Metabolic Engineering

In the Thyer lab, we consider metabolic engineering to be a key technology for growing the bioeconomy and accessing complex biomolecules which are challenging to make using traditional chemical synthesis. One of our favourite techniques for directed evolution and metabolic engineering is Compartmentalized Partnered Replication (CPR), an ultra-high-throughput emulsion-PCR selection platform. CPR relies on linking the activity of your gene of interest to either expression or activity of a thermostable DNA polymerase, where cells containing more active gene variants produce more DNA polymerase. Individual cells are emulsified in PCR buffer containing dNTPs and oligonucleotide primers which flank the gene library resulting in preferential amplification of highly active mutants. These amplicons are recovered and subjected to multiple rounds of selection. CPR has two key advantages compared to other methods for directed evolution; it decouples protein function from host fitness (all the cells are killed during the PCR step) and it enables exponential enrichment of desirable variants during both positive and negative (counter) selections.

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Shown above is a CPR workflow used to engineer a novel biosensor which responds to a new small molecule. Each emulsion bubble containing a bacterial cell can be thought of as its own microscopic PCR tube, ensuring each reaction only amplifies a single gene variant. This circuit can be expanded and new layers introduced in order to evolve biosynthetic enzymes or even entire pathways. CPR is a platform technology in the Thyer lab and we are broadly interested in biosensor and biosynthesis pathway engineering for high-value biomolecules - particularly plant secondary metabolites such as terpenes and alkaloids.