*3.2. Structural Identification of Unknown Polymer Formed by KfoC in Absence of GalNAc*

Compound **1** produced by strain EcDGCø as well as chondroitin produced by EcDGCA were purified and subjected to various analyses. The HPSEC-MALS analysis of compound **1** revealed a very low molecular weight, compared to chondroitin samples (Table 1).


**Table 1.** HPSEC-MALS analysis of chondbiuronan and chondroitin in this work. Error is the mean of two independent experiments.

We then acquired the <sup>1</sup>H NMR (Figure 3A) and <sup>13</sup>C NMR (Figure 4A) spectra of compound **1**. They showed characteristic signals of glucuronic acid and galactose linked in β1-3 and β1-4 respectively. Indeed, this data proved that KfoC incorporated galactose in absence of UDP-GalNAc, enough to lead to the synthesis of a small chondroitin-like polysaccharide we named "chondbiuronan."

Considering the very low size of chondbiuronan, we decided to run a mass spectrometry analysis. MALDI-TOF-MS analysis in linear negative mode allowed to obtain several molecular ion clusters separated by 338.3 daltons which correspond to the [Gal-GlcA] motif (Figure 5).

β β

**Figure 3.** <sup>1</sup>H NMR spectra of chondbiuronan (**A**) and chondroitin obtained using 0.5% (**B**) and 2% (**C**) arabinose induction. Inset: chemical structure of the polysaccharides.

**Figure 4.** <sup>13</sup>C NMR spectra of chondbiuronan (**A**) and chondroitin obtained using 0.5% (**B**) and 2% (**C**) arabinose induction. Inset: chemical structure of the polysaccharides.

Although this analysis does not reflect the true size repartition of the sample as signal detection decreases with size, we clearly see that main molecular ions belong to odd-numbered polymers, indicating that they contain GlcA at non-reducing termini and lactosyl motif at the reducing termini. The fact that GlcA residues terminate elongating chains suggests that the glucuronyltransferase activity

of KfoC was not limiting in the polymerization reaction, and confirms that the GalNAc-T activity was the limiting reaction instead, as suggested before.
