E. coli K5 fermentation and the preparation of heparosan, a bioengineered heparin precursor

Abstract

Heparosan is an acidic polysaccharide natural product, which serves as the critical precursor in heparin biosynthesis and in the chemoenzymatic synthesis of bioengineered heparin. Heparosan is also the capsular polysaccharide of Escherichia coli K5 strain. The current study was focused on the examination of the fermentation of E. coli K5 with the goal of producing heparosan in high yield and volumetric productivity. The structure and molecular weight properties of this bacterial heparosan were determined using polyacrylamide gel electrophoresis (PAGE) and Fourier transform mass spectrometry. Fermentation of E. coli K5 in a defined medium using exponential fed-batch glucose addition with oxygen enrichment afforded heparosan at 15 g/L having a number average molecular weight of 58,000 Da and a weight average molecular weight of 84,000 Da.

Figure 1

Structure of heparosan. A. repeating disaccharide unit of heparosan B. structure of heparosan chain resulting from the action of heparosan K5 lyase.

Figure 2

¹H-NMR spectra (600 MHz) of heparosan prepared in shake flask culture. A. stack plot of heparosan recovered from: 1. LB medium, 2. LB medium having MW_Avg > 3,000, 3. LB medium having MW_Avg < 3,000, and inset showing PAGE analysis of samples 1, 2 and 3. B. heparosan recovered from M9 medium. C. heparosan recovered from glycerol defined medium. D. heparosan recovered from glucose defined medium.

Figure 3

Production of heparosan in a 7 L fermentor. Panel A shows the glucose feed curve (black), the pH curve (blue) and the dissolved oxygen (% DO) curve (red) as a function of fermentation time (h). Panel B shows the cell growth curve (total DCW g, ▴ ) and heparosan production (g, ■) as a function of fermentation time (h).

Figure 4

Characterization of heparosan purified from the supernatant of a 7 L fermentation. A. ¹H-NMR (600 MHz) of heparosan. B. ¹³C-NMR of heparosan prepared on M9 medium containing ¹³C glucose and ¹⁵N ammonia sulfate. C. FT-MS of a heparosan chain of average molecular weight 4551.81 (degree of polymerization =24) purified by preparative PAGE (Ly et al., 2010).

Figure 5

PAGE used for MW analysis of heparosans with Alcian blue stain. A. molecular standards spanning the range of the gradient gel. Lanes contain: 1. HA molecular markers (30 kD-310 kD), 2. heparosan molecular markers (6.4 kD-14.1 kD). B. heparosans prepared in shake flasks in different media. Lanes contain: 1. heparosan from M9 medium, 2. heparosan from glycerol synthetic medium, 3. heparosan from glucose synthetic medium, and 4. heparosan from LB medium. C. heparosans prepared in a 7 L fermentor sampled at different times. Lanes contain: 1–6. heparosan sampled from the fermentor at 4.5 h; 12.6 h; 14.5 h; 20 h; 32.9 h; and 37.6 h after the start of the fermentation.

Figure 6

Time course of molecular weight properties of heparosan produced in 7 L fermentor. Shown are the trends of M_N ( ), M_W () and PDI (◆).