Electrochemical High-Throughput Discovery of Novel Lytic Polysaccharide Monooxygenase Enzymes

Lignin and cellulose are carbohydrate molecules which are produced by plants to give their cell walls structure and protection from degradation. Both lignin and cellulose are therefore highly stable, unreactive molecules and in order to chemically degrade them into their smaller sugar building blocks harsh chemical reaction conditions are required. As a result of this, it is more cost effective to use enzyme cocktails to convert plant matter into biofuel, and biocatalysts which are useful in this process have a significant commercial value.

Prozomix (http://www.prozomix.com/) is a British Biotech company based in Northumberland specializing in biocatalyst enzyme production and sales. Currently LPMOs are the only family of glycosidic bond cleavage enzymes missing from Prozomix's catalogue because (i) a rapid LPMO activity-screening method has not been previously available to support LPMO enzyme discovery via the companies unique, proprietary in-house metagenome library and expertise in sequence searches and rapid, commercial protein production; and (ii) in LPMO biochemistry, the role of active site ligands in tuning the energetics of the copper chemistry is not known so as-yet it is impossible to rationally relate LPMO sequence to substrate-reactivity/specificity. This project combines complementary expertise in enzyme electrochemistry (Parkin), structure (Davies) and spectroscopy (Walton) studies with industrial enzyme discovery and commercialization (Prozomix) to find and characterize new lytic polysaccharide monooxygenase (LPMO) carbohydrate-activating enzymes.

In ground-breaking work, Davies and Walton (York) proved that the lytic polysaccharide monooxygenase (LPMO) enzymes which catalyze the first glycosidic bond cleavages of cellulose are copper enzymes. Parkin (York) has worked with Davies, Walton and collaborators from Brazil to show that her group's expertise in protein film electrochemistry enables purified enzymes to be screened for LPMO electron-transfer catalytic activity in a low-sample requiring (mcg), rapid (approx. 15 min) and high throughput, multiplexed manner.
The project objectives will be to:
(i) Identify (CaZy sequence analysis and AlphaFold2) and clone out new bacterial LPMOs from the Prozomix metagenome library
(ii) Use electrochemistry to rapidly screen for electron-transfer activity and identify oxidative LPMO activity in the putative enzymes
(iii) Test the carbohydrate substrate specificity of new LPMOs, searching for highly lignin and cellulose active enzymes of potential bio-fuel interest
(iv) Large scale production of up to 5 lead enzymes with novel activity, i.e., high temperature stability or unusual stereo-selectivity
(v) Further structurally characterize the lead enzymes and use molecular biology to probe structure-function hypotheses
(vii) Identify high-stability, high-activity cellulose- and/or lignin-degrading LPMO, Prozomix to produce enzyme for scaled industrial testing

The student will be provided with expert training in protein purification and molecular biology and learn to purify an LPMO. Expert support will also be provided in electrochemistry and EPR. While on placement, the student will attend appropriate training sessions organised by Prozomix, covering all aspects of novel enzyme discovery, development and production.