Engineering of Bacillus thuringiensis Cry Proteins to Enhance the Activity against Western Corn Rootworm

Abstract

A novel <i>Bacillus thuringiensis</i> Cry protein, Cry8Hb, active against <i>Diabrotica virgifera virgifera</i> (Western corn rootworm, WCRW) was discovered. Unexpectedly, the anti-rootworm activity of the Cry8Hb toxin was enhanced significantly by fusing <i>Escherichia coli</i> maltose binding protein (MBP) to this Cry toxin. While the exact mechanism of the activity enhancement remains indefinite, it is probable that the enhancement is a result of increased solubility of the MBP-Cry8Hb fusion in the rootworm midgut. This hypothesis was examined using a synthetic Cry3 protein called IP3-1, which was not soluble at a neutral pH like Cry8Hb and marginally active to WCRW. When IP3-1 was fused to MBP, its anti-WCRW activity was enhanced 13-fold. To further test the hypothesis, DNA shuffling was performed on IP3-1 to increase the solubility without MBP. Screening of shuffled libraries found six new IP3 variants showing very high anti-WCRW activity without MBP. Sequence and 3D structure analysis of those highly active, shuffled IP3 variants revealed several charge-altering mutations such as Lys to Glu on the putative MBP-attaching side of the IP3 molecule. It is likely that those mutations make the protein acidic to substitute the functions of MBP including enhancing the solubility of IP3 at a neutral pH.