Sandra Ziemons, Katerina Koutsantas, Kordula Becker, Tim Dahlmann and Ulrich Kück

MC Biotechnology 16 February 2017; DOI: 10.1186/s12896-017-0335-8

Abstract

Background

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. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster.

Results

We 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.

Conclusions

Here 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.

See: http://bmcbiotechnol.biomedcentral.com/articles/10.1186/s12896-017-0335-8

Fig. 1

Determination of the copy-number of different P. chrysogenum strains using PFGE. (A) Genetic map of the duplicated 110 kb pen cluster region, based on the recently published genome sequence [23]. The penicillin biosynthesis genes are marked in red and recombination sites are indicated in bold face (A-H) and are identical to those reported for other Penicillium strains [9]. (B) Simplified map of the penicillin genes and adjacent regions showing the probes (adh-like, pcbC, rco3 and exo84) for Southern hybridization experiments. In addition restriction sites for PmeI, PacI and SwaI are given. (C) Pulsed field gel electrophoresis (PFGE) of PmeI, PacI or SwaI restricted genomic DNA. For these experiments, the original cantaloupe strain NRRL 1951 [C] and the high-producer P2niaD18 [P] were used. The duplicated area is given as a grey bar in AB, and fragments that indicate the proposed duplications are marked with a red asterisk in C