title: Engineering an enhanced, thermostable, monomeric bacterial luciferase gene as a reporter in plant protoplasts.
abstract: The application of the luxCDABE operon of the bioluminescent bacterium Photorhabdus luminescens as a reporter has been published for bacteria, yeast and mammalian cells. We report here the optimization of fused luxAB (the bacterial luciferase heterodimeric enzyme) expression, quantum yield and its application as a reporter gene in plant protoplasts. The fused luxAB gene was mutated by error prone PCR or chemical mutagenesis and screened for enhanced luciferase activity utilizing decanal as substrate. Positive luxAB mutants with superior quantum yield were subsequently shuffled by DNase I digestion and PCR assembly for generation of recombinants with additional increases in luciferase activity in bacteria. The coding sequence of the best recombinant, called eluxAB, was then optimized further to conform to Arabidopsis (Arabidopsis thaliana) codon usage. A plant expression vector of the final, optimized eluxAB gene (opt-eluxAB) was constructed and transformed into protoplasts of Arabidopsis and maize (Zea mays). Luciferase activity was dramatically increased for opt-eluxAB compared to the original luxAB in Arabidopsis and maize cells. The opt-eluxAB driven by two copies of the 35S promoter expresses significantly higher than that driven by a single copy. These results indicate that the eluxAB gene can be used as a reporter in plant protoplasts. To our knowledge, this is the first report to engineer the bacterium Photorhabdus luminescens luciferase luxAB as a reporter by directed evolution which paved the way for further improving the luxAB reporter in the future.
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Microbiology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Boyu
authors.initials: B
authors.lastname: Cui
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Biochemistry and Molecular Biology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Lifeng
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Microbiology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yunhong
authors.initials: Y
authors.lastname: Song
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Biochemistry and Molecular Biology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Jinsong
authors.initials: J
authors.lastname: Wei
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Biochemistry and Molecular Biology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Changfu
authors.initials: C
authors.lastname: Li
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Microbiology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Tietao
authors.initials: T
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Biochemistry and Molecular Biology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Biochemistry and Molecular Biology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Tianyong
authors.initials: T
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China; Department of Microbiology, College of Life Sciences, Northwest A&F University, Yangling, Shaanxi, China.
authors.forename: Xihui
authors.initials: X
authors.lastname: Shen
mesh_terms: D003062:Codon; D032621:DNA Shuffling; D004795:Enzyme Stability; D015870:Gene Expression; D023061:Gene Order; D017343:Genes, Plant; D017930:Genes, Reporter; D006863:Hydrogen-Ion Concentration; D049453:Luciferases, Bacterial / Q000737:chemistry / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010944:Plants / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D011523:Protoplasts / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D003313:Zea mays / Q000235:genetics / Q000378:metabolism
chemical_list: D003062:Codon; D011993:Recombinant Fusion Proteins; D049453:Luciferases, Bacterial