthesis of levan- and inulin-type FOS. Modifications were introduced at positions R370, K373 and F419, which are either part of the oligosaccharide elongation pathway or are located in the vicinity of residues that modulate polymerization. These amino acids were exchanged by residues of different characteristics, some of them being extremely low- or non-represented in enzymes of the levansucrase family (Glycoside Hydrolase 68, GH68). F419 seemed to play a minor role in FOS binding. However, changes at R370 abated the levansucrase capacity to synthesize levan-type oligosaccharides, with some mutations turning the product specificity towards neo-FOS and the inulin-like sugar 1-kestose. Although variants retaining the native R370 produced efficiently levan-type tri-, tetra- and pentasaccharides, their capacity to elongate these FOS was hampered by including the mutation K373H or K373L. Mutant K373H, for instance, generated 37- and 5.6-fold higher yields of 6-kestose and 6-nystose, respectively, than the wild-type enzyme, while maintaining a similar catalytic activity. The effect of mutations on the levansucrase product specificity is discussed.
Highlights
The French gastronome Jean Anthelme Brillat-Savarin wrote in his Physiologie du Gout: “Dis-moi ce que tu manges, je te dirai ce que tu es” (Tell me what you eat and I will tell you what you are)
The levansucrase library explores the modification of residues R370, K373 and F419, which are situated in the central sucrose/oligosaccharide binding pocket
Residues R370 and K373 constitute some of the earlier binding subsites in the path of polymer elongation and their role in defining the oligosaccharide product size has been demonstrated with the levansucrase from B. megaterium and other enzymes from family GH6810,18,19,21 (Supplementary Information, Fig. 1)
Summary
The French gastronome Jean Anthelme Brillat-Savarin wrote in his Physiologie du Gout: “Dis-moi ce que tu manges, je te dirai ce que tu es” (Tell me what you eat and I will tell you what you are). 6-kestose is the first product of levansucrase-catalyzed transfructosylation reactions and is as well the best acceptor of the fructosyl-moiety during polymerization. It does not accumulate in substantial amounts during the reaction course, this being the case for other short FOS with β2-6 linkages. A number of in vitro studies showed that many species of Bifidobacteria, one of the major bacterial genera in mammalian microbiomes, utilize FOS of DP 2–8 rather than larger oligosaccharides[15]. We report the creation of a library of B. megaterium levansucrase variants with the capacity to synthesize predominately FOS of DP 2–8, in contrast to the wild-type enzyme that produces low amounts of FOS with DP 2–2016,17. Substitution of residue F419 could indirectly provoke changes in the +1 and +2 subsites
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