Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains

Dahlmann, Tim A.; Böhm, Julia; Becker, Kordula; Kück, Ulrich

doi:10.1007/s00294-015-0497-7

Cited by 12 publications

(9 citation statements)

References 37 publications

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“…We previously showed via pulsed-field gel electrophoresis analysis that strains with different cephalosporin C production rates have undergone major genomic reorganizations [75]. This is reminiscent of genome rearrangements that were recently demonstrated for P. chrysogenum P2niaD18 [20, 76] and other industrial penicillin producer strains [77, 78]. These major chromosome rearrangements are most likely the result of extensive random mutagenesis that occurred during repeated strain improvement programs.…”

Section: Resultsmentioning

confidence: 98%

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

2017

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BackgroundCephalosporins and penicillins are the most frequently used β-lactam antibiotics for the treatment of human infections worldwide. The main industrial producers of these antibiotics are Acremonium chrysogenum and Penicillium chrysogenum, two taxonomically unrelated fungi. Both were subjects of long-term strain development programs to reach economically relevant antibiotic titers. It is so far unknown, whether equivalent changes in gene expression lead to elevated antibiotic titers in production strains.ResultsUsing the sequence of PcbC, a key enzyme of β-lactam antibiotic biosynthesis, from eighteen different pro- and eukaryotic microorganisms, we have constructed a phylogenetic tree to demonstrate the distant relationship of both fungal producers. To address the question whether both fungi have undergone similar genetic adaptions, we have performed a comparative gene expression analysis of wild-type and production strains. We found that strain improvement is associated with the remodeling of the transcriptional landscape in both fungi. In P. chrysogenum, 748 genes showed differential expression, while 1572 genes from A. chrysogenum are differentially expressed in the industrial strain. Common in both fungi is the upregulation of genes belonging to primary and secondary metabolism, notably those involved in precursor supply for β-lactam production. Other genes not essential for β-lactam production are downregulated with a preference for those responsible for transport processes or biosynthesis of other secondary metabolites. Transcriptional regulation was shown to be an important parameter during strain improvement in different organisms. We therefore investigated deletion strains of the major transcriptional regulator velvet from both production strains.We identified 567 P. chrysogenum and 412 A. chrysogenum Velvet target genes. In both deletion strains, approximately 50% of all secondary metabolite cluster genes are differentially regulated, including β-lactam biosynthesis genes. Most importantly, 35-57% of Velvet target genes are among those that showed differential expression in both improved industrial strains.ConclusionsThe major finding of our comparative transcriptome analysis is that strain improvement programs in two unrelated fungal β-lactam antibiotic producers alter the expression of target genes of Velvet, a global regulator of secondary metabolism. From these results, we conclude that regulatory alterations are crucial contributing factors for improved β-lactam antibiotic titers during strain improvement in both fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-017-3663-0) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 98%

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

2017

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“…This analysis can be extended by using rare cutting restriction enzymes, thus allowing determination of the chromosomal structure of a region of interest [ 29 ]. This approach is in particular reasonable for P. chrysogenum , which has only four chromosomes, because the two larger chromosomes with a size above 10 Mb are difficult to separate electrophoretically [ 30 ]. Here, we performed PFGE (Fig.…”

Section: Resultsmentioning

confidence: 99%

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes

et al. 2017

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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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0335-8) contains supplementary material, which is available to authorized users.

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“…M. thermophila is not capable of self-crossing and does not cross with close relative Myceliophthora heterothallica , which has been experimentally demonstrated to have a sexual life cycle [ 57 , 58 ]. An additional advantage of the homothallic T. aurantiacus is that crossing does not require strains with different mating types as in heterothallic fungi like T. reesei [ 23 ] or P. chrysogenum [ 59 ]. Ascospores are the only means of propagation in T. aurantiacus and are produced in as little as 4–5 days.…”

Section: Discussionmentioning

confidence: 99%

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

Gabriel

Prinz

Jecmenica

et al. 2020

Biotechnol Biofuels

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Background Fungal enzymes are vital for industrial biotechnology, including the conversion of plant biomass to biofuels and bio-based chemicals. In recent years, there is increasing interest in using enzymes from thermophilic fungi, which often have higher reaction rates and thermal tolerance compared to currently used fungal enzymes. The thermophilic filamentous fungus Thermoascus aurantiacus produces large amounts of highly thermostable plant cell wall-degrading enzymes. However, no genetic tools have yet been developed for this fungus, which prevents strain engineering efforts. The goal of this study was to develop strain engineering tools such as a transformation system, a CRISPR/Cas9 gene editing system and a sexual crossing protocol to improve the enzyme production. Results Here, we report Agrobacterium tumefaciens-mediated transformation (ATMT) of T. aurantiacus using the hph marker gene, conferring resistance to hygromycin B. The newly developed transformation protocol was optimized and used to integrate an expression cassette of the transcriptional xylanase regulator xlnR, which led to up to 500% increased xylanase activity. Furthermore, a CRISPR/Cas9 gene editing system was established in this fungus, and two different gRNAs were tested to delete the pyrG orthologue with 10% and 35% deletion efficiency, respectively. Lastly, a sexual crossing protocol was established using a hygromycin B- and a 5-fluoroorotic acid-resistant parent strain. Crossing and isolation of progeny on selective media were completed in a week. Conclusion The genetic tools developed for T. aurantiacus can now be used individually or in combination to further improve thermostable enzyme production by this fungus.

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Sexual recombination as a tool for engineering industrial Penicillium chrysogenum strains

Cited by 12 publications

References 37 publications

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

Transcriptome analysis of the two unrelated fungal β-lactam producers Acremonium chrysogenum and Penicillium chrysogenum: Velvet-regulated genes are major targets during conventional strain improvement programs

Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes

Development of genetic tools for the thermophilic filamentous fungus Thermoascus aurantiacus

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