RESUMO
The discovery of the bioluminescence pathway in the fungus Neonothopanus nambi enabled engineering of eukaryotes with self-sustained luminescence. However, the brightness of luminescence in heterologous hosts was limited by performance of the native fungal enzymes. Here we report optimized versions of the pathway that enhance bioluminescence by one to two orders of magnitude in plant, fungal and mammalian hosts, and enable longitudinal video-rate imaging.
Assuntos
Eucariotos , Luminescência , Animais , MamíferosRESUMO
We report a systematic comparison of 19 plant promoters and 20 promoter-terminator combinations in two expression systems: agroinfiltration in Nicotiana benthamiana leaves, and Nicotiana tabacum BY-2 plant cell packs. The set of promoters tested comprised those not present in previously published work, including several computationally predicted synthetic promoters validated here for the first time. The expression of EGFP driven by different promoters varied by more than two orders of magnitude and was largely consistent between two tested Nicotiana systems. We confirmed previous reports of significant modulation of expression by terminators, as well as synergistic effects of promoters and terminators. Additionally, we observed non-linear effects of gene dosage on expression level. The dataset presented here can inform the design of genetic constructs for plant engineering and transient expression assays.
Assuntos
Nicotiana , Plantas , Nicotiana/genética , Regiões Promotoras Genéticas , Plantas/genética , Folhas de Planta/genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genéticaRESUMO
An aerobic facultatively methylotrophic bacterium, designated strain Das4.1T, was isolated from a root of Daucus carota L. The cells of this strain were observed to be Gram-stain negative, asporogenous, non-motile short rods multiplying by binary fission. Strain Das4.1T can utilise methanol, methylamine and a variety of polycarbon compounds as carbon and energy sources. C1-compounds were found to be assimilated via the isocitrate lyase-negative variant of the serine pathway. On medium with 0.5% methanol, growth of strain Das4.1T was observed at pH 5.5-9.0 (optimum, pH 6.0-7.0) and 18-37 °C (optimum, 24-29 °C) and in the presence of 0-2% (w/v) NaCl (optimum, 0.05%). Cells are catalase and oxidase positive and synthesise indole from L-tryptophan. The major fatty acids of methanol-grown cells were identified as C18:1ω7c, C18:0 and 11-methyl-C18:1ω7c. The predominant phospholipids were found to be phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine and phosphatidylmonomethylethanolamine. The major respiratory quinone was identified as Q-10. The DNA G + C content of strain Das4.1T was determined to be 67.3 mol% (Tm). Phylogenetic analysis based on 16S rRNA gene sequence comparison revealed that strain Das4.1T belongs to the genus Methylopila and shows high sequence similarity to Methylopila oligotropha 2395AT (98.4%) and Methylopila capsulata IM1T (98.0%). However, the DNA-DNA relatedness of strain Das4.1T with M. oligotropha 2395AT was only 22 ± 3%. Based on genotypic, chemotaxonomic and physiological characterisation, the isolate can be classified as a novel species of the genus Methylopila, for which the name Methylopila carotae sp. nov. is proposed. The type strain is Das4.1T (= VKM B-3244T = CCUG 72399T).
Assuntos
Daucus carota/microbiologia , Methylocystaceae/classificação , Methylocystaceae/isolamento & purificação , Técnicas de Tipagem Bacteriana , Composição de Bases , Análise por Conglomerados , Citosol/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Ácidos Graxos/análise , Methylocystaceae/genética , Methylocystaceae/fisiologia , Hibridização de Ácido Nucleico , Fosfolipídeos/análise , Filogenia , Raízes de Plantas/microbiologia , Quinonas/análise , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
The fungal bioluminescence pathway can be reconstituted in other organisms allowing luminescence imaging without exogenously supplied substrate. The pathway starts from hispidin biosynthesis-a step catalyzed by a large fungal polyketide synthase that requires a posttranslational modification for activity. Here, we report identification of alternative compact hispidin synthases encoded by a phylogenetically diverse group of plants. A hybrid bioluminescence pathway that combines plant and fungal genes is more compact, not dependent on availability of machinery for posttranslational modifications, and confers autonomous bioluminescence in yeast, mammalian, and plant hosts. The compact size of plant hispidin synthases enables additional modes of delivery of autoluminescence, such as delivery with viral vectors.
Assuntos
Luminescência , Plantas , Animais , MamíferosRESUMO
Autoluminescent plants engineered to express a bacterial bioluminescence gene cluster in plastids have not been widely adopted because of low light output. We engineered tobacco plants with a fungal bioluminescence system that converts caffeic acid (present in all plants) into luciferin and report self-sustained luminescence that is visible to the naked eye. Our findings could underpin development of a suite of imaging tools for plants.
Assuntos
Luciferina de Vaga-Lumes/metabolismo , Nicotiana/genética , Plantas Geneticamente Modificadas/metabolismo , Ácidos Cafeicos/metabolismo , Fungos/genética , Fungos/metabolismoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.