Engineering a branch of the UDP‐precursor biosynthesis pathway enhances the production of capsular polysaccharide in Escherichia coli O5:K4:H4

Abstract

Escherichia coli K4 produces a capsule with a chemical structure that resembles chondroitin, a molecule with established chondro protective properties. The endogenous genes pgm and galU are involved in the biosynthesis of UDP-glucose which is a critical intermediate in carbohydrate metabolism and biochemical precursor of UDP-glucuronic acid. Together with UDP-N-acetylgalactosamine, UDP-glucuronic acid is used as sugar donor for capsule biosynthesis. The aim of the study was to evaluate how a change in the pathways leading to UDP-glucuronic acid biosynthesis affected capsular polysaccharide production. One additional copy of pgm and galU was introduced in E. coli K4 and in the previously described recombinant strain EcK4r3. A microbioreactor was used to analyse strain performance with parallel batch experiments, demonstrating increased polysaccharide concentrations and providing data that are comparable to those obtained in larger fermenters. Further experiments on a glutamine enriched medium showed an additional 45% increase of capsule production, maybe indicating the need to balance both branches leading to polymer biosynthesis in order to maximize yields. In the effort towards the establishment of a feasible bio-chondroitin production process this study provides information on how the availability of sugar precursors impacts polysaccharide biosynthesis in E. coli K4, a complex unexplored aspect of a multifaceted process.

Physiological characterization and quantitative proteomic analyses of metabolically engineered E. coli K4 strains with improved pathways for capsular polysaccharide biosynthesis

“…Among these, UDP-Glu, the precursor of UDP-GlcA, is obtained by the sequential conversion of Glc-6P to glucose-1-phosphate (Glc-1P), and further into UDP-Glu, by the concerted action of a phosphoglucomutase (Pgm), and of a pyrophosphorylase (GalU). We previously demonstrated that higher concentrations of Pgm and GalU significantly influenced polysaccharide production and UDP-sugar pools in a recombinant E. coli K4 strain also overexpressing a positive regulator of the CPS operon (Cimini et al, 2015). During the exponential phase, higher titers of both UDP-Glu and UDP-GlcA, and an unexpected reduction of UDP-GlcNAc and UDP-GalNAc pools were observed combined to a significant increase of CPS (Cimini et al, 2015).…”

Increased 3′‐Phosphoadenosine‐5′‐phosphosulfate Levels in Engineered Escherichia coli Cell Lysate Facilitate the In Vitro Synthesis of Chondroitin Sulfate A

“…In this article, we focus on getting a step closer to preparing CS from simple sugars like glucose in an Escherichia coli cell‐based system. Previous reports show that chondroitin can be produced using E. coli K4 and engineered BL21 strains . Other reports also show successful expression of active sulfotransferase enzymes in E. coli .…”

Engineering S. equi subsp. zooepidemicus towards concurrent production of hyaluronic acid and chondroitin biopolymers of biomedical interest

“…The crystal structure of chondroitin polymerase suggested a higher binding affinity for UDP-GalNAc (Zanfardino et al 2010) and, a simulated implementation of the UDP-GalNAc productive branch by glutamine addition boosted capsular polysaccharide production in recombinant E. coli K4 (Cimini et al 2015). However, in the present work addition of GalNAc only resulted in a slight reduction of HA titers, leaving the production of chondroitin unchanged and a higher (80%) concentration of the latter inside the cells compared to that found in control conditions.…”

“…K4 and K5 polysaccharides are consolidated precursors of chondroitin and heparin; however, CS is still obtained from limited and potentially harmful, animal sources such as chicken keel, shark fins, and swine or bovine trachea. Several efforts have been made to use E. coli K4, or its biosynthetic machinery in a different background, to generate cell factories for the production of chondroitin, and encouraging results were obtained (Cimini et al 2013, 2015; He et al 2015; Jin et al 2016). The reason lies in the fact that the capsular polysaccharide of this uropathogenic strain, composed of alternating residues of N -acetylgalactosamine (GalNAc) and glucuronic acid (GlcA), has a structure that closely resembles the backbone of CS.…”