Biosynthesis of high-purity amylopectin for high-performance bioplastic.
Plastic is one of the most common materials in our daily lives, but discarded plastic products also pose a serious environmental hazard to us. They are difficult to degrade in the natural
environment, and during their slow degradation process and incineration treatment, they release a variety of toxic substances, polluting the soil, water, and atmosphere and posing a serious
threat to plants, animals, and even humans. With the development and progress of society, the demand for non-polluting or low-polluting biodegradable plastics is increasing.
Due to the wide and inexpensive source of plant starch in nature, starch-based bio plastics are particularly important among various biodegradable plastics. However， because the plant starch
includes both amylopectin and amylose, their different content ratios have different effects on the performance of starch-based bioplastics. It is difficult to obtain starch-based bioplastics
with high performance using existing plant starches as raw materials. This situation can be changed by adjusting the proportions of the two components in raw starch-which requires the use of
high-purity amylopectin and amylose.
The regulatory protein for sweet potato amylose synthesis is granule-bound starch synthase I (IbGBSSI). Therefore, we can use CRISPR/cas9 technology to knockout the IbGBSSI gene so that sweet
potatoes no longer synthesize amylose and obtain high-purity amylopectin.
This allows us to save a series of complex purification processes and easily obtain pure amylopectin, which can better be used to adjust the ratio of amylopectin to amylose in raw starch and
produce different performance starch-based biodegradable plastic products.
In order to obtain high-purity amylopectin, the starch synthesis pathway in the plant sweet potato (Ipomoea batatas) was modified using CRISPR/Cas9 system. Firstly, sgRNA oligos targeting the
IbGBSSI gene was designed and annealed, and was inserted into the plasmid psgR-Cas9-At to construct the backbone vector of sgRNA (IbGBSSI), which was combined with the binary vector pCAMBIA1301s
to obtain the expression vector of sgRNA (IbGBSSI). Afterwards, this knockout construct was transformed into embryogenic calli of sweet potato using the method of Agrobacterium-mediated
transformation and then IbGBSSI-deficient mutants were received after further culturing and screening. After detections of the storage root starch of the mutants, it was showed that, compared to
the wild-type, the total starch content in the storage roots of the mutants was hardly changed, but the proportion of amylose was significantly reduced to between 0.5% and 0.8% while it was
23.33% of the wild-type.
It was indicated that sweet potato lines with high-purity amylopectin was obtained. In the future, similar methods would be used to obtain lines with high-purity amylose for better use in the
production of different performance starch-based biodegradable plastics.