title: A synthetic suicide riboswitch for the high-throughput screening of metabolite production in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Korea University, Seongbuk-gu, Seoul 136-713, Republic of Korea.
authors.forename: Sang-Woo
authors.initials: SW
authors.lastname: Lee
authors.affiliation: Department of Chemical and Biological Engineering, Korea University, Seongbuk-gu, Seoul 136-713, Republic of Korea. Electronic address: mkoh@korea.ac.kr.
authors.forename: Min-Kyu
authors.initials: MK
authors.lastname: Oh
mesh_terms: D043525:Cytosine Deaminase / Q000096:biosynthesis / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D041722:Genes, Transgenic, Suicide; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing) / Q000096:biosynthesis / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism; D058928:Riboswitch; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D058928:Riboswitch; D029701:Saccharomyces cerevisiae Proteins; C098848:GFA1 protein, S cerevisiae; D005945:Glutamine-Fructose-6-Phosphate Transaminase (Isomerizing); D043525:Cytosine Deaminase
keywords: Evolutionary engineering; Random mutagenesis; Riboswitch; glmS ribozyme