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

BACKGROUND: Cyclic ß-1,2-glucans (CßG) 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 CßG 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 CßG 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 CßG. RESULTS: We present the production of CßG in specially designed E. coli strains by means of the deletion of intrinsic polysaccharide biosynthetic genes and the heterologous expression of enzymes involved in CßG synthesis, transport and succinilation. These strains produce different types of CßG: unsubstituted CßG, anionic CßG and CßG of high size. Unsubstituted CßG 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 CßG /L in culture medium within 48 h culture. CONCLUSIONS: We have developed a new recombinant bacterial system for the synthesis of cyclic ß-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 CßG in E. coli with high yields and the highest volumetric productivity reported to date. We expect this new highly scalable system facilitates CßG availability for further research and the widespread use of these promising molecules across many application fields.

New two-stage pH combined with dissolved oxygen control strategy for cyclic ß-1,2 glucans synthesis.

On the basis of a novel two-stage pH combined with dissolved oxygen (DO) control strategy in fed-batch fermentation, this research addresses the influence of pH on cyclic ß-1,2-glucans (CßGs) biosynthesis and melanin accumulation during the production of CßGs by Rhizobium radiobacter ATCC 13,333. Under these optimal fermentation conditions, the maximum cell concentration and CßGs concentration in a 7-L stirred-tank fermenter were 7.94 g L-1 and 3.12 g L-1, which were the maximum production reported for R. radiobacter. The melanin concentration of the fermentation broth was maintained at a low level, which was beneficial to the subsequent separation and purification of the CßGs. In addition, a neutral extracellular oligosaccharide (COGs-1) purified by the two-stage pH combined with DO control strategy fermentation medium was structurally characterized. Structural analyses indicated that COGs-1 was a family of unbranched cyclic oligosaccharides composed of only ß-1,2-linked D-glucopyranose residues with degree of polymerization between 17 and 23, namely CßGs. This research provides a reliable source of CßGs and structural basis for further studies of biological activity and function. KEY POINTS: • A two-stage pH combined with DO control strategy was proposed for CßGs production and melanin biosynthesis by Rhizobium radiobacter. • The final extracellular CßGs production reached 3.12 g L-1, which was the highest achieved by Rhizobium radiobacter. • The existence of CßGs could be detected by TLC quickly and accurately.

Enhanced solubility of curcumin by complexation with fermented cyclic ß-1,2-glucans.

Curcumin (CUR) is a low-solubility polyphenolic compound with many physiological functions. Cyclic ß-1,2-glucans (cyclosophoraoses [Cys]), which contain rings of different sizes with degrees of polymerization ranging from 17 to 23, were obtained from Rhizobium radiobacter ATCC 1333, a soil microorganism. The complexation ability and solubility enhancement of cyclic ß-1,2-glucans with insoluble curcumin were investigated. Phase-solubility analysis revealed that the stoichiometric ratio of the inclusion complexes was 1:1. The stability constant of Cys was 930 M-1, which was 7.68 times that of α-cyclodextrin (α-CD) and 2.09 times that of ß-cyclodextrin (ß-CD). The characteristics of the curcumin/Cys inclusion complexes were successfully determined by using Fourier transform infrared (FTIR) spectrometry, differential scanning calorimetry (DSC), nuclear magnetic resonance (1H NMR) spectroscopy, and scanning electron microscopy (SEM). Moreover, a 1:1 molecular model of the curcumin/Cys inclusion complexes was established through molecular docking analysis. These findings indicated that cyclic ß-1,2-glucans successfully formed complexes with curcumin, which suggested that they could be used as solubility-increasing agents. To the best of our knowledge, this is the first report in which curcumin has been embedded into cyclic ß-1,2-glucans resulting in an increase in its aqueous solubility.