Sequence analysis and gene amplification study of the penicillin biosynthesis gene cluster from different strains of <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> |
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Authors: | Roman Šmidák Martina Kralovičová Beatrica Ševčíková Mária Jakubčová Ján Kormanec Jozef Timko Ján Turňa |
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Institution: | 1.Institute of Molecular Biology,Slovak Academy of Sciences,Bratislava 45,Slovakia;2.Slovenská ?up?a 566,Slovakia;3.Faculty of Natural Sciences,Comenius University, Department of Molecular Biology, Mlynská dolina,Bratislava,Slovakia |
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Abstract: | Industrial strains of Penicillium chrysogenum possess many genomic changes leading to higher levels of penicillin. In this work several production and wild-type strains
of Penicillium chrysogenum were used in comparative nucleotide sequence analysis of the biosynthesis cluster. The alignments confirmed sequence conservation
not only in promoter regions of the biosynthesis genes but also throughout the entire 44.7-kbp genomic fragment comprising
the whole biosynthesis cluster with 15.5-kbp and 13.1-kbp flanking regions. As another titre-enhancing mechanism we subsequently
examined gene dosage in two production strains introduced here, NMU2/40 and B14. Quantitative real-time PCR and Southern blot
analysis showed the amplification of the biosynthesis genes in both these strains. Through the real-time PCR method the exact
copy number was estimated for each of the pcbAB, pcbC and penDE genes. The equal pool of all three genes per genome was confirmed for the both production strains indicating that in these
strains the entire penicillin cluster has been amplified as an intact element. Penicillium chrysogenum NMU2/40 was found to carry four copies of the cluster, while six copies were estimated for B14. This also proves the contribution
of the additional titre-enhancing mechanisms in both strains, since the industrial data referred much higher production of
these strains compared with the single copy reference strain NRRL 1951. |
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