High-Throughput Screening of Metabolite Producers Using Synthetic Suicide Riboswitch in Saccharomyces Cerevisiae

Utilization of microorganisms has been drastically diversified since the invention of various methods for phenotypic alteration such as the recombinant DNA technology and novel synthetic tools developed recently. Artificial devices such as synthetic riboswitch have shown potential for distinct phenotypic perturbation by its synthetic nature. Riboswitch, a small regulatory element found in RNAs, was conducted for reprogramming of microorganism to produce valuable metabolites in this work. Natural riboswitches can sense intracellular concentration of metabolite and regulate the expression of gene in response to metabolite level. To mimicking this feature, we utilized a self-cleaving ribozyme glmS found in Gram-positive bacteria. glmS ribozyme cleaves its own transcript in response to intracellular glucosamine 6-phosphate(GlcN6P) concentration. glmS ribozyme was integrated into 3’-untranslated region of FCY1, which encodes cytosine deaminase, in Saccharomyces cerevisiae to construct suicide riboswitch for evolutionary engieering. The strain BY4742 FGU contains suicide riboswitch showed reprogrammed metabolite level-dependent growth as we expected. By using this riboswitch, we isolated a N-acetyl glucosamine(GlcNAc) producer strains by screening a mutant library of glutamine-fructose-6-phosphate transaminase (Gfa1p) and a library of haloacid dehalogenase-like phosphatases (HAD phosphatases) from Escherichia coli which cannot be screened by conventional evolutionary engineering. Since the mechanisms used in this work are universal in microorganisms, our synthetic suicide riboswitch can be applied to wide range of organisms and can aid efficient and high-throughput screening of inconspicuous phenotypes.