Elizabeth Gillam

Engineering enzymes for bioremediation of microplastics

Plastics such as polyethylene (PE) are major pollutants in both terrestrial and aquatic environments because they are not easily degraded in nature. Physico-chemical methods for PE remediation are energy intensive and not economically sustainable, raising the possibility of bioremediation instead. The larvae of the greater wax moth (GWM, Galleria mellonella) feed on beeswax, which is rich in long-chain alkanes, and have recently been shown to consume chemically similar low-density PE at considerably higher rates than those currently reported for PE-degrading microbes. Recent work suggests that P450 enzymes may be involved but the mechanism by which this occurs is not yet clear and there is no consensus on how the degradation is achieved biochemically, or even whether it is carried out entirely by the caterpillars themselves or with a contribution from the gut microbiota. Intriguingly, PE breakdown appears to involve a shift to high pH in the lumen of the insect gut, suggesting these enzymes may operate extracellularly and under very alkaline conditions, both of which are highly unusual for P450 enzymes. This project will involve expressing these enzymes and analysing their activity under the high pH and low oxygen conditions of the gut environment. We will then engineer them by ancestral sequence reconstruction (ASR) to identify thermostable and more pH-neutral forms of the PE-degrading P450s to develop a system in which these enzymes can be used for breakdown of microplastics in wastes.

How did koalas evolve to exist entirely on eucalyptus leaves, which are toxic to most mammals?

The diet of koalas is unique in comprising effectively 100% eucalyptus leaves, which contain a variety of toxic terpenes. Despite the interest in koala conservation and many years of study, we still do not understand how koalas can exist on such a this toxic diet. However a clue has come in the sequencing of the koala genome: compared to other marsupials and mammals more generally, koalas show a dramatic expansion in the CYP2C subfamily of cytochrome P450 enzymes. P450s are regarded as responsible for the metabolism of dietary and other environmental xenobiotics, so we hypothesise that the CYP2C forms in koalas have expanded to deal with the terpenes present in their diet and can oxidise these chemicals to facilitate their clearance from the koala’s circulation.

This project will test this hypothesis by synthesising the CYP2C enzymes from koalas then expressing them in E. coli with the extant reductase accessory enzyme. We will determine how well the recombinant enzymes metabolise cineole and other eucalyptus terpenes. In so doing, we hope to answer a fundamental question about the biology of this iconic Australian animal, and one that has implications for koala conservation.

If the hypothesis is proven to be correct (i.e. the extant koala CYP2C forms metabolise terpenes), selected ancestors of these CYP2C enzymes will be inferred, reconstructed and expressed to determine how the ability to metabolise eucalyptus terpenes arose during koala evolution, a model of how proteins evolve new functions.

Engineering a sustainable source of strigolactone hormones to improve food security across the world.

Strigolactones (SLs) are a class of plant hormones that control many traits important for agriculture including shoot and root architecture, nutrient uptake and responses to parasitic weeds. Parasitic weeds stimulated by plant-derived SLs are widespread in arable lands of many developing countries and have devastating impacts on food production. Application of synthetic SLs to infested soils would provide a way to clear arable land of parasitic weeds and greatly enhance food security in the third world. Biotechnological sources of natural or chemically modified SLs would also improve agricultural crop yield and reduce manual labour costs in horticultural industries. The overall objective of this project is to develop means for SL production in biofactories and to improve the potency of synthetic and/or biofactory/engineered SLs. We will do so by analysing the evolution of naturally occurring SL-synthesising enzymes and leveraging ancestral sequence reconstruction to engineer robust novel 'designer' enzymes with specific desired activities.

Crystallisation of thermostable ancestral cytochrome P450 enzymes

We have developed ancestral P450 enzymes that are extremely thermostable compared to modern enzymes, making them potentially very useful in industry, since they can withstand long incubations at elevated temperatures. They can be used as ‘off the shelf’ reagents to catalyse useful chemistry, such as in in drug discovery and development, fine chemicals synthesis, and cleaning up the environment. Working with drug companies, we are exploring how they can be best deployed in chemical processes and what structural features make them efficient, robust and specialized. Key to this is obtaining crystal structures of the enzymes to determine why they are more stable.

We have already obtained crystals of a number of different thermostable P450s so this project would allow a student to make accelerated progress towards the goal of obtaining a structure for high impact publications that would be of great interest to industry as well as the field of protein structural biology and engineering.