This project will aim to develop both synthetic biology and hardware tools to better understand and utilise the pectic polysaccharides.
Plant cell walls are an intricate assembly of polysaccharides and phenolic compounds. There is a significant interest in the use of plant cell walls as a source of energy and to extract compounds which may have industrial application. One of the biggest hurdles in the development of cell wall derived products is our poor understanding of cell wall biosynthesis. Synthesis of polysaccharides occurs mainly through the activity of Glycosyltransferase (GT) enzymes that transfer an activated nucleotide-sugar onto a specific growing polysaccharide acceptor. The ability to manufacture sugar nucleotides is therefore of profound importance to the development of novel technologies for study and engineering of polysaccharide biosynthesis. While many sugar nucleotides are commercially available, some of the key sugar nucleotides employed in the synthesis of pectin are not available. Better understanding of cell wall biosynthesis, enabled through experiments using manufactured sugar nucleotides, may enable us to engineer the polysaccharides to have desired properties. This project will focus on pectin, which is a heterogenous polysaccharide found in plant primary cell walls. Pectin is abundant in food waste and as a highly negatively charged polymer it might find industrial application. We will aim to develop a synthetic biology toolbox to synthetise nucleotide sugars, which is required for in vitro analysis of pectin biosynthesis enzymes and is currently not available commercially.
We propose to develop the technology to manufacture a range of novel sugar nucleotides that are presently not available commercially. This will include sugar nucleotides that are required to study pectin biosynthesis enzymes. Sugar nucleotide biosynthesis occurs in the Golgi apparatus and is performed by a range of different enzymes. As a part of our project we propose to synthetise GoldenGate compatible modules encoding sugar nucleotide biosynthetic enzymes and express those in E. coli. Expressed enzymes will be used to convert commercially available sugar nucleotides into sugar nucleotides that are not available commercially, or that are prohibitively expensive. Sugar nucleotides will then be purified through a combination of enzyme treatments and chromatographic separations.
By doing so we will enable further study into the structure and function of glycosyl transferases and the manufacture of novel pectic saccharides.
This project is due to report in 2018