High cell density cultivation of Escherichia coli K4 in a microfiltration bioreactor: a step towards improvement of chondroitin precursor production

Abstract

BackgroundThe bacteria Escherichia coli K4 produces a capsular polysaccharide (K4 CPS) whose backbone is similar to the non sulphated chondroitin chain. The chondroitin sulphate is one of the major components of the extra-cellular matrix of the vertebrate connective tissues and a high value molecule, widely employed as active principle in the treatment of osteoarthritis. It is usually obtained by extraction from animal tissues, but the risk of virus contaminations, as well as the scarceness of raw material, makes this productive process unsafe and unable to satisfy the growing market demand. In previous studies a new biotechnological process to produce chondroitin from Escherichia coli K4 capsular polysaccharide was investigated and a 1.4 g·L-1 K4 CPS concentration was reached using fed-batch fermentation techniques. In this work, on the trail of these results, we exploited new fermentation strategies to further improve the capsular polysaccharide production.ResultsThe inhibitory effect of acetate on the bacterial cells growth and K4 CPS production was studied in shake flask conditions, while a new approach, that combined the optimization of the feeding profiles, the improvement of aeration conditions and the use of a microfiltration bioreactor, was investigated in three different types of fermentation processes. High polysaccharide concentrations (4.73 ± 0.2 g·L-1), with corresponding average yields (0.13 ± 0.006 gK4 CPS·gcdw-1), were obtained; the increase of K4 CPS titre, compared to batch and fed-batch results, was of 16-fold and 3.3-fold respectively, while average yield was almost 3.5 and 1.4 fold higher.ConclusionThe increase of capsular polysaccharide titre confirmed the validity of the proposed fermentation strategy and opened the way to the use of the microfiltration bioreactor for the biotechnological production of chondroitin.

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

“…Previous fermentation experiments on the wild type E. coli K4 performed by our group showed a significant improvement of the final K4 CPS concentration by using a membrane bioreactor combined with a fermentation strategy based on in situ product removal [25]. ECK4r3 would allow the use of well scalable fed-batch processes that do not require additional devices inside the fermentation vessel, which would be a step towards the actual production-scale of tons per year of chondroitin sulfate.…”

The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life

“…248 This demonstrates the power of plant expression systems for this kind of production. Besides plants, algae that naturally produce very valuable fatty acids and lipids 366 have recently received attention for their great potential as heterologous host for the biosynthesis of complex molecules, 367–370 and should be further investigated for application in biofuel production.…”

S-Layer Protein Self-Assembly

“…In archaea, special isolation and purification procedures for the S-layers, including treatments with Triton X-100 and SDS, changes in pH and ionic strength, or extraction with organic solvents, have been applied [50]. In addition, it has also been shown that recombinant S-layer proteins may be secreted into the culture medium and were able to form self-assembly products in suspension but did not recrystallize on the surface of the cells [51]. …”