Microbial starch-binding domains as a tool for modifying starch biosynthesis

Modification of the starch biosynthesis pathway holds an enormous potential for tailoring novel starches in planta . In this thesis, we have explored the possibility of anchoring effector proteins in potato starch granules during starch biosynthesis by using starch-binding domains (SBDs) of starch degrading enzymes. In this way, starches with new or improved functionalities may be generated. For this, a family 20 SBD derived from the Bacillus circulans cyclodextrin glycosyltransferase (CGTase) was used as an anchor for making granule-bound proteins.A separate SBD (thus without a protein attached to it) was expressed in the tubers of two potato cultivars (Kardal and Karnico) and the amylose-free ( amf ) potato mutants under the control of the potato granule-bound starch synthase I (GBSSI) promoter. The potato GBSSI transit peptide mediated amyloplast entry of the SBD. SBDs accumulated inside starch granules and not at the granule surface. Amylose-free granules contained 8 times more SBD than the two amylose-containing ones. The physicochemical properties of the transgenic starches were not affected by SBD accumulation. Our results demonstrate that the SBD can be used as anchor for making granule-bound proteins.The SBD was fused to the C- or N-terminus of the luciferase (LUC) gene via a PT-linker. A similar construct without the linker and SBD was used as a control. After introduction of these genes into potato plants, it was observed that the SBD-containing fusion proteins could be accumulated inside the granules with retention of luciferase activity, whereas the luciferase alone did not. These results indicate that SBDs can anchor proteins (without affinity for starch granules) to granules with retention of the activity of effector protein. Furthermore, the C-terminal SBD can also be used as an N-terminal anchor for attaching luciferase to granules, although it seemed to be more efficient at the C-terminus.The efficiency of two granule-targeting sequences, SBD and GBSSI, for attaching active luciferase protein to amf granules during starch biosynthesis was investigated. The GBSSI was fused to the C- or N-terminus of luciferase. The luciferase activity in the most positive transformant of each fusion protein was much lower than that of SBD-containing ones when expressing these genes in amf potato plants. In addition, the amount of GBSSI protein accumulated in these granules was much less than that in amf granules complemented with the native potato GBSSI. It appeared the SBD was superior to GBSSI as a targeting sequence for luciferase to starch granules.An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered and used to investigate whether it is a higher-affinity anchor than SBD. The affinity of SBD and SBD2 for starch was tested both in vitro and in planta . Both genes were expressed in E. coli , and the affinity for soluble starch of purified proteins was determined. SBD2 had an approximately 10-fold higher binding affinity for starch than SBD, indicating that two appened SBDs act in synergy when binding to their target ligand. The increased affinity of SBD2 for starch was also observed inplanta . Higher levels of SBD2 could be accumulated in the Kardal and amf granules (KDSS and amf SS series, respectively) compared with SBD in the two backgrounds. These results demonstrate that SBD2 is a higher-affinity anchor than SBD. However, it was observed that accumulation of SBD2 in potato starch granules to interfered with certain aspects of the starch biosynthesis process, which resulted in changes in granule packing, morphology (Kardal and amf ), size (only observed in amf ), crystallinity, and the formation of growth rings (only appeared in Kardal). However, the accumulation of SBD2 in the KDSS and amf SS starch granules did not affect the starch content and primary structure of the constituent starch molecules. Our results demonstrate that the SBD technology has a great potential for tailoring starches with novel properties in planta

Bibliographic Details

Main Author:	Ji, Q.
Other Authors:	Visser, Richard
Format:	Doctoral thesis biblioteca
Language:	English
Subjects:	biosynthesis, gene expression, genetic engineering, plant breeding, potatoes, solanum tuberosum, starch, aardappelen, biosynthese, genetische modificatie, genexpressie, plantenveredeling, zetmeel,
Online Access:	https://research.wur.nl/en/publications/microbial-starch-binding-domains-as-a-tool-for-modifying-starch-b
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