Functional and Molecular Characterization of the Halomicrobium sp. IBSBa Inulosucrase

Abstract

Fructans are fructose-based (poly)saccharides with inulin and levan being the best-known ones. Thanks to their health-related benefits, inulin-type fructans have been under the focus of scientific and industrial communities, though mostly represented by plant-based inulins, and rarely by microbial ones. Recently, it was discovered that some extremely halophilic Archaea are also able to synthesize fructans. Here, we describe the first in-depth functional and molecular characterization of an Archaeal inulosucrase from Halomicrobium sp. IBSBa (HmcIsc). The HmcIsc enzyme was recombinantly expressed and purified in Escherichia coli and shown to synthesize inulin as proven by nuclear magnetic resonance (NMR) analysis. In accordance with the halophilic lifestyle of its native host, the enzyme showed maximum activity at very high NaCl concentrations (3.5 M), with specific adaptations for that purpose. Phylogenetic analyses suggested that Archaeal inulosucrases have been acquired from halophilic bacilli through horizontal gene transfer, with a HX(H/F)T motif evolving further into a HXHT motif, together with a unique D residue creating the onset of a specific alternative acceptor binding groove. This work uncovers a novel area in fructan research, highlighting unexplored aspects of life in hypersaline habitats, and raising questions about the general physiological relevance of inulosucrases and their products in nature.

Exploring Synergistic Effects of Levan and Levan‐MetabolizingBacillaceae in Promoting Growth and Enhancing Immunity of Tomato and Wheat

“…This urges further research into the biological effects of levans on other cereals and monocot species and with other types of pathogens. Moreover, it raises the question whether microbial inulin with high DP, for instance those derived from archaeal species (Abaramak et al, 2021; Aragón‐León et al, 2023), may then also be efficient priming agents for monocot species, which never produce endogenous high‐DP inulin by themselves. Nevertheless, B. velezensis GA1 and B. subtilis NCD‐2, isolated from the rhizosphere of eudicot plants, were able to counteract MoT in wheat during seed defense priming.…”

“…3.2.1.7) are involved in degradation of levan and inulin to levan oligosaccharides (LOS) and inulin oligosaccharides (IOS), respectively, usually in concert with exo‐type fructanases (E.C. 3.2.1.80) (Arrieta et al, 2004; Porras‐Domínguez et al, 2014; Mardo et al, 2017; Abaramak et al, 2021; Nasir et al, 2022; Alamäe et al, 2023). Besides other exopolymers, these microbes also incorporate fructans as extracellular carbohydrate polymers in their outer layers, serving as protective compounds and/or as (temporal) carbohydrate reserves (Dogsa et al, 2013).…”

Improving the Thermostability and Catalytic Activity of an Inulosucrase by Rational Engineering for the Biosynthesis of Microbial Inulin

“…21,22 To date, Lactobacillus species have been the primary sources of ISs, though recently, novel ISs have also been found in Archaea. 12,23,24 Studies on ISs are mainly focused on enzymatic characterization and chain-length modulation of produced inulin by rational design. Chain-length modulation of the product is a hot topic for LSs, and tailor-made levan was produced by controlling reaction conditions and altering structural features of LSs in some studies.…”

“…The primary purposes of molecular modification of sucrose-utilizing enzymes are improving their catalytic activity and thermostability, altering product distribution, and extending the utilization of promiscuous acceptor substrates. , To date, Lactobacillus species have been the primary sources of ISs, though recently, novel ISs have also been found in Archaea. ,, Studies on ISs are mainly focused on enzymatic characterization and chain-length modulation of produced inulin by rational design. Chain-length modulation of the product is a hot topic for LSs, and tailor-made levan was produced by controlling reaction conditions and altering structural features of LSs in some studies .…”