13TSB_CRD - Flexible Engineered Solutions for Xylose Metabolism Using Synthetic Biology (FLEX)

The development of biofuels to replace our reliance on petrochemical-based fuels is a challenging economic problem. Basic science can make significant contributions to this problem by making the process more efficient and hence more economically competitive. The replacement of starch-based feedstock for the production of biofuels like biobutanol with more sustainable lignocellulose-based material is challenging due to the complexity of this material to efficient breakdown into forms that can used by the biofuel producing bacteria. In this project we wish to use new synthetic biology methods to engineer a whole new biological property into two biotechnologically important bacteria, namely the ability to grow on hemicellulose-derived xylo-oligomers.

The formation of xylo-oligomers such as xylotetraose (X4), xylotriose (x3) and xylobiose(X2) occurs during the enzymatic breakdown of the hemicelluloses by xylanases. Further breakdown to the monosaccharide xylose (X1) requires the action of beta-xylosidases and then the free xylose is transported into bacterial cell via active transporters. The utilisation of the xylose monosaccharide has been the focus on most attention in the engineering of microbes for efficient hemicelluloses utilisation and the use of the xylo-oligomers directly has not been considered very widely. In this project we wish to engineer bacteria to use this material more efficiently. By using transporters for X2-X4, which take up one of these molecules for one unit of energy, the bacteria are much more efficient than those which have to each 2-4 units of energy to take up the same amount of sugar present in the monomeric form. There is no energetic costs to break down the X2-X4 to X1 on the inside. Bacteria growing under anaerobic fermentatative conditions used to make biobutanol are energy limited and so this will make a demonstrable increase in their growth rates as oligo-xylans are the primary carbon source available when growing on hemi-cellulose and hence these reactions have high flux. We will also investigate using a more efficient secondary transporter versus a primary ABC transporter to further improve growth yields. At a bioprocess level the use of xylo-oligomers rather than the fully hydrolysed xylose allow the use of milder chemical hydrolysis steps in the hemicelluloses pretreatment which also results in lower rates of production of chemicals that subsequently inhibit the fermentation.

The xylo-oligomer clusters, Xylo4, will be created and tested in Escherichia coli and then transferred to industrial Clostridium strains used by Green Biologics that are currently being used in China by partners of Green Biologics to make butanol from lignocellulose.

The development of biofuels to replace our reliance on petrochemical-based fuels is a challenging economic problem. Basic science can make significant contributions to this problem by making the process more efficient and hence more economically competitive. The replacement of starch-based feedstock for the production of biofuels like biobutanol with more sustainable lignocellulose-based material is challenging due to the complexity of this material and difficulties in efficient breakdown into forms that can used by the biofuel producing bacteria. Current commercial processes require the use of extreme heat and harsh chemicals to convert agricultural waste material to simple fermentable sugars. This not only reduces the environmental benefits of renewables but also makes the fermentation of waste feedstocks less economic when compared with easier substrates such as starch. In this project we will use new synthetic biology methods to engineer a new biological properties into biotechnologically important bacteria. The aim is to improve their ability to grow on hemicellulose-derived sugars released during less intensive pre-treatment procedures to produce bio-butanol with high efficiency, thereby reducing the environmental impact of harsh chemical and high temperature processes and reducing the competition for food crops as fermentation substrates.

The work described in this application clearly has an applied nature and as such its impact is direct and immediate, the beneficiaries being the UK biotechnology sector. In this short-term project we communicate our work through two specific activities. The PI, Dr Thomas, will deliver a lecture to the Yorkshire Philosophical Society on the applications of microbes in bioenergy and biofuels. This could also be delivered to local secondary schools. Dr Thomas has recently given a public lecture at the York Castle Museum on microbiology in the early 20th, which included Weismann's work on the acetone-butanol ethanol (ABE) fermentation. Our second activity will be to work with the Society of General Microbiology to prepare short briefing documents/factsheets about the use of microbes in biofuel production.

If the approach is successful we have the infrastructure in York for commercialisation of work from this project and we will build strong links with Green Biologics. One immediate impact is that Green Biologics are providing material for a White Rose PhD network application in this area that is in preparation and we anticipate further collaborative work.

Although only an 18 month position for the PDRA, he/she will gain practical experience in the synthesis and assembly of large DNA fragments for functional work, which is complemented with more traditional microbiological, genetic, biochemical and physiological methods. He/she will also benefit from regular meeting with Green Biologics and exposure to the industrial environment.