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

Among capsulated bacteria, some produce polysaccharides with unique properties that have been shown to possess relevant industrial applications and commercial value. The capsular polysaccharide (CPS) produced by Escherichia coli K4 is similar to chondroitin sulphate, and recent efforts focused on the development of genetic and fermentation strategies to increase its production titers up to technologically attractive levels. However, the control of the metabolic pathways leading to CPS synthesis together with the effect of varying the concentration of pathway intermediates on CPS final titers, is still quite unexplored, and not fully understood. In the present study four genes involved in the biosynthesis of UDP-sugar CPS precursors, namely kfoA, kfoF, pgm, and galU, were overexpressed in different combinations, and diversely affected the biosynthetic machinery. At the physiological level, results revealed a central role for kfoF, coding for UDP-glucose dehydrogenase, that increased CPS production mostly. In the attempt to unravel the molecular mechanisms regulating CPS biosynthesis, an in depth analysis of the proteome of the recombinant strains overexpressing respectively pgm and galU, and pgm, galU, and kfoF was performed and compared to the wild-type. Although, interestingly, in both strains the impact of the genetic manipulation seemed rather limited at the proteome level, results obtained from the triple mutant indicated a crosstalk between the two pathways leading to UDP-sugar precursors biosynthesis, and also an unexpected link with the purine biosynthetic pathway. Overall our results present new insights into the role of metabolic intermediates for the formation of capsular polysaccharides, utilizing a systematic approach of metabolic engineering, combined with state-of-the-art quantitative proteomic approaches, as well as genetic and physiological information.

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

Glycosaminoglycans, such as hyaluronic acid and chondroitin sulphate, are not only more and more required as main ingredients in cosmeceutical and nutraceutical preparations, but also as active principles in medical devices and pharmaceutical products. However, while biotechnological production of hyaluronic acid is industrially established through fermentation of Streptococcus spp. and recently Bacillus subtilis, biotechnological chondroitin is not yet on the market. A non-hemolytic and hyaluronidase negative S. equi subsp. zooepidemicus mutant strain was engineered in this work by the addition of two E. coli K4 genes, namely kfoA and kfoC, involved in the biosynthesis of chondroitin-like polysaccharide. Chondroitin is the precursor of chondroitin sulphate, a nutraceutical present on the market as anti-arthritic drug, that is lately being studied for its intrinsic bioactivity. In small scale bioreactor batch experiments the production of about 1.46 ± 0.38 g/L hyaluronic acid and 300 ± 28 mg/L of chondroitin with an average molecular weight of 1750 and 25 kDa, respectively, was demonstrated, providing an approach to the concurrent production of both biopolymers in a single fermentation.

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

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.

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

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.

Monosaccharide precursors for boosting chondroitin-like capsular polysaccharide production.

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.

Improved fructosylated chondroitin production by kfoC overexpression in E. coli K4.

Escherichia coli K4 is one of the bacteria expressing a surface polysaccharide, indicated as capsular polysaccharide (K-antigen), showing a chemical structure that resembles that of metabolites commonly used in pharmaceutical applications. In this study we provide evidence that homologous overexpression of the chondroitin polymerase, encoded by the kfoC gene, acts on a potential bottleneck for production of capsular polysaccharide, and increases productivity by 100%. However, we also demonstrate that genetic engineering and scale-up of the production process with E. coli K4 is not straight forward due to genetic instability of recombinant strains, partly overcome by multiple additions of antibiotic throughout fermentation that prove to increase plasmid maintenance inside the cells. A lower resistance to the antibiotic was nevertheless highlighted in the stationary phase suggesting other concomitant causes for plasmid instability. The latter might partly be related to a newly discovered endogenous mobile element that we indicate as pK4EC05. Sequencing and analysis of a 1900 bp fragment of pK4EC05 shows a high percentage of sequence similarity to large conjugative plasmids isolated from Shigella, Salmonella and E. coli strains.