Microbial starch-binding domains as a tool for targeting proteins to granules during starch biosynthesis.

Abstract

Modification of starch biosynthesis pathways holds an enormous potential for tailoring granules or polymers with new functionalities. In this study, we explored the possibility of engineering artificial granule-bound proteins, which can be incorporated in the granule during biosynthesis. The starch-binding domain (SBD)-encoding region of cyclodextrin glycosyltransferase from Bacillus circulans was fused to the sequence encoding the transit peptide (amyloplast entry) of potato granule-bound starch synthase I (GBSS I). The synthetic gene was expressed in the tubers of two potato cultivars (cv. Kardal and cv. Karnico) and one amylose-free (amf) potato mutant. SBDs accumulated inside starch granules, not at the granule surface. Amylose-free granules contained 8 times more SBD (estimated at ca. 1.6% of dry weight) than the amylose-containing ones. No consistent differences in physicochemical properties between transgenic SBD starches and their corresponding controls were found, suggesting that SBD can be used as an anchor for effector proteins without having side-effects. To test this, a construct harbouring the GBSS I transit peptide, the luciferase reporter gene, a PT-linker, and the SBD (in frame), and a similar construct without the linker and the SBD, were introduced in cv. Kardal. The fusion protein accumulated in starch granules (with retainment of luciferase activity), whereas the luciferase alone did not. Our results demonstrate that SBD technology can be developed into a true platform technology, in which SBDs can be fused to a large choice of effector proteins to generate potato starches with new or improved functionalities.