Constitutive expression of genes relevant to xylose catabolism in ethanol-producing E. coli KO11 for improvement of xylose utilization

Ethanologenic E. coli strain KO11 utilizes glucose preferentially as compared to xylose due to carbon catabolite repression when grown in a mixture of the two sugars. The purpose of this work is to improve the xylose utilization of the KO11 by constitutive expression of genes relevant to xylose catabolism. Two copies of promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene derived from E. coli JM109 were inserted at the EcoRI/SacI sites and the SalI/HindIII sites of plasmid pUC18 to give pUC18comp. First, the xylA gene encoding d-xylose isomerase and xylB gene encoding d-xylulose kinase were inserted at the SacI/KpnI sites of the pUC18comp to give pXOP201. Subsequently, the xylFGH genes encoding components of d-xylose ABC transporter were further cloned at the HindIII site of the pXOP201 to give pXOP503. Constructs KO11(pXOP201) containing xylAB genes and KO11(pXOP503) containing xylAB genes and xylFGH genes as well as the vector control KO11 (pUC18comp) were grown on an orbital shaker (150 rpm) at 30°C for 4 days in the M9 medium (80 ml) supplemented with 6% (w/v) glucose and 4% (w/v) xylose. Glucose in the medium was exhausted in 3 days during the fermentation by the constructs and KO11(pUC18comp). While the control strain KO11(pUC18comp) consumed 30% of the xylose, KO11(pXOP201) and KO11(pXOP503) consumed 50 and 56% of the xylose, respectively. KO11(pUC18comp), KO11(pXOP201), and KO11(pXOP503) produced 4.4, 5.1, and 5.3% (v/v) ethanol, respectively. These results suggest that constitutive expression of xylAB genes and xylFGH genes has partially relieved carbon catabolite repression in the E. coli KO11.