Homologous overexpression of rfaH in E. coli K4 improves the production of chondroitin-like capsular polysaccharide

Abstract

Background: Glycosaminoglycans, such as hyaluronic acid, heparin, and chondroitin sulfate, are among the top ranked products in industrial biotechnology for biomedical applications, with a growing world market of billion dollars per year. Recently a remarkable progress has been made in the development of tailor-made strains as sources for the manufacturing of such products. The genetic modification of E. coli K4, a natural producer of chondroitin sulfate precursor, is challenging considering the lack of detailed information on its genome, as well as its mobilome. Chondroitin sulfate is currently used as nutraceutical for the treatment of osteoarthritis, and several new therapeutic applications, spanning from the development of skin substitutes to live attenuated vaccines, are under evaluation.Results: E. coli K4 was used as host for the overexpression of RfaH, a positive regulator that controls expression of the polysaccharide biosynthesis genes and other genes necessary for the virulence of E. coli K4. Various engineering strategies were compared to investigate different types of expression systems (plasmid vs integrative cassettes) and integration sites (genome vs endogenous mobile element). All strains analysed in shake flasks on different media showed a capsular polysaccharide production improved by 40 to 140%, compared to the wild type, with respect to the final product titer. A DO-stat fed-batch process on the 2L scale was also developed for the best performing integrative strain, EcK4r3, yielding 5.3 g • L -1 of K4 polysaccharide. The effect of rfaH overexpression in EcK4r3 affected the production of lipopolysaccharide and the expression of genes involved in the polysaccharide biosynthesis pathway (kfoC and kfoA), as expected. An alteration of cellular metabolism was revealed by changes of intracellular pools of UDP-sugars which are used as precursors for polysaccharide biosynthesis.

A Unique Capsular Polysaccharide Structure from the Psychrophilic Marine Bacterium Colwellia psychrerythraea 34H That Mimics Antifreeze (Glyco)proteins

“…44 As a negative control we used chondroitin, a capsular polysaccharide produced by an engineered E. coli K4. 46 All particle solutions (CPS, AFP and chondroitin) were measured at the same concentration of 6.6 μM. Furthermore, we measured a particle-free solution of MilliQ water with 9 ‰ NaCl.…”

A microbial–enzymatic strategy for producing chondroitin sulfate glycosaminoglycans

“…Naturally, Escherichia coli K4 produces the capsular polysaccharide K4, a fructosylated chondroitin (Cress, Greene, Linhardt, & Koffas, ) which is structurally similar to chondroitin. Many studies on optimization of the metabolic pathway (Cimini, De Rosa, Carlino, Ruggiero, & Schiraldi, ; Cimini et al, ) and cultivation process (Restaino, di Lauro, Di Nuzzo, De Rosa, & Schiraldi, ; Restaino et al, ; Wu et al, ) have been carried out to improve the chondroitin production. Nevertheless, E. coli K4 is a pathogenic strain, which causes urinary tract infection (Nash et al, ).…”

Expression of the Yersinia enterocolitica O:3 LPS O-antigen and outer core gene clusters is RfaH-dependent

“…The rfaH gene and the ops sequence are conserved amongst gammaproteobacteria, and RfaH orthologues were able to complement an E. coli rfaH mutant, suggesting functional homology (Bailey et al, 1997;Carter et al, 2004). Overproduction of RfaH, however, increased the production of capsular polysaccharide in E. coli K4 (Cimini et al, 2013). Furthermore, in E. coli, RfaH and NusG are known to interact with the ribosomal protein S10 coupling transcription with translation (Burmann et al, 2010(Burmann et al, , 2012NandyMazumdar & Artsimovitch, 2015).…”