Directed Evolution of Energy-Efficient Non-Suicidal THi4s - a Next-Gen Synbio Strategy

Around half the carbon captured by photosynthesis is lost by respiration and half of that respiratory carbon loss is driven by protein turnover and other maintenance processes. Scaling back maintenance costs by slowing the turnover rates of fast-turnover proteins could therefore save carbon and energy for allocation to biomass yield. Until now, SynBio approaches to increasing crop biomass yield have overlooked carbon losses and have instead focused on carbon gain (photosynthesis) because molecular targets can be defined; molecular targets to reduce carbon loss have been elusive. The situation has changed thanks to advances in proteomics that enable measurement of turnover rates of individual proteins. For instance, the thiamin synthesis enzyme THI4 is now known to turn over extremely fast, and bioenergetic calculations indicate that this turnover could account for up to 5% of maintenance costs, or 1% of carbon fixed by photosynthesis. An efficient THI4 substitute is therefore a Next-Gen SynBio target for crop improvement. Canonical plant THI4s are suicide enzymes that use an active-site cysteine residue as sulfur donor and so are irreversibly inactivated after a single reaction cycle. Certain anaerobic prokaryotes have THI4s that use sulfide as sulfur donor and so are energy-efficient catalysts; these enzymes have histidine or another residue in place of the active-site cysteine. Screening such THI4s by functional complementation of an Escherichia coli thiamin auxotroph identified some that can function in aerobic conditions similar to those in plant cells. However, the low prevailing sulfide levels in planta may limit the activity of such sulfide-utilizing THI4s. Applying directed evolution, e.g., with the EvolvR system, can potentially modify candidate THI4s such as that of Thermovibrio ammonificans to function efficiently in aerobic, low-sulfide conditions. Combining parts-prospecting for energy-efficient SynBio target THI4s with a directed evolution approach is thus an innovative path to boost crop productivity.