Continuous Directed Evolution of Short-Lived Plant Enzymes for Reduced Maintenance Respiration Costs

To ensure adequate nutrition for a fast-growing world population, overall production of major food crops must rise substantially. Conventional breeding may be too limited in potential and too slow to satisfy this rising demand, making it important to deploy also metabolic engineering and synthetic biology to increase crop biomass and harvested yields. One unexplored metabolic engineering strategy is to decrease carbon loss by slowing protein turnover (degradation and resynthesis) and hence reducing the maintenance component of respiration. The carbon saved thereby could be channeled into biomass. In plants, diverse enzyme are candidates for reduction in their turnover rate. An extreme example is the suicidal THI4 thiazole synthase that forms the thiazole moiety of thiamin (vitamin B1) and is an abundant protein in all plants. THI4 is not a true catalyst because one enzyme molecule is required per reaction cycle due to destruction of an active-site cysteine residue to provide the sulfur atom for the thiazole ring. Destruction of the cysteine irreversibly inactivates the enzyme. Based on CCR (‘catalytic cycles until replacement’) values, which provide an estimate of an enzyme’s energy cost in relation to its operation, we selected abundant, short-lived enzyme candidates in Arabidopsis. Besides suicide THI4s, the selected candidates include methionine synthase 2 and phosphomethylpyrimidine synthase as well as cereal THI4 isoforms that have no active-site cysteine and may therefore be truly catalytic instead of suidical. Using the yeast OrthoRep continuous directed evolution system, which has been successfully used to evolve microbial THI4s, we are evolving the selected candidates for longevity and improved performance. When used to replace the endogenous gene in its native host, the improved enzymes are expected to decrease maintenance respiration costs leading to increased biomass.