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Applied Microbiology and Biotechnology

, Volume 102, Issue 1, pp 319–332 | Cite as

Comparative proteomic analysis: SclR is importantly involved in carbohydrate metabolism in Aspergillus oryzae

  • Feng-Jie Jin
  • Pei Han
  • Miao Zhuang
  • Zhi-Min Zhang
  • Long Jin
  • Yasuji Koyama
Genomics, transcriptomics, proteomics

Abstract

The helix-loop-helix (HLH) family of transcriptional factors is a key player in a wide range of developmental processes in organisms from mammals to microbes. We previously identified the bHLH transcription factor SclR in Aspergillus oryzae and found that the loss of SclR function led to significant phenotypic changes, such as rapid protein degradation and cell lysis in dextrin-polypeptone-yeast extract liquid medium. The result implied that SclR is potentially important in both traditional fermentative manufacturing and commercial enzyme production in A. oryzae because of its effect on growth. Therefore, this study presents a comparative assessment at the proteome level of the intracellular differences between an sclR-disrupted strain and a control strain using isobaric tandem mass tag (TMT) labeling for quantification. A total of 5447 proteins were identified, and 568 were differentially expressed proteins (DEPs). Of the DEPs, 251 proteins were increased by 1.5-fold, and 317 proteins were decreased by 1.5-fold in an sclR-disrupted strain compared to the control. The comparison of the quantitative TMT results revealed that SclR was mainly involved in carbon metabolism, especially carbohydrate metabolism. In addition, an enzyme profile by a semi-quantitative method (API-ZYM) indicated that three enzymes (β-galactosidase, α-glucosidase, and α-mannosidase) were significantly less active in the ∆sclR strain than in the control. Moreover, quantitative RT-PCR showed that the expression of certain genes was changed similarly to their corresponding proteins. These results suggested that a possible function of SclR during growth of A. oryzae is its important involvement in carbohydrate metabolism.

Keywords

Aspergillus oryzae Comparative proteomic analysis bHLH transcription factor API-ZYM assay 

Notes

Funding information

This study was supported by the Natural Science Foundation of China (31570107) and the project funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD). We also thank for the help of bioinformatics analysis from SuZhou BioNovoGene (http://www.bionovogene.com).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

253_2017_8588_MOESM1_ESM.pdf (1.8 mb)
ESM 1 (PDF 1804 kb)

References

  1. Adav SS, Li AA, Manavalan A, Punt P, Sze SK (2010) Quantitative iTRAQ secretome analysis of Aspergillus niger reveals novel hydrolytic enzymes. J Proteome Res 9:3932–3940CrossRefPubMedGoogle Scholar
  2. Adav SS, Ravindran A, Sze SK (2015) Quantitative proteomic study of Aspergillus fumigatus secretome revealed deamidation of secretory enzymes. J Proteome 119:154–168CrossRefGoogle Scholar
  3. Atchley WR, Fitch WM (1997) A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci U S A 94:5172–5176CrossRefPubMedPubMedCentralGoogle Scholar
  4. Atchley WR, Terhalle W, Dress A (1999) Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J Mol Evol 48:501–516CrossRefPubMedGoogle Scholar
  5. Beutler E (2007) PGK deficiency. Br J Haematol 136:3–11CrossRefPubMedGoogle Scholar
  6. Budzyńska A, Sadowska B, Więckowska-Szakiel M, Różalska B (2014) Enzymatic profile, adhesive and invasive properties of Candida albicans under the influence of selected plant essential oils. Acta Biochim Pol 61:115–121PubMedGoogle Scholar
  7. Caruso ML, Litzka O, Martic G, Lottspeich F, Brakhage AA (2002) Novel basic-region helix-loop-helix transcription factor (AnBH1) of Aspergillus nidulans counteracts the CCAAT-binding complex AnCF in the promoter of a penicillin biosynthesis gene. J Mol Biol 323:425–439CrossRefPubMedGoogle Scholar
  8. Castilhos G, Lazzarotto F, Spagnolo-Fonini L, Bodanese-Zanettini MH, Margis-Pinheiro M (2014) Possible roles of basic helix-loop-helix transcription factors in adaptation to drought. Plant Sci 223:1–7CrossRefPubMedGoogle Scholar
  9. Chaves DF, Carvalho PC, Lima DB, Nicastro H, Lorenzeti FM, Siqueira-Filho M, Hirabara SM, Alves PH, Moresco JJ, Yates JR 3rd, Lancha AH Jr (2013) Comparative proteomic analysis of the aging soleus and extensor digitorum longus rat muscles using TMT labeling and mass spectrometry. J Proteome Res 12:4532–4546CrossRefPubMedGoogle Scholar
  10. Dutton JR, Johns S, Miller BL (1997) StuAp is a sequence-specific transcription factor that regulates developmental complexity in Aspergillus nidulans. EMBO J 16:5710–5721CrossRefPubMedPubMedCentralGoogle Scholar
  11. Fischer A, Leimeister C, Winkler C, Schumacher N, Klamt B, Elmasri H, Steidl C, Maier M, Knobeloch KP, Amann K, Helisch A, Sendtner M, Gessler M (2002) Hey bHLH factors in cardiovascular development. Cold Spring Harb Symp Quant Biol 67:63–70CrossRefPubMedGoogle Scholar
  12. Imayoshi I, Ishidate F, Kageyama R (2015) Real-time imaging of bHLH transcription factors reveals their dynamic control in the multipotency and fate choice of neural stem cells. Front Cell Neurosci 9:288CrossRefPubMedPubMedCentralGoogle Scholar
  13. Janecek S (1997) Alpha-amylase family: molecular biology and evolution. Prog Biophys Mol Biol 67:67–97CrossRefPubMedGoogle Scholar
  14. Jin FJ, Takahashi T, Machida M, Koyama Y (2009) Identification of bHLH-type transcription regulator gene by systematically deleting large chromosomal segments in Aspergillus oryzae. Appl Environ Microbiol 75:5943–5951CrossRefPubMedPubMedCentralGoogle Scholar
  15. Jin FJ, Takahashi T, Matsushima K, Hara S, Maruyama J, Kitamoto K, Koyama Y (2011a) SclR, a basic helix-loop-helix transcription factor, regulates hyphal morphology and promotes sclerotial formation in Aspergillus oryzae. Eukaryot Cell 10:945–955CrossRefPubMedPubMedCentralGoogle Scholar
  16. Jin FJ, Nishida M, Hara S, Koyama Y (2011b) Identification and characterization of a putative basic helix-loop-helix transcription factor involved in the early stage of conidiophore development in Aspergillus oryzae. Fungal Genet Biol 48:1108–1115CrossRefPubMedGoogle Scholar
  17. Jin FJ, Katayama T, Maruyama JI, Kitamoto K (2016) Comparative genomic analysis identified a mutation related to enhanced heterologous protein production in the filamentous fungus Aspergillus oryzae. Appl Microbiol Biotechnol 100:9163–9174CrossRefPubMedGoogle Scholar
  18. Kawai S, Mukai T, Mori S, Mikami B, Murata K (2005) Hypothesis: structures, evolution, and ancestor of glucose kinases in the hexokinase family. J Biosci Bioeng 99:320–330CrossRefPubMedGoogle Scholar
  19. Kitamoto N, Go M, Shibayama T, Kimura T, Kito Y, Ohmiya K, Tsukagoshi N (1996) Molecular cloning, purification and characterization of two endo-1,4-beta-glucanases from Aspergillus oryzae KBN616. Appl Microbiol Biotechnol 46:538–544CrossRefPubMedGoogle Scholar
  20. Kobayashi T, Abe K, Asai K, Gomi K, Juvvadi PR, Kato M, Kitamoto M, Takeuchi M, Machida M (2007) Genomics of Aspergillus oryzae. Biosci Biotechnol Biochem 71:646–670CrossRefPubMedGoogle Scholar
  21. Ledent V, Vervoort M (2001) The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis. Genome Res 11:754–770CrossRefPubMedPubMedCentralGoogle Scholar
  22. Leone R, Buonomo S, Nakamura K, Aoki S, Vidotto V (1998) Enzymatic profile of Cryptococcus neoformans strains by using the API-ZYM system. Rev Iberoam Micol 15:136–140PubMedGoogle Scholar
  23. Li X, Duan X, Jiang H, Sun Y, Tang Y, Yuan Z, Guo J, Liang W, Chen L, Yin J, Ma H, Wang J, Zhang D (2006) Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis. Plant Physiol 141:1167–1184CrossRefPubMedPubMedCentralGoogle Scholar
  24. Li Y, Wei Y, Guo J, Cheng Y, He W (2015) Interactional role of microRNAs and bHLH-PAS proteins in cancer (review). Int J Oncol 47:25–34CrossRefPubMedGoogle Scholar
  25. Liu JY, Men JL, Chang MC, Feng CP, Yuan LG (2017) iTRAQ-based quantitative proteome revealed metabolic changes of Flammulina velutipes mycelia in response to cold stress. J Proteome 156:75–84CrossRefGoogle Scholar
  26. Machida M, Asai K, SanoM TK, Kumagai T, Terai G, Kusumoto K, Arima T, Akita O, Kashiwagi Y, Abe K, Gomi K, Horiuchi H, Kitamoto K, Kobayashi T, Takeuchi M, Denning DW, Galagan JE, Nierman WC, Yu J, Archer DB, Bennett JW, Bhatnagar D, Cleveland TE, Fedorova ND, Gotoh O, Horikawa H, Hosoyama A, Ichinomiya M, Igarashi R, Iwashita K, Juvvadi PR, Kato M, Kato Y, Kin T, Kokubun A, Maeda H, Maeyama N, Maruyama J, Nagasaki H, Nakajima T, Oda K, Okada K, Paulsen I, Sakamoto K, Sawano T, Takahashi M, Takase K, Terabayashi Y, Wortman JR, Yamada O, Yamagata Y, Anazawa H, Hata Y, Koide Y, Komori T, Koyama Y, Minetoki T, Suharnan S, Tanaka A, Isono K, Kuhara S, Ogasawara N, Kikuchi H (2005) Genome sequencing and analysis of Aspergillus oryzae. Nature 438:1157–1161CrossRefPubMedGoogle Scholar
  27. Majumdar S, Ghatak J, Mukherji S, Bhattacharjee H, Bhaduri A (2004) UDPgalactose 4-epimerase from Saccharomyces cerevisiae. A bifunctional enzyme with aldose 1-epimerase activity. Eur J Biochem 271:753–759CrossRefPubMedGoogle Scholar
  28. Mazurek S (2011) Pyruvate kinase type M2: a key regulator of the metabolic budget system in tumor cells. Int J Biochem Cell Biol 43:969–980CrossRefPubMedGoogle Scholar
  29. Murre C, McCaw PS, Baltimore D (1989) A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56:777–783CrossRefPubMedGoogle Scholar
  30. Murre C, Bain G, van Dijk MA, Engel I, Furnari BA, Massari ME, Matthews JR, Quong MW, Rivera RR, Stuiver MH (1994) Structure and function of helix-loop-helix proteins. Biochim Biophys Acta 1218:129–135CrossRefPubMedGoogle Scholar
  31. Nesi N, Debeaujon I, Jond C, Pelletier G, Caboche M, Lepiniec L (2000) The TT8 gene encodes a basic helix-loop-helix domain protein required for expression of DFR and BAN genes in Arabidopsis siliques. Plant Cell 12:1863–1878CrossRefPubMedPubMedCentralGoogle Scholar
  32. Nitsche BM, Burggraaf-van Welzen AM, Lamers G, Meyer V, Ram AF (2013) Autophagy promotes survival in aging submerged cultures of the filamentous fungus Aspergillus niger. Appl Microbiol Biotechnol 97:8205–8218CrossRefPubMedGoogle Scholar
  33. Patterson MC (2005) Metabolic mimics: the disorders of N-linked glycosylation. Sem Pediatr Neurol 12:144–151CrossRefGoogle Scholar
  34. Pruss B, Meyer HE, Holldorf AW (1993) Characterization of the glyceraldehyde 3-phosphate dehydrogenase from the extremely halophilic archaebacterium Haloarcula vallismortis. Arch Microbiol 160:5–11PubMedGoogle Scholar
  35. Roach PJ, Depaoli-Roach AA, Hurley TD, Tagliabracci VS (2012) Glycogen and its metabolism: some new developments and old themes. Biochem J 441:763–787CrossRefPubMedPubMedCentralGoogle Scholar
  36. Rodrigues JR, Couto A, Cabezas A, Pinto RM, Ribeiro JM, Canales J, Costas MJ, Cameselle JC (2014) Bifunctional homodimeric triokinase/FMN cyclase: contribution of protein domains to the activities of the human enzyme and molecular dynamics simulation of domain movements. J Biol Chem 289:10620–10636CrossRefPubMedPubMedCentralGoogle Scholar
  37. Schurig H, Beaucamp N, Ostendorp R, Jaenicke R, Adler E, Knowles JR (1995) Phoglycerate kinase and triosephosphate isomerase from the hyperthermophilic bacterium Thermotoga maritima form a covalent bifunctional enzyme complex. EMBO J 14:442–451PubMedPubMedCentralGoogle Scholar
  38. Sirover MA (2011) On the functional diversity of glyceraldehyde-3-phosphate dehydrogenase: biochemical mechanisms and regulatory control. Biochim Biophys Acta 1810:741–751CrossRefPubMedGoogle Scholar
  39. Sloothaak J, Odoni DI, de Graaff LH, Martins Dos Santos VA, Schaap PJ, Tamayo-Ramos JA (2015) Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters. Biotechnol Biofuels 8:150CrossRefPubMedPubMedCentralGoogle Scholar
  40. Suzuki K, Tanaka M, Konno Y, Ichikawa T, Ichinose S, Hasegawa-Shiro S, Shintani T, Gomi K (2014) Distinct mechanism of activation of two transcription factors, AmyR and MalR, involved in amylolytic enzyme production in Aspergillus oryzae. Appl Microbiol Biotechnol 99:1805–1815CrossRefPubMedGoogle Scholar
  41. Toledo-Ortiz G, Huq E, Quail PH (2003) The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15:1749–1770CrossRefPubMedPubMedCentralGoogle Scholar
  42. Tüncher A, Reinke H, Martic G, Caruso ML, Brakhage AA (2004) A basic-region helix-loop-helix protein-encoding gene (devR) involved in the development of Aspergillus nidulans. Mol Microbiol 52:227–241CrossRefPubMedGoogle Scholar
  43. Valiante V, Baldin C, Hortschansky P, Jain R, Thywißen A, Straßburger M, Shelest E, Heinekamp T, Brakhage AA (2016) The Aspergillus fumigatus conidial melanin production is regulated by the bifunctional bHLH DevR and MADS-box RlmA transcription factors. Mol Microbiol 102:321–335CrossRefPubMedGoogle Scholar
  44. Vizcaíno JA, Csordas A, del-Toro N, Dianes JA, Griss J, Lavidas I, Mayer G, Perez-Riverol Y, Reisinger F, Ternent T, Xu QW, Wang R, Hermjakob H (2016) 2016 update of the PRIDE database and related tools. Nucleic Acids Res 44(D1):D447–D456CrossRefPubMedGoogle Scholar
  45. Wada R, Jin FJ, Koyama Y, Maruyama J, Kitamoto K (2014) Efficient formation of heterokaryotic sclerotia in the filamentous fungus Aspergillus oryzae. Appl Microbiol Biotechnol 98:325–334CrossRefPubMedGoogle Scholar
  46. Wang C, Lv Y, Wang B, Yin C, Lin Y, Pan L (2015) Survey of protein-DNA interactions in Aspergillus oryzae on a genomic scale. Nucleic Acids Res 43:4429–4446CrossRefPubMedPubMedCentralGoogle Scholar
  47. Xie Y, Wang G (2015) Mechanisms of fatty acid synthesis in marine fungus-like protists. Appl Microbiol Biotechnol 99:8363–8375CrossRefPubMedGoogle Scholar
  48. Yang LT, Qi YP, YB L, Guo P, Sang W, Feng H, Zhang HX, Chen LS (2013) iTRAQ protein profile analysis of Citrus sinensis roots in response to long-term boron-deficiency. J Proteome 93:179–206CrossRefGoogle Scholar
  49. Zhang F, Zhong H, Han X, Guo Z, Yang W, Liu Y, Yang K, Zhuang Z, Wang S (2015) Proteomic profile of Aspergillus flavus in response to water activity. Fungal Biol 119:114–124CrossRefPubMedGoogle Scholar
  50. Zhao G, Hou L, Yao Y, Wang C, Cao X (2012) Comparative proteome analysis of Aspergillus oryzae 3.042 and A. oryzae 100-8 strains: towards the production of different soy sauce flavors. J Proteome 75:3914–3924CrossRefGoogle Scholar
  51. Żukiewicz-Sobczak WA, Cholewa G, Sobczak P, Silny W, Nadulski R, Wojtyła-Buciora P, Zagórski J (2016) Enzymatic activity of fungi isolated from crops. Postepy Dermatol Alergol 33:457–463CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  1. 1.Co-Innovation Center for Sustainable Forestry in Southern China, College of Biology and the EnvironmentNanjing Forestry UniversityNanjingChina
  2. 2.Technology and Engineering Center for Space UtilizationChinese Academy of SciencesBeijingChina
  3. 3.Noda Institute for Scientific ResearchNoda CityJapan

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