Carbohydrate engineering

Workpackage G: Carbohydrate engineering

Plants provide unrivalled opportunities for provision of sugars and polysaccharides for biorefining, biofuels, animal feed, food and other industrial uses. The main goal of this workpackage is to improve the quality and increase the yield of target polymers, and to alter their structure for higher value applications. The targets will be plant cell wall polymers that important to these applications: xylan, mannan, and novel digestible glucans. The objectives will be achieved by building a registry of polysaccharide synthesis pathway genes and transcription factors that can be co-ordinately expressed using tested promoters from this and other workpackages.

OpenPlant is developing a library of DNA parts for engineering of carbohydrates in plants.

Engineering mannan and xylan in fibre cells

Section through Gnetum wood, confocal                image: Jim Haseloff

Section through Gnetum wood, confocal                image: Jim Haseloff

In 2017 work is starting on a project in the Dupree lab to produce the following resources for public release:

  1. Inducible expression systems in fibre cells
  2. Genes for engineering of ectopic mannan synthesis
  3. Genes for engineering of xylan synthesis

A complementary project in the Leverhulme Natural Material Innovation Centre will study the properties of plants engineered in OpenPlant. This project aims to improve materials from plants, such as timber, for building construction.

Jan Lyczakowski (Dupree Lab) is researching xylan engineering and was awarded an OpenPlant Fund to deveop the "Whiskeroscope", a rodent whisker inspired sensor for the analysis of plant tissue structure.

A toolkit of algal glucan-active enzymes

A complete informatics analysis has been conducted on two transcriptome datasets generated by the Field lab for the photosynthetic protozoan Euglena gracilis, cultured under autotrophic and heterotrophic conditions (O’Neill et al., 2015). This identifies an unexpectedly large repertoire of carbohydrate active enzymes, including many involved in storage betaglucan metabolism and a range of what appear to be hemicellulose synthesising enzymes, although Euglena is not known to produce such glycans. The abovementioned analysis of the Euglena transcriptome has been released in the Carbohydrate-Active Enzymes (CAZy) database, with primary data in the process of being deposited through EBI. All data is available via the JIC web site: http://jicbio.nbi.ac.uk/euglena/

image: Euglenia, shared at JIC bio

image: Euglenia, shared at JIC bio

Advanced bioinformatics and structure homology prediction approaches are being used to identify candidate algal beta1,3 glucan phosphorylases.

Further analyses of algae, such as Emiliania and Prymnesium, is ongoing together with an Innovate UK funded project, to assess their repertoire of polysaccharide and natural product glycosylation capabilities to feed into synthetic biology and industrial biotechnology studies.

Engineering carbohydrate content in potatoes

Aytug Tuncel (Smith Lab, JIC) is working with the BRACT transformation group at the John Innes Centre to establish a transformation method for potatoes. This work is supported by additional funding from the Norwich Research Park Innovation fund.

Aytug Tuncel is applying and testing the genome editing tools and technologies developed in the Patron lab to generate novel, commercially or nutritionally valuable glucans in model plant and crop species. The primary objective is the creation of potatoes that contain digestion-resistant starches with two major nutritional benefits: reduced calorie intake from consumption of chips, crisps and other potato-based snack foods and increased supply of complex carbohydrates to the microbiota of the lower gut that reduces risk of several diseases including colorectal cancer and type II diabetes. Constructs encoding RNAguided Cas9 to target starch branching enzymes in the potato genome have been assembled and delivered to potatoes by the BRACT group. Several potato plantlets have successful editings in different isoforms of the starch branching enzymes and are being grown up to be re-examined for increased gene editing. In addition, new vectors were constructed which are expected to improve editing efficiency in a second generation of mutants.

In an alternative approach, a protocol to isolate protoplasts from potato is being implemented and optimized. Potato protoplast will then be transformed with the new constructs and regenerated into plants with a high chance obtaining starch branching enzyme null mutants.

References

O'Neill, E.C., Trick, M., Hill, L., Rejzek, M., Dusi, R.G., Hamilton, C.J., Zimba, P.V., Henrissat, B., and Field, R.A., 2015. The transcriptome of Euglena gracilis reveals unexpected metabolic capabilities for carbohydrate and natural product biochemistry. Mol. BioSyst., 11, p2808-2820. DOI: 10.1039/c5mb00319a.