Artificial devices such as the synthetic riboswitch have shown potential to introduce unnatural phenotypic perturbation because its synthetic traits are distinct from that of innate metabolism. In this study, a riboswitch, a small regulatory element found in RNAs, was employed to reprogram microorganisms to produce valuable metabolites. A self-cleaving ribozyme glmS, found in gram-positive bacteria, cleaves its own transcript in response to the intracellular glucosamine 6-phosphate (GlcN6P) concentration. The glmS ribozyme was integrated into the 3'-untranslated region of FCY1, which encodes cytosine deaminase in Saccharomyces cerevisiae to construct a suicide riboswitch for evolutionary engineering. Growth of the strain harboring the suicide riboswitch was hampered by the addition of fluorocytosine, and was recovered as metabolite level increased. By using this riboswitch, we isolated a N-acetyl glucosamine (GlcNAc) producer strain by screening an efficient glutamine-fructose-6-phosphate transaminase (Gfa1p) and haloacid dehalogenase-like phosphatases (HAD phosphatases) originated from Escherichia coli. The suicide riboswitch was also applied to different metabolite by using artificial allosteric ribozyme. Since the mechanisms used in this work are universal in microorganisms, our synthetic suicide riboswitch can be applied to a wide range of organisms and can be exploited to the efficient and high-throughput screening of inconspicuous phenotypes.
Keywords: Evolutionary engineering; Random mutagenesis; Riboswitch; glmS ribozyme.
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