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 substantial improvements suggests that further advance under selection will be possible by stacking mutations. This proof-of-concept study established that the performance of sulfide-dependent THI4s in aerobic conditions is evolvable and, more generally, that yeast OrthoRep provides a plant-like bridge to adapt nonplant enzymes to work better in plants.

Protein Engineering and High‐Throughput Screening by Yeast Surface Display: Survey of Current Methods

“…Through passaging of yeast under the appropriate selective pressure, directed evolution of the protein of interest toward new functions can be achieved with minimal researcher intervention. [ 82–84 ]…”

Ultrahigh-throughput screening-assisted in vivo directed evolution for enzyme engineering

“…Recently, a highly orthogonal error-prone DNAP-plasmid pair (OrthoRep) was developed for restricting mutagenesis to a linear cytoplasmic plasmid without interfering in genome replication in Saccharomyces cerevisiae [ 16 , 17 ]. OrthoRep has been applied to engineer a significant number of proteins, including dihydrofolate reductase [ 17 ], tryptophan synthase β-subunit [ 18 ] and sulfide-dependent thiazole synthase [ 19 ]. In these in vivo continuous evolution systems, user-defined genes are usually mutated by inducing the expression of mutator genes, which is conducive to reducing unexpected mutations and enabling the spatiotemporal regulation of increased mutagenesis rates.…”