Fungal artificial chromosomes for mining of the fungal secondary metabolome

BMC Genomics

Chengcang C Wu⁴  Nancy P Keller¹  Neil L Kelleher²  Paul M Thomas²  Anthony W Goering²  Jessica C Albright⁵  Amanda Krerowicz⁴  Megan Wagner⁴  David Mead⁴  Kenneth D Clevenger³  Rosa Ye⁴  Jin Woo Bok¹ 

[1] Department of Medical Microbiology and Immunology and Bacteriology, University of Wisconsin at Madison, Madison, WI, USA;Department of Molecular Biosciences, Northwestern University, Evanston, IL, USA;Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA;Lucigen Corporation, Middleton, WI, USA;Department of Chemistry, Northwestern University, Evanston, IL, USA

关键词: Natural product discovery;    Secondary metabolite (SM) gene clusters;    Functional genomics;    Fungal artificial chromosome (FAC);   

Others  :  1177575
DOI  :  10.1186/s12864-015-1561-x

 received in 2014-12-26, accepted in 2015-04-20,  发布年份 2015

【 摘 要 】

Background

With thousands of fungal genomes being sequenced, each genome containing up to 70 secondary metabolite (SM) clusters 30–80 kb in size, breakthrough techniques are needed to characterize this SM wealth.

Results

Here we describe a novel system-level methodology for unbiased cloning of intact large SM clusters from a single fungal genome for one-step transformation and expression in a model host. All 56 intact SM clusters from Aspergillus terreus were individually captured in self-replicating fungal artificial chromosomes (FACs) containing both the E. coli F replicon and an Aspergillus autonomously replicating sequence (AMA1). Candidate FACs were successfully shuttled between E. coli and the heterologous expression host A. nidulans. As proof-of-concept, an A. nidulans FAC strain was characterized in a novel liquid chromatography-high resolution mass spectrometry (LC-HRMS) and data analysis pipeline, leading to the discovery of the A. terreus astechrome biosynthetic machinery.

Conclusion

The method we present can be used to capture the entire set of intact SM gene clusters and/or pathways from fungal species for heterologous expression in A. nidulans and natural product discovery.

【 授权许可】

   
2015 Bok et al.; licensee BioMed Central.

【 图 表 】

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【 参考文献 】

• [1]Blackwell M: The fungi: 1, 2, 3 … 5.1 million species? Am J Bot 2011, 98:426-38.
• [2]Inglis DO, Binkley J, Skrzypek MS, Arnaud MB, Cerqueira GC, Shah P, et al.: Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiol 2013, 13:91. BioMed Central Full Text
• [3]Grigoriev IV, Cullen D, Goodwin SB, Hibbett D, Jeffries TW, Kubicek CP, et al.: Fueling the future with fungal genomics. Mycology 2011, 2:192-209.
• [4]Brakhage AA, Schroeckh V: Fungal secondary metabolites - strategies to activate silent gene clusters. Fungal Genet Biol 2011, 48:15-22.
• [5]Strauss J, Reyes-Dominguez Y: Regulation of secondary metabolism by chromatin structure and epigenetic codes. Fungal Genet Biol 2011, 48:62-9.
• [6]Hong SY, Roze LV, Linz JE: Oxidative stress-related transcription factors in the regulation of secondary metabolism. Toxins (Basel) 2013, 5:683-702.
• [7]Palmer JM, Keller NP: Secondary metabolism in fungi: does chromosomal location matter? Curr Opin Microbiol 2010, 13:431-6.
• [8]Itoh T, Kushiro T, Fujii I: Reconstitution of a secondary metabolite biosynthetic pathway in a heterologous fungal host. Methods Mol Biol 2012, 944:175-82.
• [9]Chiang YM, Oakley CE, Ahuja M, Entwistle R, Schultz A, Chang SL, et al.: An efficient system for heterologous expression of secondary metabolite genes in Aspergillus nidulans. J Am Chem Soc 2013, 135:7720-31.
• [10]Nielsen MT, Nielsen JB, Anyaogu DC, Holm DK, Nielsen KF, Larsen TO, et al.: Heterologous reconstitution of the intact geodin gene cluster in Aspergillus nidulans through a simple and versatile PCR based approach. PLoS One 2013., 8Article ID e72871
• [11]Tsunematsu Y, Ishiuchi K, Hotta K, Watanabe K: Yeast-based genome mining, production and mechanistic studies of the biosynthesis of fungal polyketide and peptide natural products. Nat Prod Rep 2013, 30:1139-49.
• [12]Yin WB, Chooi YH, Smith AR, Cacho RA, Hu Y, White TC, et al.: Discovery of cryptic polyketide metabolites from Dermatophytes using heterologous expression in Aspergillus nidulans. ACS Synth Biol 2013, 2:629-34.
• [13]Zhu H, Choi S, Johnston AK, Wing RA, Dean RA: A large-insert (130 kbp) bacterial artificial chromosome library of the rice blast fungus Magnaporthe grisea: genome analysis, contig assembly, and gene cloning. Fungal Genet Biol 1997, 21:337-47.
• [14]Nishimura M, Nakamura S, Hayashi N, Asakawa S, Shimizu N, Kaku H, et al.: Construction of a BAC library of the rice blast fungus Magnaporthe grisea and finding specific genome regions in which its transposons tend to cluster. Biosci Biotechnol Biochem 1998, 62:1515-21.
• [15]Adler H, Messerle M, Koszinowski UH: Cloning of herpesviral genomes as bacterial artificial chromosomes. Rev Med Virol 2003, 13:111-21.
• [16]Diener SE, Chellappan MK, Mitchell TK, Dunn-Coleman N, Ward M, Dean RA: Insight into Trichoderma reesei's genome content, organization and evolution revealed through BAC library characterization. Fungal Genet Biol 2004, 41:1077-87.
• [17]Srivastava SK, Huang X, Brar HK, Fakhoury AM, Bluhm BH, Bhattacharyya MK: The genome sequence of the fungal pathogen Fusarium virguliforme that causes sudden death syndrome in soybean. PLoS One 2014., 9Article ID e81832
• [18]Béjà O: To BAC or not to BAC: marine ecogenomics. Curr Opin Biotechnol 2004, 15:187-90.
• [19]Lorenz P, Eck J: Metagenomics and industrial applications. Nat Rev Microbiol 2005, 3:510-6.
• [20]Ongley SE, Bian X, Neilan BA, Müller R: Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat Prod Rep 2013, 30:1121-38.
• [21]Godiska R, Mead DA, Dhodda V, Hochstein R, Karsi A, Ravin N, et al.: Bias-Free Cloning of ‘Unclonable’ DNA for Simplified Genomic Finishing. In DNA Sequencing III: Dealing with Difficult Templates . Jones and Bartlett Publishers, Sudbury, MA; 2008.
• [22]Aleksenko A, Clutterbuck A: Autonomous plasmid replication in Aspergillus nidulans: AMA1 and MATE elements. J Fungal Genet Biol 1997, 21:373-87.
• [23]Zhang HB, Scheuring CF, Zhang M, Zhang Y, Wu CC, Dong JJ, et al.: Construction of BIBAC and BAC libraries from a variety of organisms for advanced genomics research. Nat Protoc 2012, 7:479-99.
• [24]Khaldi N, Seifuddin FT, Turner G, Haft D, Nierman WC, Wolfe KH, et al.: SMURF: Genomic mapping of fungal secondary metabolite clusters. Fungal Genet Biol 2010, 47:736-41.
• [25]Yin WB, Baccile JA, Bok JW, Chen Y, Keller NP, Schroeder FC: A nonribosomal peptide synthetase-derived iron(III) complex from the pathogenic fungus Aspergillus fumigatus. J Am Chem Soc 2013, 135:2064-7.
• [26]Watanabe T, Arisawa M, Narusuye K, Alam MS, Yamamoto K, Mitomi M, et al.: Alantrypinone and its derivatives: synthesis and antagonist activity toward insect GABA receptors. Bioorg Med Chem 2009, 17:94-111.
• [27]Kobayashi A, Hino T, Yata S, Itoh TJ, Sato H, Kawazu K: Unique spindle poisons, curvularin and its derivatives, isolated from Penicillium species. Agric Biol Chem 1988, 52:3119-23.
• [28]Kuno F, Otoguro K, Shiomi K, Iwai Y, Omura S: Arisugacin A and B, novel and selective acetylcholinesterase inhibitors from Penicillium sp. FO 4259. I. Screening, taxonomy, fermentation, isolation and biological activity. J Antibiot (Tokyo) 1996, 49:742-7.
• [29]Kumar CG, Mongolla P, Pombala S, Kamle A, Joseph J: Physicochemical characterization and antioxidant activity of melanin from a novel strain of Aspergillus bridgeri ICTF-201. Lett Appl Microbiol 2011, 53:350-8.
• [30]Wu MC, Law B, Wilkinson B, Micklefield J: Bioengineering natural product biosynthetic pathways for therapeutic applications. Curr Opin Biotechnol 2012, 23:931-40.
• [31]Du L, Robles AJ, King JB, Powell DR, Miller AN, Mooberry SL, et al.: Crowdsourcing natural products discovery to access uncharted dimensions of fungal metabolite diversity. Angew Chem Int Ed Engl 2014, 53:804-9.
• [32]Fang SM, Wu CJ, Li CW, Cui CB: A practical strategy to discover new antitumor compounds by activating silent metabolite production in fungi by diethyl sulphate mutagenesis. Mar Drugs 2014, 12:1788-814.
• [33]Leitão AL, Enguita FJ: Fungal extrolites as a new source for therapeutic compounds and as building blocks for applications in synthetic biology. Microbiol Res 2014, 169:652-65.
• [34]Takken FL, Van Wijk R, Michielse CB, Houterman PM, Ram AF, Cornelissen BJ: One-step method to convert vectors into binary vectors suited for Agrobacterium-mediated transformation. Curr Genet 2004, 45:242-8.
• [35]Aleksenko A, Makarova N, Nikolaev I, Clutterbuck AJ: Integrative and replicative transformation of Penicillium canescens. Curr Genet 1995, 28:474-7.
• [36]Aleksenko A, Gems D, Clutterbuck J: Multiple copies of MATE elements support autonomous plasmid replication in Aspergillus nidulans. Mol Microbiol 1996, 20:427-34.
• [37]Aleksenko A, Nikolaev I, Vinetski Y, Clutterbuck AJ: Gene expression from replicating plasmids in Aspergillus nidulans. Mol Gen Genet 1996, 253:242-6.
• [38]Fierro F, Kosalkova K, Gutierrez S, Martin JF: Autonomously replicating plasmids carrying the AMA1 region in Penicillium chrysogenum. Curr Genet 1996, 29:482-9.
• [39]Liu W, May GS, Lionakis MS, Lewis RE, Kontoyiannis DP: Extra copies of the Aspergillus fumigatus squalene epoxidase gene confer resistance to terbinafine: genetic approach to studying gene dose-dependent resistance to antifungals in A. fumigatus. Antimicrob Agents Chemother 2004, 48:2490-6.
• [40]Xue T, Nguyen CK, Romans A, Kontoyiannis DP, May GS: Isogenic auxotrophic mutant strains in the Aspergillus fumigatus genome reference strain AF293. Arch Microbiol 2004, 182:346-53.
• [41]Shizuya H, Birren B, Kim U-J, Mancino V, Slepak T, Tachiiri Y, et al.: Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci U S A 1992, 89:8794-7.
• [42]Murray AW, Szostak JW: Construction of artificial chromosomes in yeast. Nature 1983, 305:189-93.
• [43]Bird D, Bradshaw R: Gene targeting is locus dependent in the filamentous fungus Aspergillus nidulans. Mol Gen Genet 1997, 255:219-25.
• [44]Sinnemann SJ, Andrésson OS, Brown DW, Miao VP: Cloning and heterologous expression of Solorina crocea pyrG. Curr Genet 2000, 37:333-8.
• [45]Bok JW, Keller NP: LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryotic Cell 2004, 3:527-35.
• [46]Zhang M, Zhang Y, Scheuring CF, Wu CC, Dong JJ, Zhang HB: Preparation of megabase-sized DNA from a variety of organisms using the nuclei method for advanced genomics research. Nat Protoc 2012, 7:467-78.
• [47]Bok JW, Keller NP: Fast and easy method for construction of plasmid vectors using modified quick-change mutagenesis. Methods Mol Biol 2012, 944:163-74.
• [48]Bauer AW, Kirby WM, Sherris JC, Turck M: Antibiotic susceptibility testing by a standardized single disk method. Am J Clin Pathol 1966, 45:493-6.
• [49]Laatsch, H. Antibase 2011; Wiley VCH: Weinheim, Germany, 2011
• [50]Running, W. E. (1993) Chapman and Hall Dictionary of Natural-Products on Cd-Rom. J. Chem. Inf. Comput. Sci. 33, 934−935.
• [51]Caboche S, Pupin M, Leclère V, Fontaine A, Jacques P, Kucherov G: NORINE: a database of nonribosomal peptides. Nucleic Acids Res 2008, 36:D326-31.
• [52]Andersen MR, Nielsen JB, Klitgaard A, Petersen LM, Zachariasen M, Hansen TJ, et al.: Accurate prediction of secondary metabolite gene clusters in filamentous fungi. Proc Natl Acad Sci U S A 2013, 110:E99-107.