title: Stable long-term indigo production by overexpression of dioxygenase genes using a chromosomal integrated cascade expression circuit.
abstract: In our laboratory we have analyzed different factors to maximize the yield in heterologous protein expression for long-term cultivation, by combination of an efficient cascade expression system and stable integration in the bacterial chromosome. In this work, we have explored this system for the production of indigo dye as a model for biotechnological production, by expressing in Escherichia coli the thnA1A2A3A4 genes from Sphingomonas macrogolitabida strain TFA, which encode the components of a tetralin dioxygenase activity. We compared Ptac, and the Pm-based cascade expression circuit in a multicopy plasmid and stably integrated into the bacterial chromosome. Plasmid-based expression systems resulted in instability of indigo production when serially diluted batch experiments were performed without a selective pressure. This problem was solved by integrating the expression module in the chromosome. Despite the gene dosage reduction, the synergic effect of the cascade expression system produced comparable expression to the dioxygenase activity in the plasmid configuration but could be stably maintained for at least 5 days. Here, we show that the cascade amplification circuit integrated in the chromosome could be an excellent system for tight control and stable production of recombinant products.
authors.affiliation: Departamento de Ciencias Ambientales, Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide, Ctra. Utrera Km 1, 41013 Sevilla, Spain.
authors.forename: Jose Luis
authors.initials: JL
authors.lastname: Royo
authors.forename: Emilia
authors.initials: E
authors.lastname: Moreno-Ruiz
authors.forename: Angel
authors.initials: A
authors.lastname: Cebolla
authors.forename: Eduardo
authors.initials: E
authors.lastname: Santero
mesh_terms: D002876:Chromosomes, Bacterial / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism*; D004355:Drug Stability; D004789:Enzyme Activation; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology; D024861:Genetic Enhancement / Q000379:methods*; D005822:Genetic Vectors; D007203:Indigo Carmine; D007211:Indoles / Q000737:chemistry / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D015398:Signal Transduction / Q000235:genetics; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics
chemical_list: D007211:Indoles; D011994:Recombinant Proteins; D007203:Indigo Carmine; D049308:Dioxygenases