Proteins are the most structurally complex and functionally diverse class of molecules produced by Nature, yet the known proteome only samples a fraction of the available ‘protein space’. Over several decades we have built a detailed mechanistic understanding of translation and the rules that guide protein assembly. Armed with this knowledge and the tools of chemical biology, we can remove the boundaries on protein space almost entirely by introducing new, non-canonical amino acid monomers (ncAAs), drastically expanding the set of chemistries available to proteins and thus their functions. However, such freedom to explore near limitless chemical space comes with similarly daunting challenges, particularly regarding the metrology of these new chemistries. Short of low throughput analytical methods such as mass spectrometry, it is difficult to measure the presence of ncAAs, which are often imperfectly incorporated. Leveraging the full potential of ncAAs to explore protein space and develop new biological chemistry requires a complete ecosystem of capabilities beyond just the translational machinery – starting with measurement. 

No amino acid embodies these challenges like selenocysteine (Sec), the 21st amino acid. Sec is of great interest for protein engineering because the selenol moiety has a lower pKa and superior nucleophilicity than a cysteine thiol. Pairs of selenocysteine residues can also form covalent diselenide bonds, an interesting structural motif with different properties than a disulfide bond. In nature, selenocysteine is biosynthesized directly on its tRNA from a precharged serine and incorporated in response to the opal (UGA) stop codon. This process requires several unique RNA and protein factors to direct reassignment of the stop codon and is also extremely inefficient as it competes with termination of translation mediated by release factor 2 (RF2). There are many unanswered questions around selenocysteine biosynthesis and its unique incorporation pathway, and we are continually developing this technology with the goal of producing novel recombinant selenoproteins. We have developed several new methods to synthesize selenoproteins, including engineered tRNAs, and new genetic reporters to measure and investigate the biosynthesis and incorporation pathway, most recently the Selenocysteine Adjustable Ratiometric Chromophore, SeARCh. We always have several projects in this area.

Engineering new functionality into biology is inherently high-risk - we experience many failures along the way and interpreting and learning from these is an important part of the process. To mitigate the risks, and improve to scope and scale of what we can engineer, we are always experimenting with new tools and technologies for biomolecular engineering. 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. 

Shown below 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, as well as translational machinery and reporters for expanded protein chemistries. 

Using biology to access complex matural products which are challenging to make using traditional chemical synthesis is one of the key value propositions of the bioeconomy. Isoprenoids are among the most diverse natural products and include molecules which can serve as solvents, energy dense fuels, and even encompass critical materials such as natural rubber (polyisoprenes). The isoprenoid backbone is constructed using highly modular chemistry from universal five-carbon precursors. These precursors, which contain reactive pyrophosphate groups, are toxic and transient and thus difficult to measure directly within living cells. Despite the wide variety of isoprenoids in Nature (>90.000), reporters which enable high-throughput genetic measurement are lacking and most engineering relies on analytical techniques. We use transcriptional biosensors to measure terpenes as a proxy for isoprenoid pathway flux and guide engineering of new terpene synthases or enzymes involved in precursor biosynthesis.   

Actinomycetes are the 3rd largest phylum of bacteria and have untapped potential as hosts for biomanufacturing. In particular, members of the Mycolata taxon, which include Rhodococcus, Gordonia, Nocardia, and Dietzia, can assimilate a wide variety of feedstocks and are naturally competent at biosynthesizing isoprenoids and oleochemicals. We are working to develop a comprehensive set of genetic tools for these bacteria and engineering principles to enable precise metabolic control. We work with a diverse collection of strains including autotrophs and hyper-oligotrophs, carotenogenic and lipid-accumulating species, as well as isolates capable of degrading PFAS and recalcitrant hydrocarbons. Key projects include improving utilization of feedstocks derived from electrochemistry, constructing heterologous isoprenoid precursor and triacylglyceride pathways,  and resolving the biochemical basis of oligotrophic metabolism. In addition, we are investing the ability of these organisms to sequester critical metals from non-traditional sources, such as industrial wastewater. Developing environmentally friendly and cost effective methods for metal recovery from waste and industrial byproducts will play an important role in securing the supply of these materials in the face of increasing global demand.