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.

Figure 1. Diagrammatic representation of experimental layout and levansucrases used in this study.

(A) Yeast strains used to express the levansucrase M1FT; BY4742Δsuc2 and the two sucrose accumulating strains BY4742Δsuc2-SuSy and BY4742Δsuc2-SUT. (B) M1FT, a levansucrase from Leuconostoc mesenteroids and also a truncated version of M1FT, without its predicted secretion signal, was expressed in the different yeast strains.

Figure 2. Thin-layer chromatographic analysis of levan production by M1FT.

The presented TLC plates are representative of observations confirmed by at least three biological repeats. Fructose containing molecules were visualized using a urea spray after separation on TLC plates. (A) Cell extracts of strains expressing either the full-length M1FT, M1FT without signal sequence (M1FTΔsp) or the vector (YCpLac33) as control in BY4742Δsuc2 (grown in glucose and fructose containing media) and the two sucrose accumulating strains BY4742Δsuc2-SUT and BY4742Δsuc2-SuSy (grown in glucose and sucrose; and glucose and fructose containing media respectively). (B) Levan production by M1FT in BY4742Δsuc2 grown in media containing glucose as carbon source, supplemented with 5% sucrose. Cell extracts are indicated with a dashed line. The growth medium was either spotted directly from the supernatant (Media) or as a 500X concentrate of the same supernatant after ethanol precipitation (Media concentrate); in both cases, no levan production could be detected.

Figure 3. ¹³C-NMR analysis of the fructan polymer produced by M1FT.

(A) Overlay of the ¹³C-NMR spectra from the M1FT produced polymer and a commercially available levan polymer from Zymomonas mobilis. (B) Comparison of the chemical shifts for the levan polymer from Zymomonas mobilis (Levan^com.); M1FT, the polymer produced by M1FT in this study; M1FT*, previously described by Kang and colleagues [24]; Levan** and Inulin**, previously described by Shimamura and colleagues [20].

Figure 4. The potato sucrose synthase (SuSy) has lesser affinity for fructose 6-phosphate.

Thin-layer chromatogram depicting extracts of BY4742Δsuc2 and BY4742Δsuc2-SuSy strains transformed with M1FTΔsp and grown in minimal media containing either glucose or glucose and fructose to assess whether fructose 6-phosphate, originating from glucose can be used as substrate by SuSy. Strains grown in glucose and fructose are underlined with a solid line and strains grown in glucose only media with a dashed line. The decrease in sucrose formation in BY4742Δsuc2-SuSy grown in glucose, compared to glucose and fructose illustrates that fructose 6-phosphate, synthesized from glucose, is not a preferred substrate for SuSy. TLC plates are representative of observations confirmed by at least three biological repeats. Fructose containing molecules were visualized using a urea spray after separation on TLC plates.

Figure 5. Quantification of levan production by M1FT transformed strains in rich media supplemented with sucrose (YPDS).

Quantification was done by densitometric analysis of the urea stained levan polymers after visualization on TLC plates. Produced levan was either quantified from cell extracts (Intracellular) or from the growth medium (Media). Error bars indicate standard deviation (in g/L) within a triplicate sample set. (A) Levan produced by M1FT expressed in the BY4742Δsuc2 genetic background. (B) Levan production by M1FT expressed in the BY4742Δsuc2-SUT genetic background.

Figure 6. Extracellular polysaccharide (EPS) production by the M1FT expressing strains.

(A) Slime production by the BY4742Δsuc2-SUT-M1FT yeast strain is dependent on growth medium. Cultures where grown on either rich media without sucrose (YPD), rich media with sucrose (YPDS), minimal media with sucrose (SCDS) or media containing 1% yeast extract and molasses, which was diluted to provide the same sucrose (5%) concentration as YPDS (YE-Molasses). Slime is only visible on rich media containing sucrose (YPDS). (B) The effect of pH on extracellular polymer accumulation. The production of levan as an extracellular polysaccharide (EPS) is reduced when the pH of the growth medium (YPDS) is changed from pH 6.5 (the unadjusted pH of YPDS) to pH 5.5 (which is the same as the pH of the SCDS media). Plate pictures are a representative of observations confirmed by at least three separate biological repeats.