Role of xylR gene on the construction of xylose-inducible expression vectors using xylF promoter of escherichia coli

For construction of xylose-inducible expression vector, 428 bp of xylF promoter was ligated between AatII and HindIII of pUC18, and named pXF428. To investigate the role of XylR protein encoded by xylR on xylF promoter, 1,988 bp of xylR including its promoter was ligated downstream of multiple cloning site (MCS) in the opposite direction of xylF promoter, and named pXFR428. For the measurement of the expression level, 3,048 bp of lacZ was inserted into MCS downstream of the xylF promoter in plasmids pXF428 and pXFR428 as reporter genes and named pXF428- lacZ and pXFR428-lacZ, respectively. The recombinant plasmids were transformed into Escherichia coli JM109 and DH60, which contains a single point mutation in xylR of E. coli JM109. The constructed plasmids pXF428-lacZ and pXFR428-lacZ inductively produced β-galactosidase by addition of xylose. In β-galactosidase assays of pXF428-lacZ and pXFR428-lacZ in JM109, β- galactosidase activity of pXFR428-lacZ/JM109 induced with xylose was 2.2-fold higher than that of pXF428-lacZ/JM109 with no induction. In xylR mutant E. coli DH60, β-galactosidase activity of pXFR428-lacZ was 7.6 times higher by induction with xylose than that of pXF428-lacZ with no induction. Expression level of lacZ gene in pXF428-lacZ/DH60 was very low, because xylR was defective in both chromosome and plasmid. XylR protein was confirmed to act as an activator on xylF promoter. Inductive production of enzymes in pXF428-lacZ/JM109 and pXFR428-lacZ/ JM109 showed over 90% repression by addition of 0.5% glucose in the culture broth. xynA of Streptomyces thermocyaneoviolaceus was successfully expressed in pXF428 and pXFR428 in E. coli BLR(DE3). These results show that both pXF428 and pXFR428 using xylF promoter are sufficient as new expression systems controlled by xylR, and are essential for the gene expression on xylF promoter of the constructed vectors.