Talk Summary by Harry Crook
Recently the world reached the one-degree centigrade rise predicted by the Intergovernmental Panel on Climate Change as the first step in irreparable global warming; this has led to much research into CO2 emissions reduction. Initially this involved methods such as sequestration underground or simply planting trees, but more recently the research has focused on manipulating natural machines which are already capable of removing CO2: enzymes.
Therefore, perhaps one of the most interesting talks of this week’s symposium was presented by Dr. Godwin Aleku. In just 30 minutes he condensed several painstaking years of research into a whistle-stop tour of how to engineer your own enzyme to help slow the planet’s march towards a permanent and global Death Valley-like environment.
Aleku’s research set out aiming to either find or engineer an enzyme which could help to recycle CO2 made in industrial processes by reducing it to form solvents, fuels or (perhaps more usefully) organic molecules. These molecules could then be used in further synthesis by inserting CO2 into ‘base scaffolds’ to produce alcohols, carboxylic acids or even the amines and amides craved by the chemical synthesis industry.
Whilst some may argue that many natural CO2-fixing enzymes, such as RuBisCo, already exist to fulfil part of Aleku’s aims, these are often inefficient for the synthetic substrates desired as starting points in chemical synthesis. Instead, Aleku turned to (de)carboxylase enzymes which are often more robust and stable than naturally CO2-fixing enzymes.
Although the decarboxylases are more forgiving towards synthetic substrates, they present a thermodynamic limitation as their equilibrium reactions strongly favour removing CO2. In order to achieve his goals, Aleku would therefore need to both overcome these thermodynamic problems and produce the relevant scaffolds needed in industry utilising decarboxylases.
To identify a specific decarboxylase for his research Aleku scoured the tree of life, searching across various genomes and known metabolic/synthesis pathways to identify sample enzymes which could then be further improved. This eventually led to the identification of fungal ferulic acid decarboxylase (FDC1) as a candidate ‘biocatalyst’. FDC1 was particularly suitable due to its stability and its cofactor (needed for catalytic activity) being bound to the enzyme; this removed the lengthy and expensive step of reconstituting the enzyme and cofactor, making FDC1 easier to manipulate in the future.
Having found a suitable enzyme Aleku needed to identify substrates which could be used as the scaffold. This was achieved through microtiter plate screening, which helps to identify competitive inhibitors of the reaction. Crystallographic analysis of these inhibitors revealed that indole-2-carboxylic acid (ICA) had weak productive activity via binding, although it seemed to be skewed in the active site; this lead Aleku to believe that simple steric issues were responsible for the problems.
He therefore used alanine and serine scanning of the active site to identify catalytic residues and those he could replace in order to move the ICA into a more favourable position. With the role of residues established he replaced specific residues with all possible combinations using an in vivo system to assess the conversion rate of ICA with CO2. This led to the discovery of a mutant which had a >99% conversion rate. Crystallographic studies also confirmed that the insertion of bulkier side chains had indeed pushed the ICA further towards a key catalytic residue.
Aleku now had an enzyme which could utilise synthetic substrates; however, it still strongly favoured decarboxylating substrates. In order to overcome this, he designed reaction cascades which would remove the carboxylated product through irreversible conversion into other substrates such as aldehydes using ‘CAR’ enzymes. Different secondary steps can also be used to produce other key scaffolds such as amines. Overall, through his research Aleku has taken the first steps in reducing CO2 emissions in the chemical industry – for example, even from the basic scaffolds synthetic antifungals can be produced. Furthermore, he postulates that these pathways could, with further research, be incorporated into host cells themselves to produce scaffolds.
This fascinating research is, I believe, part of a field which will continue to grow and come to dominate industrial chemistry in the future as we improve on natural machines which have been in development for millions of years.
This talk gives a report of the talk hosted by Dr. Godwin Aleku on the 24th of September-Day 4 of the Oxbridge Varsity Sci Symposium.