**Misincorporation of Galactose by Chondroitin Synthase of** *Escherichia coli* **K4: From Traces to Synthesis of Chondbiuronan, a Novel Chondroitin-Like Polysaccharide**

### **Mélanie Leroux 1,2, Julie Michaud <sup>2</sup> , Eric Bayma <sup>2</sup> , Sylvie Armand <sup>2</sup> , Sophie Drouillard <sup>2</sup> and Bernard Priem 2,\***


Received: 25 November 2020; Accepted: 10 December 2020; Published: 12 December 2020 -

**Abstract:** Chondroitin synthase KfoC is a bifunctional enzyme which polymerizes the capsular chondroitin backbone of *Escherichia coli* K4, composed of repeated β3N-acetylgalactosamine (GalNAc)-β4-glucuronic acid (GlcA) units. Sugar donors UDP-GalNAc and UDP-GlcA are the natural precursors of bacterial chondroitin synthesis. We have expressed KfoC in a recombinant strain of *Escherichia coli* deprived of 4-epimerase activity, thus incapable of supplying UDP-GalNAc in the bacterial cytoplasm. The strain was also co-expressing mammal galactose β-glucuronyltransferase, providing glucuronyl-lactose from exogenously added lactose, serving as a primer of polymerization. We show by the mean of NMR analyses that in those conditions, KfoC incorporates galactose, forming a chondroitin-like polymer composed of the repeated β3-galactose (Gal)-β4-glucuronic acid units. We also show that when UDP-GlcNAc 4-epimerase KfoA, encoded by the K4-operon, was co-expressed and produced UDP-GalNAc, a small proportion of galactose was still incorporated into the growing chain of chondroitin.

**Keywords:** chondroitin; K4 chondroitin synthase; NDP-sugar misincorporation
