Fusion proteins comprising the catalytic domain of mutansucrase and a starch-binding domain can alter the morphology of amylose-free potato starch granules during biosynthesis |
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Authors: | Farhad Nazarian Firouzabadi Géraldine A. Kok-Jacon Jean-Paul Vincken Qin Ji Luc C. J. M. Suurs Richard G. F. Visser |
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Affiliation: | (1) Graduate School Experimental Plant Sciences, Laboratory of Plant Breeding, Wageningen University, 386, 6700 AJ Wageningen, The Netherlands;(2) Agronomy and Plant Breeding Group, Faculty of Agriculture, University of Lorestan, 465, Khorramabad, Iran;(3) Present address: Laboratory of Tropical Crop Improvement, Division of Crop Biotechnics, Kasteelpark Arenberg 13-3001, Leuven, Belgium;(4) Laboratory of Food Chemistry, Wageningen University, 8129, 6700 EV Wageningen, The Netherlands;(5) Present address: Department of Biology, HuaiYin Teachers College, 223300 Huaian, China |
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Abstract: | It has been shown previously that mutan can be co-synthesized with starch when a truncated mutansucrase (GtfICAT) is directed to potato tuber amyloplasts. The mutan seemed to adhere to the isolated starch granules, but it was not incorporated in the starch granules. In this study, GtfICAT was fused to the N- or C-terminus of a starch-binding domain (SBD). These constructs were introduced into two genetically different potato backgrounds (cv. Kardal and amf), in order to bring GtfICAT in more intimate contact with growing starch granules, and to facilitate the incorporation of mutan polymers in starch. Fusion proteins of the appropriate size were evidenced in starch granules, particularly in the amf background. The starches from the various GtfICAT/SBD transformants seemed to contain less mutan than those from transformants with GtfICAT alone, suggesting that the appended SBD might inhibit the activity of GtfICAT in the engineered fusion proteins. Scanning electron microscopy showed that expression of SBD-GtfICAT resulted in alterations of granule morphology in both genetic backgrounds. Surprisingly, the amf starches containing SBD-GtfICAT had a spongeous appearance, i.e., the granule surface contained many small holes and grooves, suggesting that this fusion protein can interfere with the lateral interactions of amylopectin sidechains. No differences in physico-chemical properties of the transgenic starches were observed. Our results show that expression of granule-bound and “soluble” GtfICAT can affect starch biosynthesis differently. |
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Keywords: | Mutan Glucansucrase Starch-binding domain Granule-boundness Transgenic potato Microscopy Granule morphology |
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