Teaching Bacteria to Eat Grass: genetic engineering of Escherichia coli for complete degradation and utilisation of Miscanthus hemicellulose

Academic Lead – Gavin Thomas  (York)

The HUB network (Hemicellulose Utilisation by Bacteria) project provides a unique opportunity to bring together three separate pieces of research ongoing in the White Rose Universities, which previously had no direct connection, into an extremely timely and ambitious integrated project to engineer the model bacterium Escherichia coli for use in consolidated bioprocessing through the complete degradation and utilisation of carbon from lignocellulosic feedstocks. The improved utilisation of such feedstocks by bacteria for biofuel biosynthesis is an active area of current BBSRC-funded research, especially the development of the use of the perennial grass Miscanthus, and is of interest to UK industry. The hemicellulose component of Miscanthus consists of xylan chains highly substituted with arabinofuranose and glucuronic acid with additional cross links between the strands mediated by hydroxycinnamic acids

We will create an E. coli strain that is engineered to degrade this hemicellulose completely and then to utilise fully the resultant products for anaerobic fermentation and biofuel production. The network is based around 3 fundamental scientific breakthroughs that are united by the particular biology of Miscanthus hemicellulose . The first (project 1) builds on the extensive expertise of Paul Knox and Simon McQueen Mason on the structure and degradability of plant cell walls and we wish to develop the expertise of the Knox lab in understanding cell wall heterogeneity and also the targeting of enzymes to hemicelluloses within cell wall contexts for enzymatic degradation. We will characterise a full set of the enzymes that are required for complete and efficient breakdown of Miscanthus hemicellulose molecules and engineer these to be surface located or secreted by E. coli. In project 2, we will engineer E. coli to efficiently transport the sugars and uronic acids released from the hemicellulose, building on the recent discovery in the Thomas lab of a novel transporter specific for the furanose form of arabinose (the only form found exclusively in the hemicellulose) and expertise in the Green lab in using gene regulatory circuits to couple these genes to expression in fermentative conditions. In project 3 we will use the recent discovery in the Kelly lab of the transporters and genes responsible for catabolism of the hydroxycinnamic acids ferulate and coumarate andwill use the expertise of Baldwin and Kelly to engineer these into E. coli. The removal and potential catabolism of these hydroxycinnamic acids during the fermentation is a completely new component of hemicellulose utilisation not previously considered. Together these improvements in E. coli, many of which can be transferred to other relevant bacteria such as Clostridium and Geobacillus should provide significant savings in relation to pretreatment methods and reduced levels of waste from hemicellulose utilisation.

Network Project

Enzyme action and hemicellulose substrate heterogeneity in cell wall

Principal Supervisor – Paul Knox (Leeds)

Transport of hemicellulose-derived sugars and uronic acids by Escherichia coli.

Principal Supervisor  – Gavin Thomas (York)

Engineering Escherichia coli to transport and efficiently metabolise ferulate

Principal Supervisor – Dave Kelly (Sheffield)