Improvement of a Microbial Thiamin Biosynthetic Enzyme through Continuous Directed Evolution

Thiamin is the essential cofactor for various key metabolic enzymes in all kingdoms of life. Thiamin is formed from a pyrimidine moiety and from a thiazole moiety derived from NAD, glycine, and a sulfur atom. In plants and yeast, thiazole is synthesized by THI4, a suicide enzyme that destroys an active-site cysteine residue by using it as the sulfur donor for the reaction and thus self-inactivates after just one reaction cycle. The resulting continuous replacement of THI4 is energetically costly. Certain prokaryotes have THI4s that use sulfide as a sulfur donor and are truly catalytic. However, these THI4s generally come from organisms from anaerobic, high-sulfide environments and their THI4s are consequently not expected to function well in plants. Therefore, we are using continuous directed evolution to evolve the THI4 from the anerobic bacterium Mucinivorans hirudinis to function in the relatively plant-like conditions (aerobic, low-sulfide) in yeast cells. Using the OrthoRep system in a yeast thiazole auxotroph reliant upon M. hirudinis THI4 for growth we applied three evolutionary strategies: 1) cold turkey - multiple passages without thiamin; 2) gradual – multiple passages in low thiamin followed by multiple passages without thiamin; and 3) variant library building – multiple passages in luxury thiamin followed by multiple passages with low thiamin, then without thiamin. These strategies led to selection of multiple single and double mutations in THI4 that often re-occurred in independent populations. These mutations were confirmed to confer faster growth by reintroducing them into a fresh vector in fresh yeast cells. The best of the mutant THI4s allowed growth rates close to that of native yeast THI4, leading us to deploy additional strategies to further increase the selection pressure; these strategies include downregulating expression of the target THI4 and reducing THI4 activity by adding an inhibitory thiazole analog to the selection medium.