title: Engineering uroporphyrinogen III methyltransferase as a red fluorescent reporter in E. coli.
abstract: Uroporphyrinogen III methyltransferase (UMT) is a novel reporter owing to the catalytic products in the cells that emit strong red fluorescence under UV light. Here, we engineered the gene encoding the functional barley UMT (bUMT) by error-prone PCR and broadened the application UMT as a red fluorescent reporter in Escherichia coli. A variant, termed mbUMT, was selected and emitted stronger cell fluorescence than the wild type bUMT expressed in different E. coli strains, under different promoters and induction conditions respectively. The constructed mbUMT with a C-terminal ssrA tag was degraded in cells by the protease ClpXP encoded by E. coli chromosome, whereas the bUMT was expressed as active aggregates. Before they are exported to the periplasm, both proteins catalyze the substrate in the cytoplasm and emit cell fluorescence. The results suggested that the evolved bUMT is a better candidate to monitor in vivo degradation by E. coli ClpXP.
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Wang
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Si
authors.initials: S
authors.lastname: Li
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Li
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Liang
authors.initials: L
authors.lastname: Rong
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China.
authors.forename: Beijiu
authors.initials: B
authors.lastname: Cheng
authors.affiliation: School of Life Science, Anhui Agricultural University, Hefei, Anhui 230036, PR China. Electronic address: fanjun@ahau.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
mesh_terms: D000595:Amino Acid Sequence; D019020:Directed Molecular Evolution; D049071:Endopeptidase Clp / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000378:metabolism; D017343:Genes, Plant; D001467:Hordeum / Q000201:enzymology / Q000235:genetics; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D011487:Protein Conformation; D015202:Protein Engineering; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D000097573:Red Fluorescent Protein
chemical_list: D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D008780:Methyltransferases; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein; C060508:uroporphyrin-III C-methyltransferase; C483573:ClpXP protease, E coli
keywords: Engineering; Escherichia coli; Fluorescence intensities; Red fluorescent reporter; Uroporphyrinogen III methyltransferase