*3.3. Galactose Incorporation Remains in Recombinant Chondroitin*

We compared the <sup>1</sup>H and <sup>13</sup>C NMR spectra of chondbiuronan (Figures 3A and 4A) to the ones of chondroitin produced by the strain EcDGCA at different induction levels of KfoA; 0.5% (Figures 3B and 4B) and 2% arabinose (Figures 3C and 4C). In addition to the signals found with chondbiuronan, they showed NMR signals that we attributed to N-Acetylgalactosamine (GalNAc) residues as well as glucuronic acid (GlcA) residues linked to GalNAc rather than Gal. The level of incorporated Gal could be calculated according to the <sup>1</sup>H NMR signals integration: the Gal:GalNAc ratio was of 4:6 and 2:8 at 0.5% and 2% arabinose, respectively, arguing for a competition between Gal and GalNAc incorporation, related to the level of induction of KfoA: the more UDP-GalNAc, the less galactose incorporation.

The chemical shifts of the corresponding protons and carbons were determined using heteronuclear single-quantum correlation experiments (HSQC, Supplementary Figure S1) and correlation spectroscopy (COSY, Supplementary Figure S3) and are reported in Table 2.


**Table 2.** <sup>1</sup>H and <sup>13</sup>C NMR chemical shifts (δ, ppm) of chondbiuronan (EcDGCø) and chondroitin (EcDGCA) obtained using 0.5% and 2% arabinose induction.
