Directed Evolution of Aerotolerance in Sulfide-Dependent Thiazole Synthases.

ABSTRACT: Sulfide-dependent THI4 thiazole synthases could potentially be used to replace plant cysteine-dependent suicide THI4s, whose high protein turnover rates make thiamin synthesis exceptionally energy-expensive. However, sulfide-dependent THI4s are anaerobic or microoxic enzymes and hence unadapted to the aerobic conditions in plants; they are also slow enzymes (k_cat < 1 h^-1). To improve aerotolerance and activity, we applied continuous directed evolution under aerobic conditions in the yeast OrthoRep system to two sulfide-dependent bacterial THI4s. Seven beneficial single mutations were identified, of which five lie in the active-site cleft predicted by structural modeling and two recapitulate features of naturally aerotolerant THI4s. That single mutations gave su

SUBMITTER: Gelder KV 

PROVIDER: S-EPMC10127261 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature