Autophagy is important to the acidogenic metabolism of Aspergillus niger

Autoři: Baljinder Kaur aff001;  Narayan S. Punekar aff001
Působiště autorů: Metabolism and Enzymology Laboratory, Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai, India aff001
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article


Significant phenotypic overlaps exist between autophagy and acidogenesis in Aspergillus niger. The possible role of autophagy in the acidogenic growth and metabolism of this fungus was therefore examined and the movement of cytosolic EGFP to vacuoles served to monitor this phenomenon. An autophagy response to typical as well as a metabolic inhibitor-induced nitrogen starvation was observed in A. niger mycelia. The vacuolar re-localization of cytosolic EGFP was not observed upon nitrogen starvation in the A. niger Δatg1 strain. The acidogenic growth of the fungus consisted of a brief log phase followed by an extended autophagy-like state throughout the idiophase of fermentation. Mycelia in the idiophase were highly vacuolated and EGFP was localized to the vacuoles but no autolysis was observed. Both autophagy and acidogenesis are compromised in Δatg1 and Δatg8 strains of A. niger. The acidogenic growth of the fungus thus appears to mimic a condition of nutrient limitation and is associated with an extended autophagy-like state. This crucial role of autophagy in acidogenic A. niger physiology could be of value in improving citric acid fermentation.

Klíčová slova:

Autophagic cell death – Citric acid – Fermentation – Fungi – Mycelium – Vacuoles – Niger – Fungal physiology


1. Meyer V, Fiedler M, Nitsche B, King R. The Cell Factory Aspergillus Enters the Big Data Era: Opportunities and Challenges for Optimising Product Formation. Adv Biochem Eng Biotechnol. 2015;149: 91–132. doi: 10.1007/10_2014_297 25616499

2. Ciriminna R, Meneguzzo F, Delisi R, Pagliaro M. Citric acid: emerging applications of key biotechnology industrial product. Chem Cent J. 2017;11: 22. doi: 10.1186/s13065-017-0251-y 28326128

3. Papagianni M. Advances in citric acid fermentation by Aspergillus niger: biochemical aspects, membrane transport and modeling. Biotechnol Adv. 2007;25: 244–263. doi: 10.1016/j.biotechadv.2007.01.002 17337335

4. Netik A, Torres NV, Riol JM, Kubicek CP. Uptake and export of citric acid by Aspergillus niger is reciprocally regulated by manganese ions. Biochim Biophys Acta. 1997;1326: 287–294. doi: 10.1016/s0005-2736(97)00032-1 9218559

5. Karaffa L, Kubicek CP. Aspergillus niger citric acid accumulation: do we understand this well working black box? Appl Microbiol Biotechnol. 2003;61: 189–196. doi: 10.1007/s00253-002-1201-7 12698275

6. Papagianni M, Mattey M, Kristiansen B. Hyphal vacuolation and fragmentation in batch and fed-batch culture of Aspergillus niger and its relation to citric acid production. Process Biochem. 1999;35: 359–366.

7. Bizukojc M, Ledakowicz S. Morphologically structured model for growth and citric acid accumulation by Aspergillus niger. Enzyme Microb Technol. 2003;32: 268–281.

8. Kikuma T, Kitamoto K. Analysis of autophagy in Aspergillus oryzae by disruption of Aoatg13, Aoatg4, and Aoatg15 genes. FEMS Microbiol Lett. 2011;316: 61–69. doi: 10.1111/j.1574-6968.2010.02192.x 21204928

9. Kikuma T, Ohneda M, Arioka M, Kitamoto K. Functional analysis of the ATG8 homologue Aoatg8 and role of autophagy in differentiation and germination in Aspergillus oryzae. Eukaryot Cell. 2006;5: 1328–1336. doi: 10.1128/EC.00024-06 16896216

10. Richie DL, Fuller KK, Fortwendel J, Miley MD, McCarthy JW, Feldmesser M, et al. Unexpected link between metal ion deficiency and autophagy in Aspergillus fumigatus. Eukaryot Cell. 2007;6: 2437–2447. doi: 10.1128/EC.00224-07 17921348

11. Legisa M, Cimerman A, Sterle M. Germination of Aspergillus niger in a high citric acid yeilding medium. FEMS Microbiol Lett. 1981;11: 149–152.

12. Kikuma T, Arioka M, Kitamoto K. Autophagy during conidiation and conidial germination in filamentous fungi. Autophagy. 2007;3: 128–129. doi: 10.4161/auto.3560 17183223

13. Ma H, Kubicek CP, Rohr M. Metabolic effects of manganese deficiency in Aspergillus niger: evidence for increased protein degradation. Arch Microbiol. 1985;141: 266–268. doi: 10.1007/bf00408070 4004449

14. Onodera J, Ohsumi Y. Autophagy is required for maintenance of amino acid levels and protein synthesis under nitrogen starvation. J Biol Chem. 2005;280: 31582–31586. doi: 10.1074/jbc.M506736200 16027116

15. Kubicek CP, Hampel W, Rohr M. Manganese deficiency leads to elevated amino acid pools in citric acid accumulating Aspergillus niger. Arch Microbiol. 1979;123: 73–79. doi: 10.1007/bf00403504 543776

16. Munafo DB, Colombo MI. A novel assay to study autophagy: regulation of autophagosome vacuole size by amino acid deprivation. J Cell Sci. 2001;114: 3619–3629. 11707514

17. Mizushima N, Yoshimori T, Ohsumi Y. The role of Atg proteins in autophagosome formation. Annu Rev Cell Dev Biol. 2011;27: 107–132. doi: 10.1146/annurev-cellbio-092910-154005 21801009

18. Wen X, Klionsky DJ. An overview of macroautophagy in yeast. J Mol Biol. 2016;428: 1681–1699. doi: 10.1016/j.jmb.2016.02.021 26908221

19. Khan IA, Lu JP, Liu XH, Rehman A, Lin FC. Multifunction of autophagy-related genes in filamentous fungi. Microbiol Res. 2012;167: 339–345. doi: 10.1016/j.micres.2012.01.004 22554685

20. Pollack JK, Harris SD, Marten MR. Autophagy in filamentous fungi. Fungal Genet Biol. 2009;46: 1–8. doi: 10.1016/j.fgb.2008.10.010 19010432

21. Shoji JY, Craven KD. Autophagy in basal hyphal compartments: A green strategy of great recyclers. Fungal Biol Rev. 2011;25: 79–83.

22. Voigt O, Pöggeler S. Self-eating to grow and kill: autophagy in filamentous ascomycetes. Appl Microbiol Biotechnol. 2013;97: 9277–9290. doi: 10.1007/s00253-013-5221-2 24077722

23. Kim Y, Islam N, Moss BJ, Nandakumar MP, Marten MR. Autophagy induced by rapamycin and carbon-starvation have distinct proteome profiles in Aspergillus nidulans. Biotechnol Bioeng. 2011;108: 2705–2715. doi: 10.1002/bit.23223 21618477

24. Nitsche BM, Jorgensen TR, Akeroyd M, Meyer V, Ram AF. The carbon starvation response of Aspergillus niger during submerged cultivation: insights from the transcriptome and secretome. BMC Genomics. 2012;13: 380. doi: 10.1186/1471-2164-13-380 22873931

25. Nitsche BM, Burggraaf-van Welzen AM, Lamers G, Meyer V, Ram AF. Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger. Appl Microbiol Biotechnol. 2013;97: 8205–8218. doi: 10.1007/s00253-013-4971-1 23700238

26. Pinar M, Pantazopoulou A, Penalva MA. Live-cell imaging of Aspergillus nidulans autophagy: RAB1 dependence, Golgi independence and ER involvement. Autophagy. 2013;9: 1024–1043. doi: 10.4161/auto.24483 23722157

27. Shoji JY, Kikuma T, Arioka M, Kitamoto K. Macroautophagy-mediated degradation of whole nuclei in the filamentous fungus Aspergillus oryzae. PLoS One. 2010;5: e15650. doi: 10.1371/journal.pone.0015650 21187926

28. Yoon J, Kikuma T, Maruyama J, Kitamoto K. Enhanced production of bovine chymosin by autophagy deficiency in the filamentous fungus Aspergillus oryzae. PLoS One. 2013;8: e62512. doi: 10.1371/journal.pone.0062512 23658635

29. Bartoszewska M, Kiel JA, Bovenberg RA, Veenhuis M, van der Klei IJ.(2011) Autophagy deficiency promotes beta-lactam production in Penicillium chrysogenum. Appl Environ Microbiol. 2011;77: 1413–1422. doi: 10.1128/AEM.01531-10 21169429

30. Dave K, Punekar NS. Utility of Aspergillus niger citrate synthase promoter for heterologous expression. J Biotechnol. 2011;155: 173–177. doi: 10.1016/j.jbiotec.2011.06.012 21723343

31. Dave KK, Punekar NS. Expression of Lactate Dehydrogenase in Aspergillus niger for L-Lactic Acid Production. PLoS One. 2015;10(12): e0145459. doi: 10.1371/journal.pone.0145459 26683313

32. Swedlow JR, Platani M. Live cell imaging using wide-field microscopy and deconvolution. Cell Struct Func. 2002;27: 335–341.

33. Shoji JY, Arioka M, Kitamoto K. Vacuolar membrane dynamics in the filamentous fungus Aspergillus oryzae. Eukaryot Cell. 2006;5: 411–421. doi: 10.1128/EC.5.2.411-421.2006 16467481

34. Sambrook J, Russell DW. Molecular Cloning: A Laboratory Manual. Cold Spring Harbour Laboratory Press, 2001.

35. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72: 248–254. doi: 10.1006/abio.1976.9999 942051

36. Barry DJ, Williams GA. Microscopic characterisation of filamentous microbes: towards fully automated morphological quantification through image analysis. J Microsc. 2011;244: 1–20. doi: 10.1111/j.1365-2818.2011.03506.x 21812778

37. Cox PW, Paul GC, Thomas CR. Image analysis of the morphology of filamentous micro-organisms. Microbiology. 1998;144: 817–827. doi: 10.1099/00221287-144-4-817 9579057

38. Hickey PC, Read ND. Imaging living cells of Aspergillus in vitro. Med Mycol. 2009;47(Suppl 1): S110–119.

39. Ohneda M, Arioka M, Nakajima H, Kitamoto K. Visualization of vacuoles in Aspergillus oryzae by expression of CPY–EGFP. Fungal Genet Biol. 2002;37: 29–38. 12223187

40. Hesse SJA, Ruijter GJG, Dijkema C, Visser J. Intracellular pH homeostasis in the filamentous fungus Aspergillus niger. Eur J Biochem. 2002;269: 3485–3494. doi: 10.1046/j.1432-1033.2002.03042.x 12135488

41. Bagar T, Altenbach K, Read ND, Bencinal M. Live-cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe. Eukaryot Cell. 2009;8: 703–712. doi: 10.1128/EC.00333-08 19286983

42. Arima K, Uozumi T, Takahashi M. Studies on the autolysis of Aspergillus oryzae. Agric Biol Chem. 1965;29: 1033–1041.

43. Trinci AP, Righelato RC. Changes in Constituents and Ultrastructure of Hyphal Compartments during Autolysis of Glucose-starved Penicillium chrysogenum. J Gen Microbiol. 1970;60: 239–249. doi: 10.1099/00221287-60-2-239 5488056

44. White S, McIntyre M, Berry DR, McNeil B. The autolysis of industrial filamentous fungi. Crit Rev Biotechnol. 2002;22: 1–14. doi: 10.1080/07388550290789432 11958333

45. Choudhury R, Noor S, Varadarajalu LP, Punekar NS. Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase. Enzyme Microb Technol. 2008;42: 151–159. doi: 10.1016/j.enzmictec.2007.08.011 22578865

46. Shiroma S, Jayakody LN, Horie K, Okamoto, K, Kitagaki H. Enhancement of ethanol fermentation in Saccharomyces cerevisiae sake yeast by disrupting mitophagy function. Appl Environ Microbiol. 2014;80: 1002–1012. doi: 10.1128/AEM.03130-13 24271183

47. Punekar NS, Vaidyanathan CS, Appaji Rao N. Mechanisms of citric acid fermentation by Aspergillus niger. J Sci Ind Res. 1984;43: 398–404.

48. Yin X, Shin H-D, Li J, Du G, Liu L, Chen J. Comparative genomics and transcriptome analysis of Aspergillus niger and metabolic engineering for citrate production. Sci Rep. 2017;7: 41040. doi: 10.1038/srep41040 28106122

Článek vyšel v časopise


2019 Číslo 10
Nejčtenější tento týden