Improving Catalytic Thi4s Using Continuous Directed Evolution Systems in Escherichia coli

Continuous directed evolution accelerates enzyme improvement, enables deep exploration of the target’s fitness landscape, and is revolutionizing metabolic engineering. Several systems to continuously evolve enzymes are now available including OrthoRep, PACE, T7-DIVA, and eMutaT7. We are applying T7-DIVA and eMutaT7 to improve Thi4 thiazole synthases, which are thiamin synthesis enzymes. Canonical plant Thi4s are suicidal (i.e. self-inactivate after one reaction cycle) because they use an active site cysteine residue as the sulfur donor for the reaction. Certain prokaryotes have catalytic Thi4s that use sulfide as sulfur donor, but these organisms are mostly strict anaerobes or thermophiles from high-sulfide environments whose Thi4s are unlikely to function efficiently in plants. Our aim is to evolve catalytic Thi4s to operate under the plant-like conditions (aerobic, mild temperature, low sulfide) in Escherichia coli. Pilot data from T7-DIVA based on GFP expression (the test for transcription via the T7-RNAP/cytidine deaminase (T7RNAP-CD) fusion) showed this system functions in MOPS minimal medium. We complemented an E. coli ΔthiG thiazole auxotroph with the Thi4 from Saccharicrinis fermentans but found a severe growth penalty from the T7-DIVA machinery that blocked efforts to evolve this enzyme. The growth penalty may reflect downregulation of the target gene by the antisense orientation of the T7 promoter. Another concern is T7-DIVA mutagenesis machinery could potentially lose mutagenic activity since the RNAP driving mutagenesis is independent from the endogenous RNAP that drives target gene expression and imposes a fitness burden that may predispose it to loss. We also explored eMutaT7, a system that uses a similar T7RNAP-CD fusion, but ties target gene expression to transcription driven by the fusion protein. We showed that the eMutaT7 system functions in MOPS minimal media, and are now using eMutaT7 to proceed with Thi4 enzyme evolution experiments.