Enhancing biofuel production through synthetic protein fusions

Lead Research Organisation: University of York
Department Name: Biology

Abstract

The development of lignocellulosic-based bioprocesses to make biofuels and other valuable chemical products is critically dependent on the efficiency with which bacteria can degrade, uptake and catabolise the sugars present in these feedstocks and then use the released energy and carbon skeleton to produce and export the desired products. While we know the transporters and enzymes used by bacteria to access some of the components of lignocellulose, such as the sugar xylose, no attempt has been made to engineer these existing pathways to enhance the rate at which catabolism occurs. We aim to achieve this by using a novel and exciting method that this based on the concept of spatial coupling of related reactions in the cell. This is to physically scaffold the proteins involved in a particular catabolic pathway into an assembly, reducing the time for diffusion of products from one reaction to be substrate for the next. This concept has recently been used successfully to increase the flux through the threonine biosynthetic pathway in E. coli by fusing the enzymes to zinc-finger proteins that then bind to a DNA scaffold (Lee et al ., 2012 Applied Environmental Microbiol 79:774-782). However, in this synthetic biology project we will primarily investigate a novel route to spatial coupling using genetically encoded rod-like protein domains, recently discovered in York in the lab of Prof. Jennifer Potts, to bring together sets of functionally related proteins. These rod-like domains can be made to a variety of different lengths to control the distance between interacting proteins. Uniquely we will also attempt to incorporate membrane transporters into the complex to more closely couple uptake to catabolism. This timely project also has the potential to apply the same approach to other pathways relevant for biofuel and chemical production.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
BB/M011151/1 30/09/2015 29/09/2023
1792408 Studentship BB/M011151/1 30/09/2016 30/03/2021 Liam Neil Chapman
 
Description I have produced a system for easily fusing membrane proteins to a downstream enzyme or fluorescent probe. From this I have determined the stability of a family of transporters, but also determined the negative impact that fusing enzymes directly to the membrane protein can have a negative impact on the downstream enzyme function. This validates my interest in the scaffold proteins to overcome such limitations.
Exploitation Route Ideally the golden gate plasmid could be used for the fusion of membrane proteins to downstream enzymes. While this may impact the activity of the downstream enzyme, when functional it could be used for easily swapping out parts of interest - i.e. studying different transporters in the membrane by fusion to fluorophores - using tagging proteins such as biotinylation proteins fused to a membrane transporter to determine what the transporter interacts with. Of industrial releveance this could be used to increase sugar metabolism for biofuel industry.
Sectors Manufacturing, including Industrial Biotechology,Other

 
Description Chemistry at work 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Demonstrated how to "cook" an egg without boiling in order to show how different chemicals react with egg white. Assisted with event planner to keep an eye on students and explain questions relating to the experiment.

Event featured multiple secondary school students, years 9-10, and took place at the University of York chemistry department. Event was organised by NYBEP.
Year(s) Of Engagement Activity 2017
 
Description YorNight - Local outreach 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Demonstrated the potential of biofuels by using them to power a toy boat in a race. We discussed production of the combustable biofuel, asked children watching to predict the winning boat and demonstrated how burning of the biofuel would facilitate motion.

Saturday evening event organised by the University of York and visited by many families, involved multiple groups from various science disciplines. Our lab hosted a series of microbiology related workshops and activities to demonstrate the role of microbes in biofuels, disease and odour.
Year(s) Of Engagement Activity 2018
URL https://www.york.ac.uk/news-and-events/events/yornight/2018/