This title appears in the Scientific Report : 2004 

Untersuchungen zur Subtilisin-Produktion mit Bacillus licheniformis und Konstruktion eines alternativen Selektionssystems
Hintz, Maren (Corresponding author)
Biotechnologie 1; IBT-1
Jülich Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag 2004
143 S.
Düsseldorf, Univ., Diss., 2003
Dissertation / PhD Thesis
Berichte des Forschungszentrums Jülich 4128
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Bacillus licheniformis is used by the detergent industry for the production of proteases of the subtilisin type. Producer strains are obtained by multiple cycles of undirected mutagenesis followed by screening for high enzyme yield in the supernatant. The genetic reason for the good secretion property is not known. In the present work, differences between the DNA sequences of the industrial strain B.licheniformis "E" and the wild-type (wt) strain B. licheniformis DSM13 that contribute to the good secretion properties of the industrial strain are to be identified. It is likely that such mutations are localized in the genes of the protein biosynthesis or the protein translocation. One possible gene locus for such mutations is the secA gene locus, which harbors one gene of the protein translocation (secA) and also one gene of the protein biosynthesis (prfB). In comparison to the wt strain, the secA gene locus of the industrial strain shows 41 point mutations. Only one of these mutations is localized within the secA gene whereas the remaining 40 mutations are found in the much shorter prfB gene. The point mutation within the secA gene is silent and has therefore no influence on the amino acid sequence of the SecA protein. Because in the late stationary growth phase the industrial strain has 4 times more SecA protein than the wt strain, it is assumed that the mutations lead to a stabilization of the bicistronic secA-prjB-mRNA. The resulting increase of the SecA concentration could be one reason for the good secretion property of the industrial strain. Two of the mutations localized within the prjB gene each lead to an amino acid change in the corresponding RF2 (release factor 2) protein. By varying the expression strength of the unchanged and the mutated prfB gene it was possible to investigate the influence of the amino acid changes as well as the influence of a higher amount of RF2 on the subtilisin yield obtained with the wt or the industrial strain. Because both the nature and the concentration of the RF2 protein have an influence on the subtilisin yield, it is very likely that the identified mutations within the secA gene locus together with additional mutations in other components of the protein biosynthesis machinery lead to an increased capacity for protein biosynthesis. It is assumed that this is one reason for the high subtilisin yields that are obtained with the industrial strain. For the industrial production of subtilisin, the subtilisin gene is expressed from a high copy plasmid, which harbors an antibiotic resistance cassette for the initial selection. In contrast, the real fermentation is carried out without adding antibiotics, therefore some of the cells from the culture will lose their plasmid. In the present work, an alternative selection system was constructed with the secA gene as a selection marker. The constructed subtilisin-SecAplasmid was stable in the secA deletion strain throughout the entire cultivation period. In shaking flask experiments this strain secrets amounts of subtilisin into the supernatant at least similar to those obtained with the original strain without selection pressure by antibiotics. The perfect stability of the subtilisin-SecA-plasmid indicates that especially under the fermentative conditions of industrial subtilisin production a significant increase in yield can be reached.