A Penicillium rubens platform strain for secondary metabolite production.
Journal
Scientific reports
ISSN: 2045-2322
Titre abrégé: Sci Rep
Pays: England
ID NLM: 101563288
Informations de publication
Date de publication:
06 05 2020
06 05 2020
Historique:
received:
21
11
2019
accepted:
08
04
2020
entrez:
8
5
2020
pubmed:
8
5
2020
medline:
1
12
2020
Statut:
epublish
Résumé
We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.
Identifiants
pubmed: 32376967
doi: 10.1038/s41598-020-64893-6
pii: 10.1038/s41598-020-64893-6
pmc: PMC7203126
doi:
Substances chimiques
Peptide Synthases
EC 6.3.2.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
7630Références
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).
pubmed: 26808821
pmcid: 4789092
doi: 10.1016/j.fgb.2016.01.012
Nielsen, J. C. et al. Global analysis of biosynthetic gene clusters reveals vast potential of secondary metabolite production in Penicillium species. Nat. Microbiol. 2 (2017).
Brakhage, A. A. Regulation of fungal secondary metabolism. Nat. Rev. Microbiol. 11, 21–32 (2013).
pubmed: 23178386
doi: 10.1038/nrmicro2916
Rutledge, P. J. & Challis, G. L. Discovery of microbial natural products by activation of silent biosynthetic gene clusters. Nat. Rev. Microbiol. 13, 509–523 (2015).
pubmed: 26119570
doi: 10.1038/nrmicro3496
Tudzynski, B. Nitrogen regulation of fungal secondary metabolism in fungi. Front. Microbiol. 5, 1–15 (2014).
doi: 10.3389/fmicb.2014.00656
Nah, H.-J., Pyeon, H.-R., Kang, S.-H., Choi, S.-S. & Kim, E.-S. Cloning and Heterologous Expression of a Large-sized Natural Product Biosynthetic Gene Cluster in Streptomyces Species. Front. Microbiol. 8, (2017).
Kim, H. U., Charusanti, P., Lee, S. Y. & Weber, T. Metabolic engineering with systems biology tools to optimize production of prokaryotic secondary metabolites. Nat. Prod. Rep. 33, 933–941 (2016).
pubmed: 27072921
doi: 10.1039/C6NP00019C
Harvey, C. J. B. et al. HEx: A heterologous expression platform for the discovery of fungal natural products. Sci. Adv. 4, (2018).
Awan, A. R. et al. Biosynthesis of the antibiotic nonribosomal peptide penicillin in baker’s yeast. Nat. Commun. 8, 1–8 (2017).
doi: 10.1038/ncomms15202
Siewers, V., Chen, X., Huang, L., Zhang, J. & Nielsen, J. Heterologous production of non-ribosomal peptide LLD-ACV in Saccharomyces cerevisiae. Metab. Eng. 11, 391–397 (2009).
pubmed: 19686863
doi: 10.1016/j.ymben.2009.08.002
Clevenger, K. D. et al. A scalable platform to identify fungal secondary metabolites and their gene clusters. Nat. Chem. Biol. 13, 895–901 (2017).
pubmed: 28604695
pmcid: 5577364
doi: 10.1038/nchembio.2408
Bok, J. W. et al. Fungal artificial chromosomes for mining of the fungal secondary metabolome. BMC Genomics 16, 1–10 (2015).
doi: 10.1186/s12864-015-1561-x
Shi, T., Liu, G., Ji, R., Shi, K. & Song, P. CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art. 7435–7443 https://doi.org/10.1007/s00253-017-8497-9 (2017).
Smanski, M. J. et al. Functional optimization of gene clusters by combinatorial design and assembly. Nat. Biotechnol. 32, 1241–1249 (2014).
pubmed: 25419741
doi: 10.1038/nbt.3063
Smanski, M. J. et al. Synthetic biology to access and expand nature’s chemical diversity. Nat. Rev. Microbiol. 14, 135–149 (2016).
pubmed: 26876034
pmcid: 5048682
doi: 10.1038/nrmicro.2015.24
Nielsen, J. C. & Nielsen, J. Development of fungal cell factories for the production of secondary metabolites: Linking genomics and metabolism. Synth. Syst. Biotechnol. 2, 5–12 (2017).
pubmed: 29062956
pmcid: 5625732
doi: 10.1016/j.synbio.2017.02.002
Chiang, Y. M. et al. Development of Genetic Dereplication Strains in Aspergillus nidulans Results in the Discovery of Aspercryptin. Angew. Chemie - Int. Ed. 55, 1662–1665 (2016).
doi: 10.1002/anie.201507097
Salo, O. V. et al. Genomic mutational analysis of the impact of the classical strain improvement program on β–lactam producing Penicillium chrysogenum. BMC Genomics 16, 937 (2015).
pubmed: 26572918
pmcid: 4647614
doi: 10.1186/s12864-015-2154-4
van den Berg, M. A. Impact of the Penicillium chrysogenum genome on industrial production of metabolites. Appl. Microbiol. Biotechnol. 92, 45–53 (2011).
pubmed: 21805169
doi: 10.1007/s00253-011-3476-z
Harris, D. M. et al. Engineering of Penicillium chrysogenum for fermentative production of a novel carbamoylated cephem antibiotic precursor. Metab. Eng. 11, 125–37 (2009).
pubmed: 19271269
doi: 10.1016/j.ymben.2008.12.003
McLean, K. J. et al. Single-step fermentative production of the cholesterol-lowering drug pravastatin via reprogramming of Penicillium chrysogenum. Proc. Natl. Acad. Sci. 112, 2847–2852 (2015).
pubmed: 25691737
doi: 10.1073/pnas.1419028112
pmcid: 4352836
van den Berg, M. A. et al. Genome sequencing and analysis of the filamentous fungus Penicillium chrysogenum. Nat. Biotechnol. 26, 1161–8 (2008).
pubmed: 18820685
doi: 10.1038/nbt.1498
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).
pubmed: 23555215
pmcid: 3605104
doi: 10.1371/journal.pcbi.1002980
de Boer, P. et al. Highly efficient gene targeting in Penicillium chrysogenum using the bi-partite approach in??lig4 or??ku70 mutants. Fungal Genet. Biol. 47, 839–846 (2010).
pubmed: 20659576
doi: 10.1016/j.fgb.2010.07.008
Pohl, C., Kiel, J. A. K. W., Driessen, A. J. M., Bovenberg, R. A. L. & Nygård, Y. CRISPR/Cas9 Based Genome Editing of Penicillium chrysogenum. ACS Synth. Biol. 5, 754–764 (2016).
pubmed: 27072635
doi: 10.1021/acssynbio.6b00082
Cepeda-García, C. et al. Direct involvement of the CreA transcription factor in penicillin biosynthesis and expression of the pcbAB gene in Penicillium chrysogenum. Appl. Microbiol. Biotechnol. 98, 7113–7124 (2014).
pubmed: 24818689
doi: 10.1007/s00253-014-5760-1
Viggiano, A. et al. Pathway for the biosynthesis of the pigment chrysogine by Penicillium chrysogenum. Appl. Environ. Microbiol. 84, 1–11 (2018).
doi: 10.1128/AEM.02246-17
Nielsen, L. et al. Chrysogine Biosynthesis Is Mediated by a Two-Module Nonribosomal Peptide Synthetase. 6–10 https://doi.org/10.1021/acs.jnatprod.6b00822 (2016).
Ali, H. et al. A branched biosynthetic pathway is involved in production of roquefortine and related compounds in Penicillium chrysogenum. PLoS One 8, e65328 (2013).
pubmed: 23776469
pmcid: 3680398
doi: 10.1371/journal.pone.0065328
Ali, H. et al. A non-canonical NRPS is involved in the synthesis of fungisporin and related hydrophobic cyclic tetrapeptides in Penicillium chrysogenum. PLoS One 9, (2014).
Salo, O. et al. Identification of a polyketide synthase involved in sorbicillin biosynthesis by Penicillium chrysogenum. Appl. Environ. Microbiol. 82, 3971–3978 (2016).
pubmed: 27107123
pmcid: 4907180
doi: 10.1128/AEM.00350-16
Salo, O. V. et al. Genomic mutational analysis of the impact of the classical strain improvement program on β-lactam producing Penicillium chrysogenum. BMC Genomics 16, 1–15 (2015).
doi: 10.1186/s12864-015-2154-4
Viggiano, A. et al. Pathway for the Biosynthesis of the Pigment Chrysogine by Penicillium chrysogenum. Appl. Environ. Microbiol. 84, e02246–17 (2017).
Salo, O. V. et al. Genomic mutational analysis of the impact of the classical strain improvement program on β–lactam producing Penicillium chrysogenum. BMC Genomics 16, 937 (2015).
pubmed: 26572918
pmcid: 4647614
doi: 10.1186/s12864-015-2154-4
Abdallah, Q. A et al. Whole - genome sequencing reveals highly specific gene targeting by in vitro assembled Cas9 - ribonucleoprotein complexes in Aspergillus fumigatus. Fungal Biol. Biotechnol. 5–12 https://doi.org/10.1186/s40694-018-0057-2 (2018).
Nødvig, C. S., Nielsen, J. B., Kogle, M. E. & Mortensen, U. H. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi. PLoS One 10, e0133085 (2015).
pubmed: 26177455
pmcid: 4503723
doi: 10.1371/journal.pone.0133085
Kovalchuk, A., Weber, S., Nijland, J., Bovenberg, R. L. & Driessen, A. M. Fungal ABC Transporter Deletion and Localization Analysis. In Plant Fungal Pathogens SE − 1 (eds. Bolton, M. D. & Thomma, B. P. H. J.) 835, 1–16 (Humana Press, 2012).
Tautenhahn, R., Patti, G. J., Rinehart, D. & Siuzdak, G. XCMS Online: A Web-Based Platform to Process Untargeted Metabolomic Data. (2012).
Righelato, R. C., Trinci, A. P. J., Pirt, S. J. & Peat, A. The Influence of Maintenance Energy and Growth Rate on the Metabolic Activity, Morphology and Conidiation of Penicillium chrysogenum. J. Gen. Microbiol. 50, 399–412 (1968).
pubmed: 5652074
doi: 10.1099/00221287-50-3-399
Jónás, Á. et al. Extra-and intracellular lactose catabolism in Penicillium chrysogenum: Phylogenetic and expression analysis of the putative permease and hydrolase genes. J. Antibiot. (Tokyo). 67, 489–497 (2014).
doi: 10.1038/ja.2014.26
Nagy, Z., Keresztessy, Z., Szentirmai, A. & Biró, S. Carbon source regulation of beta-galactosidase biosynthesis in Penicillium chrysogenum. J. Basic Microbiol. 41, 351–362 (2001).
pubmed: 11802545
doi: 10.1002/1521-4028(200112)41:6<351::AID-JOBM351>3.0.CO;2-O
Kanehisa, M. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 28, 27–30 (2000).
pubmed: 10592173
pmcid: 102409
doi: 10.1093/nar/28.1.27
Sutter, B. M., Wu, X., Laxman, S. & Tu, B. P. XMethionine inhibits autophagy and promotes growth by inducing the SAM-responsive methylation of PP2A. Cell 154, 403–415 (2013).
pubmed: 23870128
pmcid: 3774293
doi: 10.1016/j.cell.2013.06.041
Laxman, S. et al. XSulfur amino acids regulate translational capacity and metabolic homeostasis through modulation of tRNA thiolation. Cell 154, (2013).
Feng, Y., He, D., Yao, Z. & Klionsky, D. J. The machinery of macroautophagy. Cell Res. 24, 24–41 (2014).
pubmed: 24366339
doi: 10.1038/cr.2013.168
Ichimura, Y. et al. A ubiquitin-like sustem mediated protein lipidation. Nature 408, 488–492 (2000).
pubmed: 11100732
doi: 10.1038/35044114
Kraft, C. et al. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 31, 3691–3703 (2012).
pubmed: 22885598
pmcid: 3442273
doi: 10.1038/emboj.2012.225
Bartoszewska, M., Kiel, Ja. K. W., Bovenberg, Ra. L., Veenhuis, M. & van der Klei, I. J. Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum. Appl. Environ. Microbiol. 77, 1413–22 (2011).
pubmed: 21169429
doi: 10.1128/AEM.01531-10
Kleijn, R. J. et al. Cytosolic NADPH metabolism in penicillin-G producing and non-producing chemostat cultures of Penicillium chrysogenum. Metab. Eng. 9, 112–123 (2007).
pubmed: 17008114
doi: 10.1016/j.ymben.2006.08.004
Polli, F., Meijrink, B., Bovenberg, R. A. L. & Driessen, A. J. M. New promoters for strain engineering of Penicillium chrysogenum. Fungal Genet. Biol. 89, 62–71 (2016).
pubmed: 26701309
doi: 10.1016/j.fgb.2015.12.003
Langfelder, K., Streibel, M., Jahn, B., Haase, G. & Brakhage, A. A. Biosynthesis of fungal melanins and their importance for human pathogenic fungi. Fungal Genet. Biol. 38, 143–158 (2003).
pubmed: 12620252
doi: 10.1016/S1087-1845(02)00526-1
Guzmán-Chávez, F. et al. Deregulation of secondary metabolism in a histone deacetylase mutant of Penicillium chrysogenum. Microbiologyopen e00598 https://doi.org/10.1002/mbo3.598 (2018).
Hunkeler, M., Stuttfeld, E., Hagmann, A., Imseng, S. & Maier, T. The dynamic organization of fungal acetyl-CoA carboxylase. Nat. Commun. 7, 11196 (2016).
pubmed: 27073141
pmcid: 4833862
doi: 10.1038/ncomms11196
Wakil, S. J., Stoops, J. K. & Joshi, V. C. Fatty acid synthesis and its regulation. Annu. Rev. Biochem. 52, 537–79 (1983).
pubmed: 6137188
doi: 10.1146/annurev.bi.52.070183.002541
Grijseels, S. et al. Identification of the decumbenone biosynthetic gene cluster in Penicillium decumbens and the importance for production of calbistrin. Fungal Biol. Biotechnol. 5, 18 (2018).
pubmed: 30598828
pmcid: 6299560
doi: 10.1186/s40694-018-0063-4
Fujii, Y., Asahara, M., Ichinoe, M. & Nakajima, H. Fungal melanin inhibitor and related compounds from Penicillium decumbens. Phytochemistry 60, 703–708 (2002).
pubmed: 12127587
doi: 10.1016/S0031-9422(02)00196-6
Herbst, D. A., Townsend, C. A. & Maier, T. The architectures of iterative type I PKS and FAS. Nat. Prod. Rep. 35, 1046–1069 (2018).
pubmed: 30137093
pmcid: 6192843
doi: 10.1039/C8NP00039E
Livak, K. J. & Schmittgen, T. D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 25, 402–408 (2001).
pubmed: 11846609
doi: 10.1006/meth.2001.1262
Ye, J. et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13, 134 (2012).
pubmed: 22708584
pmcid: 3412702
doi: 10.1186/1471-2105-13-134
Polli, F., Meijrink, B., Bovenberg, R. A. L. & Driessen, A. J. M. New promoters for strain engineering of Penicillium chrysogenum. Fungal Genet. Biol. 89, 62–71 (2016).
pubmed: 26701309
doi: 10.1016/j.fgb.2015.12.003
Weber, S. S. & Kovalchuk, A. Bovenberg, R. a L. & Driessen, A. J. M. The ABC transporter ABC40 encodes a phenylacetic acid export system in Penicillium chrysogenum. Fungal Genet. Biol. 49, 915–21 (2012).
pubmed: 23010151
doi: 10.1016/j.fgb.2012.09.003
Grijseels, S. et al. Physiological characterization of secondary metabolite producing Penicillium cell factories. Fungal Biol. Biotechnol. 4, 8 (2017).
pubmed: 29075506
pmcid: 5644182
doi: 10.1186/s40694-017-0036-z
Douma, R. D. et al. Intracellular metabolite determination in the presence of extracellular abundance: Application to the penicillin biosynthesis pathway in Penicillium chrysogenum. Biotechnol. Bioeng. 107, 105–115 (2010).
pubmed: 20506508
doi: 10.1002/bit.22786
Iversen, J. J. L., Thomsen, J. K. & Cox, R. P. On-line growth measurements in bioreactors by titrating metabolic proton exchange. Appl. Microbiol. Biotechnol. 42, 256–262 (1994).
doi: 10.1007/BF00902726
de Jonge, L. P., Douma, R. D., Heijnen, J. J. & van Gulik, W. M. Optimization of cold methanol quenching for quantitative metabolomics of Penicillium chrysogenum. Metabolomics 8, 727–735 (2012).
pubmed: 22833711
doi: 10.1007/s11306-011-0367-3
Forsberg, E. M. et al. Data processing, multi-omic pathway mapping, and metabolite activity analysis using XCMS Online. Nat. Protoc. 13, 633–651 (2018).
pubmed: 29494574
pmcid: 5937130
doi: 10.1038/nprot.2017.151
Harris, D. M. et al. Enzymic analysis of NADPH metabolism in β-lactam-producing Penicillium chrysogenum: Presence of a mitochondrial NADPH dehydrogenase. Metab. Eng. 8, 91–101 (2006).
pubmed: 16253533
doi: 10.1016/j.ymben.2005.09.004
Garcia-Alcalde, F., Garcia-Lopez, F., Dopazo, J. & Conesa, A. Paintomics: a web based tool for the joint visualization of transcriptomics and metabolomics data. Bioinformatics 27, 137–139 (2011).
pubmed: 21098431
doi: 10.1093/bioinformatics/btq594
Priebe, S., Kreisel, C., Horn, F. & Guthke, R. Databases and ontologies FungiFun2: a comprehensive online resource for systematic analysis of gene lists from fungal species. 31, 445–446 (2015).
Ruepp, A. et al. The FunCat, a functional annotation scheme for systematic classification of proteins from whole genomes. Nucleic Acids Res. 32, 5539–5545 (2004).
pubmed: 15486203
pmcid: 524302
doi: 10.1093/nar/gkh894
Bae, S., Park, J. & Kim, J. S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473–1475 (2014).
pubmed: 24463181
pmcid: 4016707
doi: 10.1093/bioinformatics/btu048
Deatherage, D. E. & Barrick, J. E. Engineering and Analyzing Multicellular Systems. 1151, (Springer New York, 2014).
Kumar, S., Stecher, G., Li, M., Knyaz, C. & Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 35, 1547–1549 (2018).
pubmed: 29722887
pmcid: 5967553
doi: 10.1093/molbev/msy096