University of Groningen Exploring the metabolic potential of Penicillium rubens Viggiano, Annarita DOI: 10.33612/diss.126598491 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2020 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Viggiano, A. (2020). Exploring the metabolic potential of Penicillium rubens. University of Groningen. https://doi.org/10.33612/diss.126598491 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 28-09-2021 CHAPTER 3 A promoter replacement and episomal plasmid approach for the overexpression of two low-expressed PKS-NRPS hybrid genes in Penicillium rubens Annarita Viggiano1, Alka Rao1, Roel A.L. Bovenberg2,3, and Arnold J.M. Driessen1 1Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands 2DSM Biotechnology Centre, Delft, The Netherlands 3Synthetic Biology and Cell Engineering, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands ABSTRACT Filamentous fungi produce a wide range of bioactive molecules, many of which have interesting properties for human applications. Genomic data shows that the metabolic potential of these microorganisms is not fully exploited, as several biosynthetic gene clusters are silent or low-expressed under standard laboratory conditions and therefore their products are not known. A valid strategy to awaken these genes is promoter replacement, which makes the expression less dependent on environmental conditions. Here, we applied this approach to express two low-expressed polyketide synthase – non ribosomal peptide syn- thetase (PKS-NRPS) hybrid genes in Penicillium rubens. To overcome the possible epigenetic regulation in their original chromosomal locus, the genes were placed on an AMA1 (autonomous maintenance in Aspergil- lus) plasmid. This resulted in a significant overexpression of the target genes, but no new compounds could be detected in the culture broth and intracellular. Nonetheless, the approach used can be a valid tool for awakening other endogenous or heterologous biosynthetic genes, which could result in the production on novel bioactive metabolites. INTRODUCTION Filamentous fungi are an incredible source of bioactive molecules, named secondary metabolites. Secondary metabolites found many applications in medicine, agriculture and the food industry, improving significantly the expectancy and quality of our lives. Some examples include antibiotics (penicillin, cephalosporin) (1, 2), immunosuppressants (cyclosporins) (3), cholesterol-lowering drugs (statins) (4), anti-osteopo- rosis drugs (orsellinic acid derivatives) (5), antifungals (griseofulvin) (6) and food additives (carotenoids) (7). The increasing number of fungal genomes sequenced reveals that several secondary metabolite bio- synthetic genes are silent or low-expressed under standard labora- 3 tory conditions (8, 9). Therefore, many novel compounds with unique chemical structures and with potential important biological activities could be still discovered. Secondary metabolite genes are tightly regulated. Their activation is mediated by specific transcription factors as well as global and epi- genetics regulators and depends on specific conditions like carbon, nitrogen and other nutrient sources, pH, stimuli from other microor- ganisms and many other factors (10). In order to overcome the complex regulation system, the promoters of the silent genes can be exchanged with known and well characterized promoters for their expression. This approach resulted in the discovery of several new molecules. Replace- ment of the native promoter of the transcription factor apdR with the strong inducible alcohol dehydrogenase alcA promoter induced the ex- pression of a silent gene cluster containing a polyketide synthase – non ribosomal peptide synthetase (PKS-NRPS) hybrid inAspergillus nidulans. This led to the production of the toxins aspyridones (11). Another new compound was discovered few years later in the same organism using the same approach. When the native promoter of the regulator scpR was exchanged with the alcA promoter, two silent NRPSs genes and a gene cluster containing a PKS were expressed. The two NRPS genes were located nearby the regulator, while the PKS gene cluster was on another chromosome. When activated, the PKS gene cluster produced a novel compound, the asperfuranone (12). Penicillium rubens produces many diverse secondary metabolites (13, 14). Besides the well-known β-lactam antibiotics (1), several other mole- cules have been isolated, including the mycotoxins roquefortines (15) and 89 the pigments chrysogine (16) and sorbicillinoids (17). Although P. rubens has been exploited in industry for more than seventy years, its metabolic potential was revealed only one decade ago when the genome of the industrial strain Wisconsin 54-1255 was sequenced (13). The genome sequencing showed the presence of 10 NRPSs, 20 PKSs and 2 PKS-NRPS hybrid genes. Many of these secondary metabolite biosynthetic genes appeared to be silent or low expressed under the laboratory conditions tested and therefore new valuable molecules could still be uncovered in this organism (13). This situation was even more preeminent in indus- trial strains of P. rubens, as the classical strain improvement program has led to the mutational inactivation of various gene clusters as well as transcriptional down-regulation (18). Among the very low-expressed genes, the PKS-NRPS hybrids represent an intriguing class of enzymes, considering their capacity for novel and complex chemistry. PKS-NRPS hybrids are found in many filamentous fungi and have been likely acquired from bacteria through horizontal gene transfer (19). The PKS component of these enzymes has the typical organization of a highly reducing PKS (HR-PKS), having the acyl carrier protein (ACP), Penicillium rubens Penicillium ketosynthase (KS), acyltransferase (AT), dehydratase (DH), methyl- transferase (MT) and ketoreductase (KR) domains. If present, the enoyl reductase (ER) domain is not active. Its function is not essential for the formation of the molecule or can be complemented by ER proteins present in the same biosynthetic gene cluster or somewhere else in the genome (20). Interestingly, the PKS part of the hybrids shows high homology with the nonaketide synthase involved in the biosynthesis of the cholesterol-lowering agent lovastatin, suggesting they could have a common ancestor (20, 21). The PKS portion works by an iterative mechanism using acetyl coenzyme A (CoA) and malonyl-CoA as building blocks. The condensation (C) domain of the NRPS fuses the polyketide low-expressed PKS-NRPS hybrid genes in genes PKS-NRPS hybrid low-expressed chain to a specific amino acid activated by the adenylation (A) domain of the NRPS moiety (20). The mycotoxin fusarin C is the first fungal PK-NRP molecule discov- ered (22). Since then, several other hybrids have been investigated, including the toxin aspyridone (11), the immunosuppressive agent pseu- rotin A (23), the insecticidal tenellin (24). The PK-NRP metabolites have complex and unique chemical structures, which show a wide range of biological activities, making them highly attractive for human 3: A promoter replacement and episomal plasmid approach for the overexpression of two two of the overexpression for and episomal plasmid approach replacement promoter A 3: applications. In this work, we overexpressed the two low-expressed 90 PKS-NRPS hybrid genes of P. rubens. A previous attempt of overex- pression of these genes was performed by using the Q-system based transcription control device, which was proved to regulate efficiently the penicillin biosynthetic gene cluster (BGC) (25). However, this strat- egy led to an insignificant overexpression of the two PKS-NRPS hybrid genes. Therefore, we chose a strong promoter from the penicillin BGC to drive their expression and opted for an episomal plasmid approach, in order to overcome the possible epigenetic regulation. The results of the study are described in this chapter. The episomal overexpression driven by a strong promoter represents a valid approach for awakening other endogenous or heterologous secondary metabolite genes, which could lead to the discovery of novel bioactive molecules. 3 MATERIAL AND METHODS Bioinformatics tools AntiSMASH version 4.1.0 (26) was used to identify the domain orga- nization of Pc14g00080 and Pc16g13930 and predict the substrate specificity. The protein sequences of the two PKS-NRPS hybrids were blasted against a database of 19 Penicillium species using MultiGene- Blast (27) (P. rubens Wisconsin 54-1255, P. antarcticum strain IBT 31811, P. arizonense strain