Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production

Abstract

Chondroitin sulfate is a well-known bioactive molecule, widely used as an anti-osteoarthritis drug, that is nowadays mainly produced by animal tissue sources with unsafe extraction procedures. Recent studies have explored an integrated biotechnological-chemical strategy to obtain a chondroitin sulfate precursor from Escherichia coli K4 capsular polysaccharide, demonstrating the influence of environmental and growth conditions on capsule synthesis. In this research work, the flexibility of the strain biosynthetic machinery was investigated to enhance the K4 capsular polysaccharide production by supplementing the growth medium with the monosaccharides (glucuronic acid, galactosamine and fructose) that constitute the chain. Shake flask experiments were performed by adding the sugars singularly or together, by testing monosaccharide different concentrations and times of addition and by observing the bacterial sugar consumption. A K4 capsular polysaccharide production enhancement, compared to the control, was observed in all cases of supplementation and, in particular, significant 68 and 57 % increases were observed when adding 0.385 mM glucuronic acid plus galactosamine or 0.385 mM fructose, respectively. Increased expression levels of the gene kfoC, coding for a K4 polymerase, evaluated in different growth conditions, confirmed the results at the molecular level. Furthermore, batch fermentations, performed in lab-scale reactors (2 L), allowed to double the K4 capsular polysaccharide production values obtained in shake flask conditions, by means of a strict control of the growth parameters.

Masquerading microbial pathogens: capsular polysaccharides mimic host-tissue molecules

“…CS is currently harvested from animal sources (cow, pig, shark, fish, and bird cartilage; Huskisson, ), but there is a growing interest in moving to sustainable microbial production platforms to minimize contamination and to control product consistency. One such production strategy involves harvesting a CS precursor from cultures of E. coli strains biosynthesizing a K4 capsule (Zanfardino et al ., ; Restaino et al ., , ; Schiraldi et al ., , ; Trilli et al ., ). This biotechnological relevance has provoked increased interest in improving biosynthesis of K4 CPS by manipulating E. coli metabolism (Restaino et al ., ; Cimini et al ., ; Cress et al ., ; Wu et al ., ).…”

“…One such production strategy involves harvesting a CS precursor from cultures of E. coli strains biosynthesizing a K4 capsule (Zanfardino et al ., ; Restaino et al ., , ; Schiraldi et al ., , ; Trilli et al ., ). This biotechnological relevance has provoked increased interest in improving biosynthesis of K4 CPS by manipulating E. coli metabolism (Restaino et al ., ; Cimini et al ., ; Cress et al ., ; Wu et al ., ). K4 CPS is similar in structure to unsulfated CS, with the exception of an acid labile, bisecting β‐fructofuranose attached to C3 of the GlcA residue (Fig.…”

Chondroitin sulphate: a focus on osteoarthritis

“…In the first case CS is obtained enzymatically by in vitro chain elongation using UDP-sugar precursors and biotechnologically produced synthase enzymes. UDPglucose oxidized into UDP-glucuronic acid which promotes chondrogenesis and GAGs synthesis monosaccharides precursors help in boosting chondroitin-like capsular polysaccharide production [34,35], a hetero-oligomer complex of CS synthase-1 (CSS1)/chondroitin synthase-1 and CS synthase-2 (CSS2)/chondroitin polymerizing factor with the strongest polymerizing activity participate in the extension and initiation of CS [1]. In the other case CS is produced by using a biotechnological-chemical strategy consisting of three main steps: fermentative production of a chondroitin-like polysaccharide by capsulated bacteria, purification from the fermentation broth and then site specific chain sulphation.…”

Metabolic engineering of capsular polysaccharides

“…Recent studies have identified an imbalanced ratio between UDP-GalNAc and UDP-GlcA in E. coli K4 during growth, with GlcA being present in lower amounts [45]. This challenge was circumvented by adding GlcA and GalNAc monosaccharides to the medium at the beginning of growth, leading to a 64% increase in K4 CPS production [46]. Flux balance was also successfully addressed via a pfkA knockout in E. coli K4, favoring the production of fructose-6-phosphate.…”