*BBSRC EASTBIO Programme* Synthetic biology re-designing of biomass quality for biorefining using engineered polyproteins

Summary: This project aims to demonstrate the power of artificial polyproteins as tools for plant synthetic biology by using them to improve biomass quality for biorefining by the introduction of novel biochemical pathways. Self-cleaving polyprotein1 constructs will be used as a one-step method of introducing novel pathways of 2-4 enzymes into barley to alter the composition of lignin. Constructs will also be introduced into genetic backgrounds where CRISPR-Cas9 technology has been used to knock-out key genes and divert lignin precursor down specific pathway routes. These modifications will make the straw biomass easier and cheaper to process, and should yield a more uniform lignin by-product that provides a better platform for high value chemical production. Demonstration of the value of the polyprotein approach in a real biorefinery feedstock will facilitate its wider uptake across the range of plant synthetic biology opportunities.

Background: Plant synthetic biology presents enormous opportunities for industrial biotechnology, whether by engineering plant cells to be cheap production platforms for entirely novel products, or by metabolic engineering to redesign plant-based raw materials. Full exploitation of these opportunities requires development of tools for precise engineering where multiple heterologous proteins and even entire biochemical pathways, can be introduced into plants. However, achieving co-ordinate production of several introduced proteins is difficult1. Repeated use of the same promoter in an effort to achieve coordinated expression increases the risk of transgene silencing. Many viruses have solved the problem of co-ordinating protein production in infected cells by linking proteins in self-processing polyproteins. On translation, viral proteinases mediate co-translational, intramolecular cleavage of the polyprotein to yield discrete protein products. Our previous work showed that the 2A region of several picornaviruses can mediate heterologous polyprotein ‘cleavage’ in planta, into discrete protein products, despite being only 20-26 amino acids long1,2. 2A sequences could be extremely useful building blocks to add to existing standardized parts in a toolkit for the emerging plant synthetic biology sector.

An obvious target for an emerging plant synthetic biology capability is the ‘re-design’ of plant biomass to secure the economic competitiveness of integrated biorefineries.

A major current inefficiency is the expense and energy required to fractionate biomass towards different product streams. This is in part due to lignin, a rigidifying and waterproofing polymer that the carbohydrate components (cellulose/hemi-cellulose) of biomass are embedded in. Lignin’s complex structure makes it difficult to remove to allow enzymes and fermentation organisms to gain access to cell wall carbohydrates and metabolise them into fuels or other products. Much variation is tolerated in the amount and composition of lignin in different plants. This project aims to exploit that and produce plants with a modified lignin structure that makes lignin easier to remove from biomass for biorefinery applications. One target for such manipulation is to increase the proportion of syringyl (S) lignin, as this has previously been shown to improve digestibility and enzymic saccharification (release of sugars)3. Of the main lignin monomers, only S monomers with a fully substituted phenyl ring, can restrict lignin polymerization to a regular pattern.

Two genes, Ferulate-5-hydroxylase (F5H) and Caffeate-O -methyltransferase (COMT) are involved in producing S lignin monomers from guaiacyl (G) lignin monomers. Overexpression of F5H in dicot plants resulted in higher S and reduced G lignin and produced a more uniform polymer. F5H- and COMT-like genes from different plant sources can have different substrate specificities and their heterologous expression in barley could further extend the range of possible modifications to lignin. It remains to be seen whether grasses can be manipulated to increase S lignin, particularly since they contain a third lignin type, p-hyroxyphenyl (H) lignin, which does not convert to S lignin in the ‘normal’ lignin pathway.
Project methods: All techniques essential to the successful pursuit of the project are already well-established in the Halpin lab or at the James Hutton Institute. We regularly perform efficient barley transformation including the production of CRISPR-Cas9 knock outs, and are experts in downstream production of homozygote lines, and molecular and biochemical characterization, including lignin analyses.