title: Characterization of Endogenous and Reduced Promoters for Oxygen-Limited Processes Using Escherichia coli.
abstract: Oxygen limitation can be used as a simple environmental inducer for the expression of target genes. However, there is scarce information on the characteristics of microaerobic promoters potentially useful for cell engineering and synthetic biology applications. Here, we characterized the Vitreoscilla hemoglobin promoter (Pvgb) and a set of microaerobic endogenous promoters in Escherichia coli. Oxygen-limited cultures at different maximum oxygen transfer rates were carried out. The FMN-binding fluorescent protein (FbFP), which is a nonoxygen dependent marker protein, was used as a reporter. Fluorescence and fluorescence emission rates under oxygen-limited conditions were the highest when FbFP was under transcriptional control of PadhE, Ppfland Pvgb. The lengths of the E. coli endogenous promoters were shortened by 60%, maintaining their key regulatory elements. This resulted in improved promoter activity in most cases, particularly for PadhE, Ppfland PnarK. Selected promoters were also evaluated using an engineered E. coli strain expressing Vitreoscilla hemoglobin (VHb). The presence of the VHb resulted in a better repression using these promoters under aerobic conditions, and increased the specific growth and fluorescence emission rates under oxygen-limited conditions. These results are useful for the selection of promoters for specific applications and for the design of modified artificial promoters.
authors.affiliation: Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa . Av. Vasco de Quiroga 4871, Santa Fe, C.P. 05348, Mexico City, México.
authors.forename: Alvaro R
authors.initials: AR
authors.lastname: Lara
authors.affiliation: Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa . Av. Vasco de Quiroga 4871, Santa Fe, C.P. 05348, Mexico City, México.
authors.forename: Karim E
authors.initials: KE
authors.lastname: Jaén
authors.affiliation: Departamento de Procesos y Tecnología, Universidad Autónoma Metropolitana-Cuajimalpa . Av. Vasco de Quiroga 4871, Santa Fe, C.P. 05348, Mexico City, México.
authors.forename: Juan-Carlos
authors.initials: JC
authors.lastname: Sigala
authors.affiliation: RWTH Aachen University , AVT - Biochemical Engineering, Worringer Weg 1, 52074 Aachen, Germany.
authors.forename: Martina
authors.initials: M
authors.lastname: Mühlmann
authors.affiliation: RWTH Aachen University , AVT - Biochemical Engineering, Worringer Weg 1, 52074 Aachen, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Regestein
authors.affiliation: RWTH Aachen University , AVT - Biochemical Engineering, Worringer Weg 1, 52074 Aachen, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D060846:Cell Engineering / Q000379:methods; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics; D005453:Fluorescence; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics; D010100:Oxygen / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D058615:Synthetic Biology / Q000379:methods; D014158:Transcription, Genetic / Q000235:genetics; D054793:Truncated Hemoglobins / Q000235:genetics; D020589:Vitreoscilla / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D054793:Truncated Hemoglobins; C082613:hemoglobin protein, Vitreoscilla; D010100:Oxygen
keywords: FbFP expression; Vitreoscilla hemoglobin; microaerobic promoters; microbioreactors; oxygen-limited cultures; reduced promoters