Evolving thiazole synthases for expression in plants

Thiazole synthases (THI4s) catalyze the synthesis of the thiazole moiety of thiamin (vitamin B₁) from glycine, NAD, and a sulfur atom. The sulfur atom can originate from an active site cysteine residue, as is the case for cysteine-dependent THI4s (cys-THI4s), or from an external sulfide source, as it is for sulfide-dependent THI4s (noncys-THI4s). Cys-THI4s are also known as suicidal enzymes, as catalysis leads to enzyme inactivation, followed by protein degradation. These enzymes can be considered energy intensive and catalytically inefficient enzymes. Replacement of plant cys-THI4s with noncys-THI4s which are not self-inactivating though still slow catalysts, could lead to energy conservation through decreased protein turnover. However, noncys-THI4s are anaerobic or microoxic enzymes and thus would be poor replacements for plant cys-THI4s that are aerobic enzymes. Engineering a plant cys-THI4 to be a true catalyst or a noncys-THI4 to function in plant-like conditions would be necessary to achieve safe respiratory energy. In this chapter, we will provide methods outlining: (1) how to use comparative genomics to select the best candidate THI4s to be engineered via rational design or continuous directed evolution (CDE); (2) how to use rational design to engineer THI4s; and (3) how to use OrthoRep, a yeast-based CDE tool, to engineering noncys-THI4s to function under plant-like conditions.