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.

Conclusions: The present study describes the identification of a gene target and the application of a successful metabolic engineering strategy to the unconventional host E. coli K4 demonstrating the feasibility of using the recombinant strain as stable cell factory for further process implementations.

Figure 1

Analysis of rfaH, kfoC and kfoA gene expression in E.coli K4 and EcK4r3 in shake flask experiments. Histogram representing the fold overexpression of the genes under investigation in the recombinant strain EcK4r3 compared to the wild type E.coli K4.

Figure 2

Time course of 24h batch fermentations on the control medium: comparison of strains EcK4r1 and EcK4r3. (a) Production of biomass and K4 CPS (b) Consumption of glycerol and production of total acids throughout the process.

Figure 3

Production of K4 CPS in 2L DO-Stat fed-batch experiments. Time course of biomass, polysaccharide production and residual glucose.

Figure 4

Relative changes of K4 CPS, biomass, and yields on different media for EcK4r3 and E.coli K4 (wt). Percentages in boxes with the same grey tone are referred to the combination of strain and medium indicated with ref (equal to 100%). Abbreviations: Glu (Glucose), Gly (Glycerol), Ye (Yeast extract).

Figure 5

General overview on the effect of inserting additional copies of rfaH in E.coli K4. It shows that overexpression of rfaH leads to higher production of capsular polysaccharide that is probably due to an increase of expression of the genes belonging to region 2; in particular the mRNA levels of kfoA and kfoC were analysed in this study. The figure also indicates that rfaH overexpression increases lipopolysaccharide production.

Figure 6

Schematic representation of the integration cassette used for the construction of EcK4r1 and EcK4r3.