Development of a scalable recombinant system for cyclic beta-1,2-glucans production

Abstract

Background: Cyclic β-1,2-glucans (CβG) are bacterial cyclic homopolysaccharides with interesting biotechnological applications. These ring-shaped molecules have a hydrophilic surface that confers high solubility and a hydrophobic cavity able to include poorly soluble molecules. Several studies demonstrate that CβG and many derivatives can be applied in drug solubilization and stabilization, enantiomer separation, catalysis, synthesis of nanomaterials and even as immunomodulators, suggesting these molecules have great potential for their industrial and commercial exploitation. Nowadays, there is no method to produce CβG by chemical synthesis and bacteria that synthesize them are slow-growing or even pathogenic, which makes the scaling up of the process difficult and expensive. Therefore, scalable production and purification methods are needed to afford the demand and expand the repertoire of applications of CβG.

Results: We present the production of CβG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CβG synthesis, transport and succinilation. These strains produce different types of CβG: unsubstituted CβG, anionic CβG and CβG of high size. Unsubstituted CβG with a degree of polymerization of 17 to 24 glucoses were produced and secreted to the culture medium by one of the strains. Through high cell density culture (HCDC) of that strain we were able to produce 4,5 g of pure unsubstituted CβG /L in culture medium within 48 h culture.

Conclusions: We have developed a new recombinant bacterial system for the synthesis of cyclic β-1,2-glucans, expanding the use of bacteria as a platform for the production of new polysaccharides with biotechnological applications. This new approach allowed us to produce CβG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CβG availability for further research and the widespread use of these promising molecules across many application fields.

Keywords: Cyclic β-1,2-glucans; Cyclodextrin; Cyclosophoraoses; Drug solubilization; Nanomaterial; Oligosaccharides.

Fig. 1

Cyclic β-1,2-glucans (CβG) produced in E. coli. (A) Schematic representation of E. coli Δmdo strain expressing Cgs, Cgt and Cgm to produce CβG. EM, extracellular medium; OM, outer membrane; PE, periplasm; IM, inner membrane; CI, cytoplasm; CβG, Cyclic β-1,2-glucans; OPGs, Osmoregulated periplasmic glucans or β-1,6-branched-β-1,2-lineal glucans. (B) TLC analysis of ethanolic extracts from parental E. coli strain or Δmdo mutant (ΔmdoGH ΔmdoC ΔmdoB). Osmoregulated periplasmic glucans (OPGs) or anionic β-1,6-branched-β-1,2-lineal glucans of E. coli are highly decorated with anionic substituents giving sharp bands in this TLC system. The E. coli Δmdo mutant lacks OPGs. (C) TLC analysis of cellular and culture-medium fractions of CβG produced in E. coli Δmdo expressing Cgs (E. coli Δmdo^Cgs); Cgs and Cgt (E. coli Δmdo^Cgs+Cgt) or Cgs, Cgt and Cgm (E. coli Δmdo^Cgs+Cgt+Cgm). E. coli Δmdo^Cgs and E. coli Δmdo^Cgs+Cgt stains produce cell-associated unsubstituted CβG, which can be obtained from cells by ethanol extraction (left). Unsubstituted CβG produced by the strains E. coli Δmdo^Cgs+Cgt and E. coli Δmdo^Cgs+Cgt+Cgm reach the extracellular space and can be recovered from the culture medium supernatant (right). E. coli Δmdo^Cgs+Cgt+Cgm strain also produces anionic CβG that are mostly retained in the cell fraction. (D) TLC analysis of cellular and culture-medium fractions of CβG produced in E. coli Δmdo expressing Cgs mutant truncated in the phosphorylase domain (Cgs_tr, stain E. coli Δmdo^Cgstr); Cgs_tr and Cgt (E. coli Δmdo^Cgstr+Cgt) or Cgs_tr, Cgt, and Cgm (E. coli Δmdo^{Cgstr+Cgt+Cgm}). Expression of Cgs_tr resulted in the production of CβG with a reduced mobility in the TLC, indicative of a higher degree of polymerization. Unlike CβG with normal size, these bigger CβG are mostly retained in the cell fraction

Fig. 2

CβG and biomass production at different culture times and substrate concentrations. (A) Production of CβG in the culture supernatant. (B) Production of cell-associated CβG. (C) Biomass production. E. coli Δmdo^{Cgs + Cgt} strain was grown for 48 h in culture media containing 0.5%, 1.5% or 3% glycerol as carbon source. OD at 600 nm was measured after 24 and 48 h of incubation at 30 °C and agitation at 200 rpm. CβG were extracted from the cells or precipitated from the culture supernatant with ethanol and the total reducing sugars (TRS) content was measured by the anthrone-sulfuric acid method. CβG levels were expressed as mg/L glucose equivalents. Error bars represent the standard deviation of three independent experiments

Fig. 3

MALDI-TOF mass spectrometry analysis of CβG produced by E. coli Δmdo^Cgs+Cgt in Erlenmeyer and purified from culture supernatant. Signals with m/z corresponding to cyclic molecules from 17 to 24 glucose units were detected

Fig. 4

CβG production in High Cell Density Culture (HCDC) of E. coli Δmdo^Cgs+Cgt. (A) HCDC was performed on Korz medium with glycerol as carbon source and consisted of 3 steps: an initial batch step, an exponential feed batch step performed to increase biomass until oxygen transfer became the limiting factor, and a constant feed regime to reduce oxygen demand until 48 h of effective fermentation time (EFT). Biomass production was followed by DO_600nm and dry weight determination. CβG production was indirectly followed by measurement of total reducing sugars (TRS) in the culture supernatant and expressed as g/L glucose equivalents. (B) Analysis of CβG production by TLC. Equivalent volumes of culture supernatant purified by ethanol precipitation were analyzed. The results are representative of two independent experiments of HCDC. *, migration position of unsubstituted CβG; **, migration position of non-CβG sugar products

Fig. 5

Purification and characterization of CβG produced by HCDC of the E. coli Δmdo^Cgs+Cgt. (A) Purification of CβG by size exclusion chromatography (SEC). A small volume of HCDC culture supernatant was subjected to SEC on a BioGel P6 column (16/170 mm) and the resulting fractions were quantified by the anthrone-sulfuric acid method. (B) Characterization of CβG by TLC. Representative samples of each peak were subjected to qualitative analysis by TLC. *, migration position of unsubstituted CβG; **, migration position of non-CβG sugar products

Fig. 6

MALDI-TOF mass spectrometry analysis of CβG produced by E. coli Δmdo^Cgs+Cgt in stirred tank bioreactor and purified from culture supernatant. Signals with m/z corresponding to cyclic molecules from 16 to 24 glucose units were detected