*3.1. Engineering of E. Coli to Produce Chondroitin*/*Chondbiuronan Lactose*

Metabolic engineering of recombinant strains is shown in Figure 1.

The recombinant strain capable of polymerizing exogenously added lactose through the action of GAG synthases was previously described [14]. As a derivative of *E coli* DH1, the strain does not synthesize GalNAc, contrary to *E coli* K4, which expresses KfoA, an UDP-GlcNAc 4-epimerase.

Briefly, genes *lacZ* and *wcaJ* were knocked-out, resulting in the incapacity of the strain to degrade lactose (Lac), or to produce colanic acid which is a GlcA-containing polysaccharide [18]. In addition, recombinant mammal β1,3-glucuronyltransferase GlcAT-P and UDP-Glc dehydrogenase KfiD were expressed, thus allowing the production of glucuronyllactose (GlcA-Lac) upon Lac implementation [19,20]. Genes *kfiD* and *glcAT-P* were both expressed in low copy pBBR, while the gene *kfoC* encoding chondroitin synthase was expressed in high copy pBluescript.

β **Figure 1.** Synthesis of chondbiuronan and chondroitin in engineered *Escherichia coli*. Lactose (Lac) is taken up by lactose permease (LacY) and modified by the action of recombinant mouse β-1,3 glucuronyltransferase (GlcAT-P), leading to glucuronyl-lactose formation, UDP-GlcA being provided by the action of recombinant UDP-Glc dehydrogenase (KfiD) from the *E. coli* strain K5. Recombinant chondroitin synthase KfoC catalyses (i) synthesis of chondbiuronan in strain EcDGCø from UDP-GlcA and UDP-Gal constitutively produced by GalE or (ii) synthesis of chondroitin in strain EcDGCA from UDP-GlcA and UDP-GalNAc produced by recombinant epimerase KfoA. Genotypes: EcDGCø, strain DJ (pBS-kfoC, pBBR-kfiD); EcDGCA, strain DJ (pBS-kfoC, pBBR-kfiD, pBAD-kfoA).

The strain named "EcDGCø" expressing three recombinant genes *kfoC*, *glcAT-P,* and *kfiD* was constructed to examine the capacity of the enzyme KfoC to produce a polysaccharide in absence of UDP-GalNAc.

The strain "EcDGCA" was constructed in order to observe the incorporation of GalNAc while expressing UDP-GlcNAc 4-epimerase KfoA, providing UDP-GalNAc from UDP-GlcNAc epimerisation. Gene *kfoA* was cloned in medium copy pBAD33 carrying the tightly regulated promoter araBAD. Expression of the epimerase was induced by arabinose, independently of the other three recombinant genes induced by IPTG. It was thus possible to modulate the level of epimerase activity without changing the expression of the other recombinant genes. Strain EcDGCA was very similar to strain K-cho that we have described previously, capable of producing recombinant chondroitin from lactose and lactose-furyl [14].

Recombinant strains cultures were driven on minimal medium and cultivated for 72 h in fed-bach conditions (Figure 2).

**Figure 2.** Production of recombinant polysaccharide during growth of engineered strains cultivated in bioreactor. Titer is calculated on the basis of the uronic acid assay. Error bars represent standard deviation of two independent cultures.

− All cultures accumulated an ethanol-precipitated product intracellularly as the synthesis occurred from cytoplasmic lactose and *kps* genes involved in polysaccharide export were absent from K-12 strains [21]. Accordingly, strain EcDGCA accumulated several g·L <sup>−</sup><sup>1</sup> of chondroitin at the end of the culture. Interestingly enough, it could be observed that the rate of production increased with epimerase induction controlled by the addition of arabinose, suggesting that it was a limiting parameter of chondroitin synthesis. We can reasonably hypothesize that the epimerase induction level impacts the cytoplasmic concentration of UDP-GalNAc, probably limiting in the polymerization reaction. Strain EcDGCø culture lacking epimerase slightly accumulates a lower amount of unknown polysaccharide (compound **1**). However, this compound was only noticeable after 2 days of glycerol feeding.
