Needlessly Fast-Turnover Proteins As Next-Gen Synbio Targets

Carbon supply often limits or co-limits crop productivity. A quarter of carbon fixed by photosynthesis is lost via the respiration needed to supply the energy demands of protein turnover and other maintenance processes. Decreasing the energy needed by maintenance processes could therefore increase yield by making more carbon and energy available. Until now, SynBio has been used to increase yield via carbon gain (photosynthesis), not carbon loss, in part because molecular targets to reduce carbon loss have been hard to define. Proteomics now enables identification of ‘carbon loss’ targets through measuring individual protein turnover rates. One such target is THI4, the enzyme that forms the thiazole moiety of thiamin. THI4 turns over exceptionally fast, and bioenergetic calculations show this turnover could account for 5% of maintenance costs, or 1% of carbon fixed by photosynthesis. Can we convert THI4 from a high-turnover energy hog to a low-turnover, energy-efficient enzyme? Maybe yes. Plant THI4s turn over fast because they are suicide enzymes that use an essential active-site cysteine residue as sulfur donor for the reaction and are inactivated after just one reaction cycle. In contrast, some THI4s from anaerobic prokaryotes use sulfide as sulfur donor and are energy-efficient catalysts; these enzymes have no active-site cysteine. Screening such THI4s for protein expression and functional complementation of an Escherichia coli thiazole auxotroph identified some that can function in aerobic conditions like those in plant cells. However, the low sulfide levels in planta may limit the activity of such sulfide-utilizing THI4s. We are exploring directed evolution with the EvolvR and OrthoRep systems to produce THI4s that function well in aerobic, low-sulfide conditions. Replacing a suicidal THI4 with an evolved energy-efficient one could slash the energy cost of THI4 turnover by >99.9% and save enough energy to boost biomass yield by 3-4%.