Engineering new biosurfactants by harnessing the power of CRISPR and AlphaFold

This proposal is naturally aligned with Theme 1 "Advancing the frontiers of bioscience discovery" of the BBSRC's Forward Look for UK Bioscience report, being particularly relevant to the "Transformative technologies" focus area. This project aims to characterise the enzymes of the sophorolipid pathway in Starmerella bombicola and will attempt to alter the substrate specificity with the purpose of generating novel sophorolipid products, opening up a range of potential applications in formulated products.

A special focus will be given to the first glucosyltransferase as it is responsible for the coalescence reaction of the saccharide with the hydroxylated fatty acid, making it a suitable target for mutagenesis. The focus on bio-based production of an important platform chemical is highly relevant to the industrial and societal need for alternative routes to petrochemically derived molecules.

Generic skills training and public engagement experience will be provided by the main supervisor, whose volunteer outreach work has previously been recognised by The University of Manchester.

This PhD project will investigate enzymes in the native biosynthetic route in Starmerella bombicola for the production of sophorolipids. The pathway is known and in combination with the available genome sequence (Matsuzawa et al., 2015) now allows for the construction of an enzyme structure via AlphaFold. The binding of the substrates can be estimated by docking and molecular modelling.

Together with sequence alignment to find conserved residues allows us to estimate how the substrates sit in the active site. This information will then function as a guide for selecting other substrates that can be catalysed and form novel biosurfactant products. Characterisation of these new to nature substrates can be tested with varied biochemical techniques and might result in a range of novel properties, opening up a range of potential applications in formulated products.

The proposed project will deliver new production routes for modified sophorolipids, from a non-GMO origin, giving potential new functionality and hence additional market applications. Having access to structural information of the enzyme can then also be used to design mutants that potentially create new products from a GMO origin. Mutants can target binding of the sugar and/or fatty alcohol resulting in an even larger assortment of new products.

Upon successful identification of desirable products, the genome of Starmerella bombicola will be edited with state-of-the-art molecular cloning methods like CRISPR-Cas12. The final stage of the research will involve optimisation of fermentation conditions for these novel sophorolipids at 2L bioreactors scale, as larger quantities of surfactants is required for detailed characterisation of their unique properties.

Having access to a fully characterised library of alternative and novel sophorolipid molecules and their functionality, will be followed by formulation testing and evaluation of the techno-economic and environmental sustainability performance of the modified sophorolipids. The main objectives of the project are:

1. Computational modelling of substrate binding and identifying residues that can alter substrate specificity when mutated. 2. Produce pure enzyme (by recombinant expression) and develop an in vitro assay for different substrates.

3. Characterisation of kinetic parameters for wild type and mutant enzymes. Identify the new products are produced by each mutant. 4. Test physical and chemical properties of novel sophorolipids. 5. Mutants that produce interesting products can be introduced in Starmerella bombicola by CRISPR-Cas12.

6. Perform bioreactor experiments to scale the production of novel sophorolipids and optimise feedstocks.