title: Biosynthesis of levan, a bacterial extracellular polysaccharide, in the yeast Saccharomyces cerevisiae.
abstract: Levans are fructose polymers synthesized by a broad range of micro-organisms and a limited number of plant species as non-structural storage carbohydrates. In microbes, these polymers contribute to the formation of the extracellular polysaccharide (EPS) matrix and play a role in microbial biofilm formation. Levans belong to a larger group of commercially important polymers, referred to as fructans, which are used as a source of prebiotic fibre. For levan, specifically, this market remains untapped, since no viable production strategy has been established. Synthesis of levan is catalysed by a group of enzymes, referred to as levansucrases, using sucrose as substrate. Heterologous expression of levansucrases has been notoriously difficult to achieve in Saccharomyces cerevisiae. As a strategy, this study used an invertase (Δsuc2) null mutant and two separate, engineered, sucrose accumulating yeast strains as hosts for the expression of the levansucrase M1FT, previously cloned from Leuconostoc mesenteroides. Intracellular sucrose accumulation was achieved either by expression of a sucrose synthase (Susy) from potato or the spinach sucrose transporter (SUT). The data indicate that in both Δsuc2 and the sucrose accumulating strains, the M1FT was able to catalyse fructose polymerisation. In the absence of the predicted M1FT secretion signal, intracellular levan accumulation was significantly enhanced for both sucrose accumulation strains, when grown on minimal media. Interestingly, co-expression of M1FT and SUT resulted in hyper-production and extracellular build-up of levan when grown in rich medium containing sucrose. This study presents the first report of levan production in S. cerevisiae and opens potential avenues for the production of levan using this well established industrial microbe. Furthermore, the work provides interesting perspectives when considering the heterologous expression of sugar polymerizing enzymes in yeast.
authors.affiliation: Institute for Wine Biotechnology, Stellenbosch University, Stellenbosch, Western Cape, South Africa.
authors.forename: Jaco
authors.initials: J
authors.lastname: Franken
authors.forename: Bianca A
authors.initials: BA
authors.lastname: Brandt
authors.forename: Siew L
authors.initials: SL
authors.lastname: Tai
authors.forename: Florian F
authors.initials: FF
authors.lastname: Bauer
mesh_terms: D005285:Fermentation; D005630:Fructans / Q000096:biosynthesis* / Q000737:chemistry; D005636:Fructosephosphates / Q000378:metabolism; D015870:Gene Expression; D005964:Glucosyltransferases / Q000235:genetics / Q000378:metabolism; D006602:Hexosyltransferases / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D011108:Polymers / Q000737:chemistry; D011135:Polysaccharides, Bacterial / Q000096:biosynthesis* / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D013395:Sucrose / Q000378:metabolism
chemical_list: D005630:Fructans; D005636:Fructosephosphates; D026901:Membrane Transport Proteins; D011108:Polymers; D011135:Polysaccharides, Bacterial; D013395:Sucrose; C027618:fructose-6-phosphate; C072599:levan; D005964:Glucosyltransferases; D006602:Hexosyltransferases; C021513:levansucrase; C022699:sucrose synthase