title: Improvement of recombinant protein yield by a combination of transcriptional amplification and stabilization of gene expression.
abstract: We explored the use of a cascade circuit for heterologous gene expression that consists of a regulatory module with a salicylate-inducible system that controls the expression of a second regulator, xylS2, whose product is activated by common inducers. Activation and increasing the concentration of the second regulator synergistically induced heterologous genes downstream of the Pm promoter in the expression module. This module can be placed in multicopy vectors or in the chromosome of a host strain by means of minitransposons. Using reporter genes, we evaluated gene regulation capacity and gross production of the system with different configurations. The highest yield was obtained when the expression module was in a multicopy plasmid after a 6-h induction. However, expression modules in plasmids showed low stability after induction even with selective pressure. The chromosomal configuration had the lowest basal levels and induced levels comparable to those of plasmid configurations, resulting in accumulation of more than 10% of the total protein. Unlike the configurations in plasmids, the yield was maintained for at least 3 days even without selective pressure. In conclusion, the cascade system in the chromosome configuration is more efficient for long-term fermentation because of the great stability of the overexpressing phenotype in spite of the high levels of expression.
mesh_terms: D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D005819:Genetic Markers / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D007763:Lac Operon / Q000235:genetics; D010957:Plasmids; D014158:Transcription, Genetic
chemical_list: D004274:DNA, Recombinant; D005819:Genetic Markers