Abstract
BackgroundMulti-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster.ResultsWe performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains.ConclusionsHere we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.
Highlights
Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression
P2 and Wisconsin 54-1255 are two independently derived derivatives from the very early penicillin production strain Q176, which was later used for further strain improvement programs [10, 12]
The three penicillin biosynthesis genes pcbAB, pcbC, and penDE are clustered in a single 18-kb region in wild-type strains of the filamentous fungi P. chrysogenum and Aspergillus nidulans
Summary
Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. The second step is characterized by the oxidative ring closure of the linear ACV tripeptide, leading to the formation of a bicyclic ring comprising the β-lactam and thiazolidine ring This reaction is catalyzed by the isopenicillin N synthase, encoded by the pcbC gene (synonym, ipnA). This final step is catalyzed by the acyl-coenzyme A: isopenicillin N acyltransferase, and the corresponding gene is penDE (synonym, aatA) (for an overview see [2, 4, 5])
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