Construction of protein overproducer strains in Bacillus subtilis by an integrative approach.
We evaluated the effect of several genetic factors reported as having a role in the induction of the expression of significant levels of recombinant protein in Bacillus subtilis. We utilized the beta-galactosidase reporter protein from Escherichia coli as our model for measuring the overproduction of heterologous proteins in B. subtilis. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. In this study, we considered factors known to modulate the transcription and translation initiation rates and genetic and mRNA stability. We also consider the effects of different genetic backgrounds, such as degU32 and hpr2, that until now have been studied independently. By changing the native -35 promoter box to the consensus TTGACA sequence of the aprE promoter, a significant 100-fold increase in the beta-galactosidase activity was obtained. On the other hand, changes such as the GTG to ATG start codon, the construction of a consensus AAGGAGG ribosome binding site, and the addition of the cryIIIA transcription terminator at the 3' end of the lacZ gene, produced only marginal effects on the final beta-galactosidase activity.
Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos.
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D001412:Bacillus subtilis / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D015964:Gene Expression Regulation, Bacterial; D005818:Genetic Engineering; D007763:Lac Operon / Q000235:genetics / Q000502:physiology; D026901:Membrane Transport Proteins; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D014176:Protein Biosynthesis; D020871:RNA Stability; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
C088074:AprE protein, Bacteria; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; D001616:beta-Galactosidase