In tumor development, the degradation of heparan sulfate (HS) by heparanase (HPSE) is associated with cell-surface and extracellular matrix remodeling as well as the release of HS-bound signaling molecules, allowing cancer cell migration, invasion and angiogenesis. Because of their structural similarity with HS, sulfated polysaccharides are considered a promising source of molecules to control these activities. In this study, we used a depolymerisation method for producing λ-carrageenan oligosaccharides (λ-CO), with progressive desulfation over time. These were then used to investigate the influence of polymeric chain length and degree of sulfation (DS) on their anti-HPSE activity. The effects of these two features on λ-CO anticoagulant properties were also investigated to eliminate a potential limitation on the use of a candidate λ-CO as a chemotherapeutic agent. HPSE inhibition was mainly related to the DS of λ-CO, however this correlation was not complete. On the other hand, both chain length and DS modulated λ-CO activity for factor Xa and thrombin IIa inhibition, two enzymes that are involved in the coagulation cascade, and different mechanisms of inhibition were observed. A λ-carrageenan oligosaccharide of 5.9 KDa was identified as a suitable anticancer candidate because it displayed one of the lowest anticoagulant properties among the λ-CO produced, while showing a remarkable inhibitory effect on MDA-MB-231 breast cancer cell migration.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.