Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in penicillium species

ABSTRACT Filamentous fungi produce a wide range of bioactive compounds with important pharmaceutical applications, such as antibiotic penicillins and cholesterol-lowering statins. However,

less attention has been paid to fungal secondary metabolites compared to those from bacteria. In this study, we sequenced the genomes of 9 _Penicillium_ species and, together with 15

published genomes, we investigated the secondary metabolism of _Penicillium_ and identified an immense, unexploited potential for producing secondary metabolites by this genus. A total of

1,317 putative biosynthetic gene clusters (BGCs) were identified, and polyketide synthase and non-ribosomal peptide synthetase based BGCs were grouped into gene cluster families and mapped

to known pathways. The grouping of BGCs allowed us to study the evolutionary trajectory of pathways based on 6-methylsalicylic acid (6-MSA) synthases. Finally, we cross-referenced the

predicted pathways with published data on the production of secondary metabolites and experimentally validated the production of antibiotic yanuthones in Penicillia and identified a

previously undescribed compound from the yanuthone pathway. This study is the first genus-wide analysis of the genomic diversity of Penicillia and highlights the potential of these species

as a source of new antibiotics and other pharmaceuticals. Access through your institution Buy or subscribe This is a preview of subscription content, access via your institution ACCESS

OPTIONS Access through your institution Access Nature and 54 other Nature Portfolio journals Get Nature+, our best-value online-access subscription $32.99 / 30 days cancel any time Learn

more Subscribe to this journal Receive 12 digital issues and online access to articles $119.00 per year only $9.92 per issue Learn more Buy this article * Purchase on SpringerLink * Instant

access to full article PDF Buy now Prices may be subject to local taxes which are calculated during checkout ADDITIONAL ACCESS OPTIONS: * Log in * Learn about institutional subscriptions *

Read our FAQs * Contact customer support SIMILAR CONTENT BEING VIEWED BY OTHERS COMPREHENSIVE ANALYSIS OF BIOSYNTHETIC GENE CLUSTERS IN BACTERIA AND DISCOVERY OF _TUMEBACILLUS_ AS A

POTENTIAL PRODUCER OF NATURAL PRODUCTS Article 29 March 2023 GENOME MINING REVEALS NOVEL BIOSYNTHETIC GENE CLUSTERS IN ENTOMOPATHOGENIC BACTERIA Article Open access 25 November 2023

COMPENDIUM OF SPECIALIZED METABOLITE BIOSYNTHETIC DIVERSITY ENCODED IN BACTERIAL GENOMES Article 02 May 2022 REFERENCES * Aminov, R. I. A brief history of the antibiotic era: lessons learned

and challenges for the future. _Front. Microbiol._ 1, 134 (2010). PubMed  PubMed Central  Google Scholar  * Keller, N. P., Turner, G. & Bennett, J. W. Fungal secondary metabolism—from

biochemistry to genomics. _Nat. Rev. Microbiol._ 3, 937–947 (2005). CAS  PubMed  Google Scholar  * Nielsen, J. C. & Nielsen, J. Development of fungal cell factories for the production of

secondary metabolites: linking genomics and metabolism. _Synth. Syst. Biotechnol._ http://dx.doi.org/10.1016/j.synbio.2017.02.002 (2017). * Ziemert, N., Alanjary, M. & Weber, T. The

evolution of genome mining in microbes—a review. _Nat. Prod. Rep._ 33, 988–1005 (2016). CAS  PubMed  Google Scholar  * Medema, M. H. & Fischbach, M. A. Computational approaches to

natural product discovery. _Nat. Chem. Biol._ 11, 639–648 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Visagie, C. M. _et al._ Identification and nomenclature of the genus

_Penicillium_. _Stud. Mycol._ 78, 343–371 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Barrios-González, J. & Miranda, R. U. Biotechnological production and applications of

statins. _Appl. Microbiol. Biotechnol._ 85, 869–883 (2010). PubMed  Google Scholar  * Fang, X., Shen, Y., Zhao, J., Bao, X. & Qu, Y. Status and prospect of lignocellulosic bioethanol

production in China. _Bioresour. Technol._ 101, 4814–4819 (2010). CAS  PubMed  Google Scholar  * García-Estrada, C. & Martín, J.-F. Biosynthetic gene clusters for relevant secondary

metabolites produced by _Penicillium roqueforti_ in blue cheeses. _Appl. Microbiol. Biotechnol._ 100, 8303–8313 (2016). PubMed  Google Scholar  * Chai, B., Wu, Y., Liu, P., Liu, B. &

Gao, M. Isolation and phosphate-solubilizing ability of a fungus, _Penicillium_ sp. from soil of an alum mine. _J. Basic Microbiol._ 51, 5–14 (2011). CAS  PubMed  Google Scholar  *

Richardson, A. E. & Simpson, R. J. Soil microorganisms mediating phosphorus availability update on microbial phosphorus. _Plant Physiol._ 156, 989–996 (2011). CAS  PubMed  PubMed Central

  Google Scholar  * Puel, O., Galtier, P. & Oswald, I. P. Biosynthesis and toxicological effects of patulin. _Toxins_ 2, 613–631 (2010). CAS  PubMed  PubMed Central  Google Scholar  *

Grijseels, S . _et al._ _Penicillium arizonense_, a new, genome sequenced fungal species, reveals a high chemical diversity in secreted metabolites. _Sci. Rep_. 6, 35112 (2016). CAS  PubMed

  PubMed Central  Google Scholar  * Park, M. S., Lee, E. J., Fong, J. J., Sohn, J. H. & Lim, Y. W. A new record of _Penicillium antarcticum_ from marine environments in Korea.

_Mycobiology_ 42, 109–113 (2014). PubMed  PubMed Central  Google Scholar  * Houbraken, J., Wang, L., Lee, H. B. & Frisvad, J. C. New sections in _Penicillium_ containing novel species

producing patulin, pyripyropens or other bioactive compounds. _Persoonia_ 36, 299–314 (2015). Google Scholar  * Weber, T. _et al._ antiSMASH 3.0—a comprehensive resource for the genome

mining of biosynthetic gene clusters. _Nucleic Acids Res._ 43, W237–W243 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Kroken, S., Glass, N. L., Taylor, J. W., Yoder, O. C. &

Turgeon, B. G. Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. _Proc. Natl Acad. Sci. USA_ 100, 15670–15675 (2003). CAS  PubMed  PubMed

Central  Google Scholar  * Rausch, C., Hoof, I., Weber, T., Wohlleben, W. & Huson, D. H. Phylogenetic analysis of condensation domains in NRPS sheds light on their functional evolution.

_BMC Evol. Biol._ 7, 78 (2007). PubMed  PubMed Central  Google Scholar  * Ziemert, N. _et al._ Diversity and evolution of secondary metabolism in the marine Actinomycete genus _Salinispora_.

_Proc. Natl Acad. Sci. USA_ 111, E1130–E1139 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Medema, M. H. _et al._ Minimum information about a biosynthetic gene cluster. _Nat. Chem.

Biol._ 11, 625–631 (2015). CAS  PubMed  PubMed Central  Google Scholar  * Klejnstrup, M. L. _et al._ Genetics of polyketide metabolism in _Aspergillus nidulans_. _Metabolites_ 2, 100–133

(2012). CAS  PubMed  PubMed Central  Google Scholar  * Artigot, M. P. _et al._ Molecular cloning and functional characterization of two CYP619 cytochrome P450s involved in biosynthesis of

patulin in _Aspergillus clavatus_. _Microbiology_ 155, 1738–1747 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Holm, D. K. _et al._ Molecular and chemical characterization of the

biosynthesis of the 6-MSA-derived meroterpenoid yanuthone D in _Aspergillus niger_. _Chem. Biol._ 21, 519–529 (2014). CAS  PubMed  Google Scholar  * Frisvad, J. C., Smedsgaard, J., Larsen,

T. O. & Samson, R. A. Mycotoxins, drugs and other extrolites produced by species in _Penicillium_ subgenus _penicillium_. _Stud. Mycol._ 49, 201–241 (2004). Google Scholar  *

Vansteelandt, M. _et al._ Patulin and secondary metabolite production by marine-derived _Penicillium_ strains. _Fungal Biol._ 116, 954–961 (2012). CAS  PubMed  Google Scholar  * Boysen, M.,

Skouboe, P., Frisvad, J. & Rossen, L. Reclassification of the _Penicillium roqueforti_ group into three species on the basis of molecular genetic and biochemical profiles. _Microbiology_

142, 541–549 (1996). CAS  PubMed  Google Scholar  * Ballester, A. _et al._ Genome, transcriptome, and functional analyses of _Penicillium expansum_ provide new insights into secondary

metabolism and pathogenicity. _Mol. Plant–Microbe Interact._ 28, 232–248 (2015). CAS  PubMed  Google Scholar  * Medema, M. H., Cimermancic, P., Sali, A., Takano, E. & Fischbach, M. A. A

systematic computational analysis of biosynthetic gene cluster evolution: lessons for engineering biosynthesis. _PLoS Comput. Biol._ 10, e1004016 (2014). PubMed  PubMed Central  Google

Scholar  * Banani, H. _et al._ Genome sequencing and secondary metabolism of the postharvest pathogen _Penicillium griseofulvum_. _BMC Genomics_ 17, 19 (2016). PubMed  PubMed Central  Google

Scholar  * Itoh, T. _et al._ Reconstitution of a fungal meroterpenoid biosynthesis reveals the involvement of a novel family of terpene cyclases. _Nat. Chem._ 2, 858–864 (2010). CAS  PubMed

  Google Scholar  * Petersen, L. M., Holm, D. K., Gotfredsen, C. H., Mortensen, U. H. & Larsen, T. O. Investigation of a 6-MSA synthase gene cluster in _Aspergillus aculeatus_ reveals

6-MSA-derived aculinic acid, aculins A-B and Epi-Aculin A. _ChemBioChem_ 16, 2200–2204 (2015). CAS  PubMed  Google Scholar  * Guo, C.-J., Sun, W.-W., Bruno, K. S. & Wang, C. C. C.

Molecular genetic characterization of terreic acid pathway in _Aspergillus terreus_. _Org. Lett._ 16, 5250–5253 (2014). CAS  PubMed  PubMed Central  Google Scholar  * Bacha, N. _et al._

Cloning and characterization of novel methylsalicylic acid synthase gene involved in the biosynthesis of isoasperlactone and asperlactone in _Aspergillus westerdijkiae_. _Fungal Genet.

Biol._ 46, 742–749 (2009). CAS  PubMed  Google Scholar  * Brakhage, A. A. Regulation of fungal secondary metabolism. _Nat. Rev. Microbiol._ 11, 21–32 (2013). CAS  PubMed  Google Scholar  *

Wisecaver, J. H. & Rokas, A. Fungal metabolic gene clusters—caravans traveling across genomes and environments. _Front. Microbiol._ 6, 161 (2015). * Chae, L., Kim, T., Nilo-Poyanco, R.

& Rhee, S. Y. Genomic signatures of specialized metabolism in plants. _Science_ 344, 510–513 (2014). CAS  PubMed  Google Scholar  * Li, Y. F. _et al._ Comprehensive curation and analysis

of fungal biosynthetic gene clusters of published natural products. _Fungal Genet. Biol._ 89, 18–28 (2016). CAS  PubMed  PubMed Central  Google Scholar  * Gao, X. _et al._ Fungal indole

alkaloid biosynthesis: genetic and biochemical investigation of the tryptoquialanine pathway in _Penicillium aethiopicum_. _J. Am. Chem. Soc._ 133, 2729–2741 (2011). CAS  PubMed  PubMed

Central  Google Scholar  * Chooi, Y.-H., Cacho, R. & Tang, Y. Identification of the viridicatumtoxin and griseofulvin gene clusters from _Penicillium aethiopicum_. _Chem. Biol._ 17,

483–494 (2010). CAS  PubMed  PubMed Central  Google Scholar  * Petersen, L. M. _et al._ Characterization of four new antifungal yanuthones from _Aspergillus niger_. _J. Antibiot._ 68,

201–205 (2015). CAS  Google Scholar  * Li, X., Choi, H. D., Kang, J. S., Lee, C.-O. & Son, B. W. New polyoxygenated farnesylcyclohexenones, deacetoxyyanuthone A and its hydro derivative

from the marine-derived fungus _Penicillium_ sp. _J. Natural Prod._ 66, 1499–1500 (2003). CAS  Google Scholar  * Maskey, R. P., Grün-Wollny, I. & Laatsch, H. Sorbicillin analogues and

related dimeric compounds from _Penicillium notatum_. _J. Natural Prod._ 68, 865–870 (2005). CAS  Google Scholar  * Simpson, J. _et al._ ABySS: a parallel assembler for short read sequence

data. _Genome Res._ 19, 1117–1123 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Luo, R. _et al._ SOAPdenovo2: an empirically improved memory-efficient short-read _de novo_

assembler. _Gigascience_ 1, 18 (2012). PubMed  PubMed Central  Google Scholar  * Bankevich, A. _et al._ SPAdes: a new genome assembly algorithm and its applications to single-cell

sequencing. _J. Comput. Biol._ 19, 455–477 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Chevreux, B., Thomas, W. & Suhai, S. Genome sequence assembly using trace signals and

additional sequence information. _Comp. Sci. Biol._ 99, 45–56 (1999). Google Scholar  * Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: quality assessment tool for genome

assemblies. _Bioinformatics_ 29, 1072–1075 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Vezzi, F., Narzisi, G. & Mishra, B. Reevaluating assembly evaluations with feature

response curves: gAGE and assemblathons. _PLoS ONE_ 7, e52210 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Smit, A., Hubley, R. & Green, P. _RepeatMasker Open-4.0_ (2015);

http://www.repeatmasker.org. * Smit, A. & Hubley, R. _RepeatModeler Open-1.0_ (2015); http://www.repeatmasker.org. * The UniProt consortium. UniProt: a hub for protein information.

_Nucleic Acids Res._ 43, D204–D212 (2014). * Lomsadze, A., Burns, P. D. & Borodovsky, M. Integration of mapped RNA-Seq reads into automatic training of eukaryotic gene finding algorithm.

_Nucleic Acids Res._ 42, e119 (2014). PubMed  PubMed Central  Google Scholar  * Kim, D. _et al._ Tophat2: accurate alignment of transcriptomes in the presence of insertions, deletions and

gene fusions. _Genome Biol._ 14, R36 (2013). PubMed  PubMed Central  Google Scholar  * Trapnell, C. _et al._ Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts

and isoform switching during cell differentiation. _Nat. Biotechnol._ 28, 511–515 (2010). CAS  PubMed  PubMed Central  Google Scholar  * Holt, C. & Yandell, M. MAKER2: an annotation

pipeline and genome-database management tool for second-generation genome projects. _BMC Bioinformatics_ 12, 491 (2011). PubMed  PubMed Central  Google Scholar  * Zdobnov, E. M. &

Apweiler, R. InterProScan—an integration platform for the signature-recognition methods in InterPro. _Bioinformatics_ 17, 847–848 (2001). CAS  PubMed  Google Scholar  * Simão, F. A.,

Waterhouse, R. M., Ioannidis, P., Kriventseva, E. V. & Zdobnov, E. M. BUSCO: assessing genome assembly and annotation completeness with single-copy orthologs. _Bioinformatics_ 31,

3210–3212 (2015). PubMed  Google Scholar  * Li, L., Stoeckert, C. J. J. & Roos, D. S. OrthoMCL: identification of ortholog groups for eukaryotic genomes. _Genome Res._ 13, 2178–2189

(2003). CAS  PubMed  PubMed Central  Google Scholar  * Agren, R. _et al._ The RAVEN toolbox and its use for generating a genome-scale metabolic model for _Penicillium chrysogenum_. _PLoS

Comput. Biol._ 9, e1002980 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Yin, Y. _et al._ dbCAN: a web resource for automated carbohydrate-active enzyme annotation. _Nucleic Acids

Res._ 40, W445–W451 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Edgar, R. C. MUSCLE: multiple sequence alignment with high accuracy and high throughput. _Nucleic Acids Res._ 32,

1792–1797 (2004). CAS  PubMed  PubMed Central  Google Scholar  * Castresana, J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. _Mol. Biol.

Evol._ 17, 540–552 (2000). CAS  PubMed  Google Scholar  * Felsenstein, J. PHYLIP—phylogeny inference package (version 3.2). _Cladistics_ 5, 164–166 (1989). Google Scholar  * Darriba, D.,

Taboada, G. L., Doallo, R. & Posada, D. Prottest 3: fast selection of best-fit models of protein evolution. _Bioinformatics_ 27, 1164–1165 (2011). CAS  PubMed  Google Scholar  *

Stamatakis, A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. _Bioinformatics_ 22, 2688–2690 (2006). CAS  PubMed  Google Scholar  *

Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. _Mol. Biol. Evol._ 30, 772–780 (2013). CAS  PubMed  PubMed

Central  Google Scholar  * Guindon, S. _et al._ New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. _Syst. Biol._ 59, 307–321

(2010). CAS  PubMed  Google Scholar  * Huerta-Cepas, J., Serra, F. & Bork, P. ETE 3: reconstruction, analysis, and visualization of phylogenomic data. _Mol. Biol. Evol._ 33, 1635–1638

(2016). CAS  PubMed  PubMed Central  Google Scholar  * Ziemert, N . _et al._ The natural product domain seeker NaPDoS: a phylogeny based bioinformatic tool to classify secondary metabolite

gene diversity. _PLoS ONE_ 7, e34064 (2012). CAS  PubMed  PubMed Central  Google Scholar  * Smith, T. F. & Waterman, M. S. Identification of common molecular subsequences. _J. Mol.

Biol._ 147, 195–197 (1981). CAS  PubMed  Google Scholar  * Cock, P. J. A. _et al._ Biopython: freely available Python tools for computational molecular biology and bioinformatics.

_Bioinformatics_ 25, 1422–1423 (2009). CAS  PubMed  PubMed Central  Google Scholar  * Medema, M. H., Takano, E. & Breitling, R. Detecting sequence homology at the gene cluster level with

multigeneblast. _Mol. Biol. Evol._ 30, 1218–1223 (2013). CAS  PubMed  PubMed Central  Google Scholar  * Kildgaard, S. _et al._ Accurate dereplication of bioactive secondary metabolites from

marine-derived fungi by UHPLC-DAD-QTOFMS and a MS/HRMS library. _Mar. Drugs_ 12, 3681–3705 (2014). PubMed  PubMed Central  Google Scholar  * Klitgaard, A., Nielsen, J. B., Frandsen, R. J.

N., Andersen, M. R. & Nielsen, K. F. Combining stable isotope labeling and molecular networking for biosynthetic pathway characterization. _Anal. Chem._ 87, 6520–6526 (2015). CAS  PubMed

  Google Scholar  * Nielsen, K. F., Månsson, M., Rank, C., Frisvad, J. C. & Larsen, T. O. Dereplication of microbial natural products by LC-DAD-TOFMS. _J. Natural Prod._ 74, 2338–2348

(2011). CAS  Google Scholar  * Tannous, J. _et al._ Sequencing, physical organization and kinetic expression of the patulin biosynthetic gene cluster from _Penicillium expansum_. _Int. J.

Food Microbiol._ 189, 51–60 (2014). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the European Commission Marie Curie Initial Training Network

Quantfung (FP7-People-2013-ITN, grant no. 607332), the Novo Nordisk Foundation and the Knut and Alice Wallenberg Foundation. The computations were performed using resources at the Chalmers

Centre for Computational Science and Engineering (C3SE) provided by the Swedish National Infrastructure for Computing (SNIC). Sequencing support was provided by the Science for Life

Laboratory (SciLifeLab), National Genomics Infrastructure (NGI) and UPPMAX (UPPNEX project ID no. b2014081). Support on genome annotation by the National Bioinformatics Infrastructure Sweden

(NBIS) is acknowledged. Agilent Technologies is acknowledged for the Thought Leader Donation of the 6545 UHPLC-QTOF. The authors thank H. Wang for comments on the manuscript. AUTHOR

INFORMATION AUTHORS AND AFFILIATIONS * Department of Biology and Biological Engineering, Chalmers University of Technology, SE412 96 Gothenburg, Sweden Jens Christian Nielsen, Sylvain

Prigent, Boyang Ji & Jens Nielsen * Department of Biotechnology and Biomedicine, Technical University of Denmark, DK2800 Kgs. Lyngby, Denmark Sietske Grijseels, Kristian Fog Nielsen, 

Jens Christian Frisvad & Mhairi Workman * Department of Medical Biochemistry and Microbiology, Uppsala University, 751 23 Uppsala, Sweden Jacques Dainat * National Bioinformatics

Infrastructure Sweden (NBIS), SciLifeLab, Uppsala University, 752 37 Uppsala, Sweden Jacques Dainat * Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark,

DK2800 Kgs. Lyngby, Denmark Jens Nielsen Authors * Jens Christian Nielsen View author publications You can also search for this author inPubMed Google Scholar * Sietske Grijseels View author

publications You can also search for this author inPubMed Google Scholar * Sylvain Prigent View author publications You can also search for this author inPubMed Google Scholar * Boyang Ji

View author publications You can also search for this author inPubMed Google Scholar * Jacques Dainat View author publications You can also search for this author inPubMed Google Scholar *

Kristian Fog Nielsen View author publications You can also search for this author inPubMed Google Scholar * Jens Christian Frisvad View author publications You can also search for this

author inPubMed Google Scholar * Mhairi Workman View author publications You can also search for this author inPubMed Google Scholar * Jens Nielsen View author publications You can also

search for this author inPubMed Google Scholar CONTRIBUTIONS J.C.N., J.C.F., M.W. and J.N. conceived the study. J.C.N. designed and performed the bioinformatics computations, and analysed

and interpreted the data. S.P. and B.J. assisted with bioinformatics design and interpretation. J.D. carried out the annotation of the genomes. S.G. and K.F.N. generated culture extracts and

performed LC–MS analysis. J.C.N., S.G. and J.N. wrote the manuscript. All authors read and approved the final version of the manuscript. CORRESPONDING AUTHOR Correspondence to Jens Nielsen.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing financial interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION Supplementary Figures 1–12, Supplementary

Table 1, Supplementary References. (PDF 13841 kb) SUPPLEMENTARY DATA 1 AND 2 Supplementary Data 1: Detected PKS containing BGCs mapped to BGCs in the MIBiG database. Supplementary Data 2:

Detected NRPS containing BGCs mapped to BGCs in the MIBiG database. (XLSX 51 kb) RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Nielsen, J., Grijseels,

S., Prigent, S. _et al._ Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in _Penicillium_ species. _Nat Microbiol_ 2, 17044 (2017).

https://doi.org/10.1038/nmicrobiol.2017.44 Download citation * Received: 03 December 2016 * Accepted: 02 March 2017 * Published: 03 April 2017 * DOI:

https://doi.org/10.1038/nmicrobiol.2017.44 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative