Skip to main content
SpringerLink
Log in

Apc.LaeA and Apc.VeA of the velvet complex govern secondary metabolism and morphological development in the echinocandin-producing fungus Aspergillus pachycristatus

  • Genetics and Molecular Biology of Industrial Organisms - Original Paper
  • Published:
Journal of Industrial Microbiology & Biotechnology

Abstract

The impact of the global secondary metabolite regulators LaeA and VeA on echinocandin B production and morphological development was evaluated in the industrial production strain Aspergillus pachycristatus NRRL 11440. Other representative secondary metabolites were examined as well to determine if the velvet complex functions as in A. nidulans and other species of fungi. Genetic methods used for gene manipulations in A. nidulans were applied to A. pachycristatus. Separate deletions of genes Apc.laeA and Apc.veA resulted in similar yet differing phenotypes in strain NRRL 11440. Disruption of Apc.laeA and Apc.veA significantly reduced, but did not eliminate, the production of echinocandin B. Similar to what has been observed in A. nidulans, the production of sterigmatocystin was nearly eliminated in both mutants. Quantitative reverse transcription PCR analyses confirmed that selected genes of both the echinocandin B and sterigmatocystin gene clusters were down-regulated in both mutant types. The two mutants differed with respect to growth of aerial hyphae, pigmentation, development of conidiophores, conidial germination rate, and ascospore maturation. Further functional annotation of key regulatory genes in A. pachycristatus and related Aspergillus species will improve our understanding of regulation of echinocandin production and co-produced metabolites.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Balkovec JM, Hughes DL, Masurekar P, Sable CA, Schwartz RA, Singh SB (2013) Discovery and development of first in class antifungal caspofungin (Cancidas). A case study. Nat Prod Rep 31:15–34

    Google Scholar 

  2. Vazquez JA, Sobel JD (2006) Anidulafungin: a novel echinocandin. Clin Infect Dis 43:215–222

    PubMed  Google Scholar 

  3. Sofjan AK, Mitchell A, Shah DN, Nguyen T, Sim M, Trojcak A, Beyda ND, Garey KW (2018) Rezafungin (CD101), a next-generation echinocandin: a systematic literature review and assessment of possible place in therapy. J Glob Antimicrob Resist 14:58–64

    PubMed  Google Scholar 

  4. Lakota EA, Ong V, Flanagan S, Rubino CM (2018) Population pharmacokinetic analyses for rezafungin (CD101) efficacy using phase 1 data. Antimicrob Agents Chemother 62:e02603–e02617

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Zhao Y, Perez WB, Jiménez-Ortigosa C, Hough G, Locke JB, Ong V, Bartizal K, Perlin DS (2016) CD101: a novel long-acting echinocandin. Cell Microbiol 18:1308–1316

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Wiederhold NP, Locke JB, Bartizal K, Daruwala P (2018) Rezafungin (CD101) demonstrates potent in vitro activity against Aspergillus, including azole-resistant Aspergillus fumigatus isolates and cryptic species. J Antimicrob Chemother 73:3063–3067

    CAS  PubMed  Google Scholar 

  7. Lepak AJ, Zhao M, Andes DR (2018) Pharmacodynamic evaluation of rezafungin (CD101) against Candida auris in the neutropenic mouse invasive candidiasis model. Antimicrob Agents Chemother 62:e01572–e01618

    PubMed  PubMed Central  Google Scholar 

  8. Locke JB, Almaguer AL, Donatelli JL, Bartizal KF (2018) Time-kill kinetics of rezafungin (CD101) in vagina-simulative medium for fluconazole-susceptible and fluconazole-resistant Candida albicans and non-albicans Candida species. Infect Dis Obst Gynecol 2018:10

    Google Scholar 

  9. Cushion M, Ashbaugh A (2019) Rezafungin prophylactic efficacy in a mouse model of Pneumocystis pneumonia. Biol Blood Marrow Transplant 25:S366

    Google Scholar 

  10. Benz F, Knüsel F, Nüesc J, Treichler H, Voser W, Nyfeler R, Keller-Schierlein W (1974) Stoffwechselprodukte von Mikroorganismen 143. Mitteilung. Echinocandin B, ein neuartiges Polypeptid-Antibioticum aus Aspergillus nidulans var. echinulatus: Isolierung und Bausteine. Helv Chim Act 57:2459–2477

    CAS  Google Scholar 

  11. Chen AJ, Frisvad JC, Sun BD, Varga J, Kocsubé S, Dijksterhuis J, Kim DH, Hong SB, Houbraken J, Samson RA (2016) Aspergillus section Nidulantes (formerly Emericella): polyphasic taxonomy, chemistry and biology. Stud Mycol 84:1–118

    CAS  PubMed  PubMed Central  Google Scholar 

  12. de la Cruz M, Martín J, González-Menéndez V, Pérez-Victoria I, Moreno C, Tormo JR, El Aouad N, Guarro J, Vicente F, Reyes F, Bills GF (2012) Chemical and physical modulation of antibiotic activity in Emericella species. Chem Biodivers 9:1095–1113

    PubMed  Google Scholar 

  13. Dreyfuss MM (1986) Neue Erkenntnisse aus einem pharmakologischen Pilz-screening. Sydowia 39:22–36

    Google Scholar 

  14. Mukhopadhyay T, Ganguli BN, Fehlhaber HW, Kogler H, Vertesy L (1987) Mulundocandin, a new lipopeptide antibiotic. II. Structure elucidation. J Antibiot 40:281–289

    CAS  PubMed  Google Scholar 

  15. Bills GF, Yue Q, Chen L, Li Y, An Z, Frisvad JC (2016) Aspergillus mulundensis sp. nov., a new species for the fungus producing the antifungal echinocandin lipopeptides, mulundocandins. J Antibiot 69:141–148

    CAS  PubMed  Google Scholar 

  16. Wingfield BD, Bills GF, Dong Y, Huang W, Nel WJ, Swalarsk-Parry BS, Vaghefi N, Wilken PM, An Z, de Beer ZW, De Vos L, Chen L, Duong TA, Gao Y, Hammerbacher A, Kikkert JR, Li Y, Li H, Li K, Li Q, Liu X, Ma X, Naidoo K, Pethybridge SJ, Sun J, Steenkamp ET, van der Nest MA, van Wyk S, Wingfield MJ, Xiong C, Yue Q, Zhang X (2018) Draft genome sequence of Annulohypoxylon stygium, Aspergillus mulundensis, Berkeleyomyces basicola (syn. Thielaviopsis basicola), Ceratocystis smalleyi, two Cercospora beticola strains, Coleophoma cylindrospora, Fusarium fracticaudum, Phialophora cf. hyalina, and Morchella septimelata. IMA Fungus 9:199–223

    PubMed  PubMed Central  Google Scholar 

  17. Andersen MR, Nielsen JB, Klitgaard A, Petersen LM, Zachariasen M, Hansen TJ, Blicher LH, Gotfredsen CH, Larsen TO, Nielsen KF, Mortensen UH (2013) Accurate prediction of secondary metabolite gene clusters in filamentous fungi. Proc Natl Acad Sci USA 110:E99–E107

    CAS  PubMed  Google Scholar 

  18. Inglis DO, Binkley J, Skrzypek MS, Arnaud MB, Cerqueira GC, Shah P, Wymore F, Wortman JR, Sherlock G (2013) Comprehensive annotation of secondary metabolite biosynthetic genes and gene clusters of Aspergillus nidulans, A. fumigatus, A. niger and A. oryzae. BMC Microbiol 13:23

    Google Scholar 

  19. Boeck LD (1992) New antibiotics: antifungals from Aspergillus. In: Leatham GF (ed) Frontiers in industrial mycology. Chapman & Hall, New York, pp 54–65

    Google Scholar 

  20. Boeck LD, Kastner RE (1981) Method of producing the A-30912 antibiotics. U.S. Patent 4288549

  21. Hodges RL, Hodges DW, Goggans K, Xuei X, Skatrud P, McGilvray D (1994) Genetic modification of an echinocandin B-producing strain of Aspergillus nidulans to produce mutants blocked in sterigmatocystin biosynthesis. J Ind Microbiol 13:372–381

    CAS  PubMed  Google Scholar 

  22. Hodges RL, Kelkar HS, Xuei X, Skatrud PL, Keller NP, Adams TH, Kaiser RE, Vinci VA, McGilvray D (2000) Characterization of an echinocandin B-producing strain blocked for sterigmatocystin biosynthesis reveals a translocation in the stcW gene of the aflatoxin biosynthetic pathway. J Ind Microbiol Biotechnol 25:333–341

    CAS  PubMed  Google Scholar 

  23. Cacho RA, Jiang W, Chooi YH, Walsh CT, Tang Y (2012) Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440. J Am Chem Soc 134:16781–16790

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Jiang W, Cacho RA, Chiou G, Garg NK, Tang Y, Walsh CT (2013) EcdGHK are three tailoring iron oxygenases for amino acid building blocks of the echinocandin scaffold. J Am Chem Soc 135:4457–4466

    CAS  PubMed  PubMed Central  Google Scholar 

  25. Hu Z-C, Peng L-Y, Zheng Y-G (2016) Enhancement of echinocandin B production by a UV- and microwave-induced mutant of Aspergillus nidulans with precursor- and biotin-supplying strategy. Appl Biochem Biotech 179:1213–1226

    CAS  Google Scholar 

  26. Niu K, Hu Y, Mao J, Zou S, Zheng Y (2015) Effect of microparticles on echinocandin B production by Aspergillus nidulans. Chin J Biotech 31:1082–1088

    Google Scholar 

  27. Min T, Xiong L, Liang Y, Xu R, Fa C, Yang S, Hu H (2019) Disruption of stcA blocks sterigmatocystin biosynthesis and improves echinocandin B production in Aspergillus delacroxii. World J Micrbiol Biotech 35:109

    Google Scholar 

  28. Tóth V (2012) Characterization of Aspergillus nidulans var. roseus ATCC 58397, investigation of its echinocandin B and sterigmatocystin production. PhD Thesis, University of Debrecen

  29. Yue Q, Chen L, Zhang X, Li K, Sun J, Liu X, An Z, Bills GF (2015) Evolution of chemical diversity in echinocandin lipopeptide antifungal metabolites. Euk Cell 14:698–718

    CAS  Google Scholar 

  30. Hüttel W, Youssar L, Gruning BA, Gunther S, Hugentobler KG (2016) Echinocandin B biosynthesis: a biosynthetic cluster from Aspergillus nidulans NRRL 8112 and reassembly of the subclusters Ecd and Hty from Aspergillus pachycristatus NRRL 11440 reveals a single coherent gene cluster. BMC Genom 17:570

    Google Scholar 

  31. Li Y, Lan N, Xu L, Yue Q (2018) Biosynthesis of pneumocandin lipopeptides and perspectives for its production and related echinocandins. Appl Microbiol Biotech 102:9881–9891

    CAS  Google Scholar 

  32. Oakley CE, Edgerton-Morgan H, Oakley BR (2012) Tools for manipulation of secondary metabolism pathways: rapid promoter replacements and gene deletions in Aspergillus nidulans. Methods Mol Biol 944:143–161

    CAS  PubMed  Google Scholar 

  33. Sung CT, Chang SL, Entwistle R, Ahn G, Lin TS, Petrova V, Yeh HH, Praseuth MB, Chiang YM, Oakley BR, Wang CCC (2017) Overexpression of a three-gene conidial pigment biosynthetic pathway in Aspergillus nidulans reveals the first NRPS known to acetylate tryptophan. Fungal Genet Biol 101:1–6

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Chiang Y-M, Ahuja M, Oakley CE, Entwistle R, Asokan A, Zutz C, Wang CCC, Oakley BR (2016) Development of genetic dereplication strains in Aspergillus nidulans results in the discovery of aspercryptin. Angew Chem Int Ed 55:1662–1665

    CAS  Google Scholar 

  35. Yeh HH, Ahuja M, Chiang YM, Oakley CE, Moore S, Yoon O, Hajoysky H, Bok JW, Keller NP, Wang CCC, Oakley BR (2016) Resistance gene-guided genome mining: serial promoter exchanges in Aspergillus nidulans reveal the biosynthetic pathway for fellutamide B, a proteasome inhibitor. ACS Chem Biol 11:2275–2284

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Bergmann S, Schümann J, Scherlach K, Lange C, Brakhage AA, Hertweck C (2007) Genomics-driven discovery of PKS-NRPS hybrid metabolites from Aspergillus nidulans. Nat Chem Biol 3:213–217

    CAS  PubMed  Google Scholar 

  37. Bayram O, Krappmann S, Ni M, Bok JW, Helmstaedt K, Valerius O, Braus-Stromeyer S, Kwon NJ, Keller NP, Yu JH, Braus GH (2008) VelB/VeA/LaeA complex coordinates light signal with fungal development and secondary metabolism. Science 320:1504–1506

    CAS  PubMed  Google Scholar 

  38. Sarikaya-Bayram O, Palmer JM, Keller N, Braus GH, Bayram O (2015) One Juliet and four Romeos: VeA and its methyltransferases. Front Microbiol 6:1

    PubMed  PubMed Central  Google Scholar 

  39. Kato N, Brooks W, Calvo AM (2003) The expression of sterigmatocystin and penicillin genes in Aspergillus nidulans is controlled by veA, a gene required for sexual development. Euk Cell 2:1178–1186

    CAS  Google Scholar 

  40. Bayram O, Braus GH (2012) Coordination of secondary metabolism and development in fungi: the velvet family of regulatory proteins. FEMS Microbiol Rev 36:1–24

    CAS  PubMed  Google Scholar 

  41. Bok JW, Keller NP (2004) LaeA, a regulator of secondary metabolism in Aspergillus spp. Euk Cell 3:527–535

    CAS  Google Scholar 

  42. Jain S, Keller N (2013) Insights to fungal biology through LaeA sleuthing. Fungal Biol Rev 27:51–59

    Google Scholar 

  43. Patananan AN, Palmer JM, Garvey GS, Keller NP, Clarke SG (2013) A novel automethylation reaction in the Aspergillus nidulans LaeA protein generates S-methylmethionine. J Biol Chem 288:14032–14045

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Bok JW, Keller NP (2016) Insight into fungal secondary metabolism from ten years of LaeA research. In: Hoffmeister D (ed) The mycota (a comprehensive treatise on fungi as experimental systems for basic and applied research), vol III. Springer, Berlin, pp 21–29

    Google Scholar 

  45. Estiarte N, Lawrence CB, Sanchis V, Ramos AJ, Crespo-Sempere A (2016) LaeA and VeA are involved in growth morphology, asexual development, and mycotoxin production in Alternaria alternata. Int J Food Microbiol 238:153–164

    CAS  PubMed  Google Scholar 

  46. Lan N, Zhang H, Hu C, Wang W, Calvo AM, Harris SD, Chen S, Li S (2014) Coordinated and distinct functions of velvet proteins in Fusarium verticillioides. Euk Cell 13:909–918

    Google Scholar 

  47. Wiemann P, Brown DW, Kleigrewe K, Bok JW, Keller NP, Humpf H-U, Tudzynski B (2010) FfVel1 and FfLae1, components of a velvet-like complex in Fusarium fujikuroi, affect differentiation, secondary metabolism and virulence. Mol Microbiol 77:972–994

    CAS  PubMed  PubMed Central  Google Scholar 

  48. Stanke M, Steinkamp R, Waack S, Morgenstern B (2004) AUGUSTUS: a web server for gene finding in eukaryotes. Nucl Acids Res 32:W309–W312

    CAS  PubMed  Google Scholar 

  49. Debono M (1980) Derivatives of cyclic peptide nuclei. Eur Patent Appl 0031220:A1

    Google Scholar 

  50. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408

    CAS  Google Scholar 

  51. Aghcheh RK, Nemeth Z, Atanasova L, Fekete E, Paholcsek M, Sandor E, Aquino B, Druzhinina IS, Karaffa L, Kubicek CP (2014) The VELVET A orthologue VEL1 of Trichoderma reesei regulates fungal development and is essential for cellulase gene expression. PLoS One 9:e112799

    Google Scholar 

  52. Chiang YM, Szewczyk E, Nayak T, Davidson AD, Sanchez JF, Lo HC, Ho WY, Simityan H, Kuo E, Praseuth A, Watanabe K, Oakley BR, Wang CCC (2008) Molecular genetic mining of the Aspergillus secondary metabolome: discovery of the emericellamide biosynthetic pathway. Chem Biol 15:527–532

    CAS  PubMed  PubMed Central  Google Scholar 

  53. Wiemann P, Sieber CMK, von Bargen KW, Studt L, Niehaus E-M, Espino JJ, Huß K, Michielse CB, Albermann S, Wagner D, Bergner SV, Connolly LR, Fischer A, Reuter G, Kleigrewe K, Bald T, Wingfield BD, Ophir R, Freeman S, Hippler M, Smith KM, Brown DW, Proctor RH, Münsterkötter M, Freitag M, Humpf H-U, Güldener U, Tudzynski B (2013) Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Path 9:e1003475

    CAS  Google Scholar 

  54. von Bargen KW, Niehaus E-M, Krug I, Bergander K, Würthwein E-U, Tudzynski B, Humpf H-U (2015) Isolation and structure elucidation of fujikurins A-D: products of the PKS19 gene cluster in Fusarium fujikuroi. J Nat Prod 78:1809–1815

    Google Scholar 

  55. Calvo AM (2008) The VeA regulatory system and its role in morphological and chemical development in fungi. Fungal Genet Biol 45:1053–1061

    CAS  PubMed  Google Scholar 

  56. Chang P-K, Scharfenstein LL, Ehrlich KC, Wei Q, Bhatnagar D, Ingber BF (2012) Effects of laeA deletion on Aspergillus flavus conidial development and hydrophobicity may contribute to loss of aflatoxin production. Fungal Biol 116:298–307

    CAS  PubMed  Google Scholar 

  57. Bok JW, Balajee SA, Marr KA, Andes D, Nielsen KF, Frisvad JC, Keller NP (2005) LaeA, a regulator of morphogenetic fungal virulence factors. Euk Cell 4:1574–1582

    CAS  Google Scholar 

  58. Kopke K, Hoff B, Bloemendal S, Katschorowski A, Kamerewerd J, Kück U (2013) Members of the Penicillium chrysogenum velvet complex play functionally opposing roles in the regulation of penicillin biosynthesis and conidiation. Euk Cell 12:299–310

    CAS  Google Scholar 

  59. Wu D, Oide S, Zhang N, Choi MY, Turgeon BG (2012) ChLae1 and ChVel1 regulate T-toxin production, virulence, oxidative stress response, and development of the maize pathogen Cochliobolus heterostrophus. PLoS Path 8:e1002542

    Google Scholar 

Download references

Acknowledgements

This work was supported by Cidara Therapeutics, Inc. Yan Li assisted in setting up HPLC–MS calibration curves. We thank the USDA NRRL Culture Collection for supply of strain 11440 and Philipp Weimann for helpful comments on the draft manuscript.

Author information

Authors and Affiliations

  1. Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center at Houston, 1881 East Road, 3SCR6.4676, Houston, TX, 77054, USA

    Nan Lan, Qun Yue, Zhiqiang An & Gerald F. Bills

  2. Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, China

    Qun Yue

Authors
  1. Nan Lan
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Qun Yue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhiqiang An
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Gerald F. Bills
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gerald F. Bills.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest. The opinions expressed in this article are solely those of the authors and are independent of those of Cidara Therapeutics, Inc.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (PDF 1323 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lan, N., Yue, Q., An, Z. et al. Apc.LaeA and Apc.VeA of the velvet complex govern secondary metabolism and morphological development in the echinocandin-producing fungus Aspergillus pachycristatus. J Ind Microbiol Biotechnol 47, 155–168 (2020). https://doi.org/10.1007/s10295-019-02250-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10295-019-02250-x

Keywords

Search

Navigation