Cloning of levansucrase from Leuconostoc mesenteroides and its expression in Pichia pastoris

Kang, Hee Kyoung; Yun, Sang Il; Lim, Tae Woo; Xia, Yongmei; Kim, Doman

doi:10.1007/s10068-011-0039-0

Cited by 13 publications

(6 citation statements)

References 17 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In this study, three different genetic platforms where created to assess the potential of the levansucrase, M1FT, from Leuconostoc mesenteroides to catalyse the synthesis of levan in yeast, using sucrose as a substrate. M1FT specifically presented an attractive target for expression, since it has recently been effectively expressed in Pichia pastoris [10]. The three genetic backgrounds that were constructed for M1FT expression included an invertase (Δ suc2 ) null mutant and added strategies using two different approaches to construct yeast strains with the ability to produce and accumulate intracellular sucrose.…”

Section: Discussionmentioning

confidence: 99%

“…In the latter two strains, sucrose accumulation was accomplished by expressing either a potato sucrose synthase (SuSy) or a sucrose transporter (SUT) from spinach in the Δ suc2 genetic background. These strategies where specifically selected, since prior studies have shown secretion to be a potential obstacle for levansucrases in yeast and also bacteria [10,29]. In addition, several studies using plant expression have illustrated functional intracellular expression to be consistently possible [1,6,7].…”

Section: Discussionmentioning

confidence: 99%

“…However, the recombinant enzyme only produced low molecular weight levan [9]. M1FT from, Leuconostoc mesenteroids , was also successfully expressed and secreted in P. pastoris , with yields suggesting industrial viability [10]. The M1FT gene was also expressed in Escherichia coli , with large amounts of the enzyme being retained as cytoplasmic inclusion bodies.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Biosynthesis of Levan, a Bacterial Extracellular Polysaccharide, in the Yeast Saccharomyces cerevisiae

et al. 2013

View full text Add to dashboard Cite

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.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Biosynthesis of Levan, a Bacterial Extracellular Polysaccharide, in the Yeast Saccharomyces cerevisiae

et al. 2013

View full text Add to dashboard Cite

show abstract

“…Like whole culture studies, the use of Leuconostoc spp. levansucrases for levan biosynthesis in cell-free systems is a relatively recent research topic and so far without real technological applications (Iliev et al 2018;Kang et al 2011). Nevertheless, a number of studies have confirmed that this enzyme, which has been comprehensively described for several Leuconostoc spp.…”

Section: Production Methods Strains and Optimum Conditionsmentioning

confidence: 99%

“…To obtain such recombinant enzymes, the levansucrase genes are isolated from the corresponding Leuconostoc spp. strains, then sequenced and cloned into selected heterologous expression systems, which may be of either bacterial (mostly E.coli) (Iliev et al 2018;Ishida et al 2016;Jadaun et al 2019;Kang et al 2005;Morales-Arrieta et al 2006;Xu et al 2017) or yeast (Pichia pastoris, Saccharomyces cerevisiae) origin (Franken et al 2013;Kang et al 2011). Levan synthesis is performed with isolated and purified recombinant enzyme in chemically defined media, under the optimal conditions (sucrose, enzyme and metal ion concentrations, pH, temperature) for the each particular reaction (Jadaun et al 2019;Kang et al 2005).…”

Section: Production Methods Strains and Optimum Conditionsmentioning

confidence: 99%

Extracellular polysaccharides produced by bacteria of the Leuconostoc genus

Zikmanis

Brants²,

Koļesovs

et al. 2020

World J Microbiol Biotechnol

View full text Add to dashboard Cite

Structurally diverse biopolymers, including extracellular polysaccharides (EPS), synthesized by bacteria can possess physicochemical and functional properties that make them important products of microbial synthesis with a broad and versatile biotechnological potential. Leuconostoc spp. belongs to the group of lactic acid bacteria as one of the predominant members and are relevant not only in varied food fermentations, but also can be employed in the production of extracellular homopolysaccharides (HoPS) such as α-glucans (dextran, alternan) and β-fructans (levan,inulin) from the sucrose-containing substrates. EPS are synthesized by specific Leuconostoc spp. extracellular glycosyltransferases [dextran sucrase, alternansucrase (ASR)] and fructosyltransferases (levansucrase, inulosucrase) and enzymatic reactions can be performed in whole culture systems as well as using cell-free enzymes. Both α-glucans and β-fructans have a wide range of properties, mostly depending on their pattern of linkages, which, although differing in some respects, make suitable prerequisites for their versatile application in many fields, especially in the food industry and biomedicine. As a rule, these properties (polymer type, molecular mass, rheological parameters), as well as the overall EPS yield, are strain-specific for the selected producers and depend to a large extent on the nutritional and growth conditions used, which in many cases remain not sufficiently optimized for Leuconostoc spp. This review summarizes the current knowledge on the potential of Leuconostoc spp. to produce commercially relevant EPS, including information on their applications in various fields, producer strains, production methods and techniques used, selected conditions, the productivity of bioprocesses as well as the possible use of renewable resources for their development.

show abstract