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Novel osmotic stress control strategy for improved pneumocandin B0 production in Glarea lozoyensis combined with a mechanistic analysis at the transcriptome level

  • Ping Song
  • Baoqi Huang
  • Sen Zhang
  • Ke Zhang
  • Kai Yuan
  • Xiaojun Ji
  • Lujing Ren
  • Jianping Wen
  • He Huang
Applied microbial and cell physiology
  • 6 Downloads

Abstract

Pneumocandin B0, the precursor of the antifungal drug caspofungin, is a secondary metabolite of the fungus Glarea lozoyensis. In this study, we investigated the effects of mannitol as the sole carbon source on pneumocandin B0 production by G. lozoyensis. The osmotic pressure is more important in enhancing pneumocandin B0 production than is the substrate concentration. Based on the kinetic analysis, an osmotic stress control fed-batch strategy was developed. This strategy led to a maximum pneumocandin B0 concentration of 2711 mg/L with a productivity of 9.05 mg/L/h, representing 34.67 and 6.47% improvements, respectively, over the best result achieved by the one-stage fermentation. Furthermore, G. lozoyensis accumulated glutamate and proline as compatible solutes to resist osmotic stress, and these amino acids also provided the precursors for the enhanced pneumocandin B0 production. Osmotic stress also activated ROS (reactive oxygen species)-dependent signal transduction by upregulating the levels of related genes and increasing intracellular ROS levels by 20%. We also provided a possible mechanism for pneumocandin B0 accumulation based on signal transduction. These findings will improve our understanding of the regulatory mechanisms of pneumocandin B0 biosynthesis and may be applied to improve secondary metabolite production.

Keywords

Osmotic stress Fed-batch Secondary metabolite Fungi Transcriptome analysis 

Notes

Funding

This work was supported by the Natural Science Fund for Colleges and Universities in Jiangsu Province (No. 17KJB530006), the Natural Science Foundation of Jiangsu Province (BK20161048), the National Science Foundation of China (No. 21776136), the Program for Innovative Research Teams in Universities of Jiangsu Province (2015), the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (No. PPZY2015B155), and the Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture (No. XTE1854).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

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

Supplementary material

253_2018_9440_MOESM1_ESM.pdf (686 kb)
ESM 1 (PDF 686newnbsp;kb)

References

  1. Balkovec JM, Hughes DL, Masurekar PS, Sable CA, Schwartz RE, Singh SB (2014) Discovery and development of first in class antifungal caspofungin (CANCIDAS)-a case study. Nat Prod Rep 31(1):15–34CrossRefGoogle Scholar
  2. Beck T, Hall MN (1999) The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors. Nature 402(6762):689–692CrossRefGoogle Scholar
  3. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefGoogle Scholar
  4. Causton HC, Ren B, Koh SS, Harbison CT, Kanin E, Jennings EG, Lee TI, True HL, Lander ES, Young RA (2001) Remodeling of yeast genome expression in response to environmental changes. Mol Biol Cell 12(2):323–337CrossRefGoogle Scholar
  5. Chen Z, Ricigliano JW, Klessig DF (1993) Purification and characterization of a soluble salicylic acid-binding protein from tobacco. Proc Natl Acad Sci USA 90(20):9533–9537CrossRefGoogle Scholar
  6. Chen L, Yue Q, Zhang X, Xiang M, Wang C, Li S, Che Y, Ortiz-Lopez FJ, Bills GF, Liu X, An Z (2013) Genomics-driven discovery of the pneumocandin biosynthetic gene cluster in the fungus Glarea lozoyensis. BMC Genomics 14:339CrossRefGoogle Scholar
  7. Chen L, Yue Q, Li Y, Niu X, Xiang M, Wang W, Bills GF, Liu X, An Z (2015) Engineering of Glarea lozoyensis for exclusive production of the pneumocandin B0 precursor of the antifungal drug caspofungin acetate. Appl Environ Microbiol 81(5):1550–1558CrossRefGoogle Scholar
  8. Chen L, Li Y, Yue Q, Loksztejn A, Yokoyama K, Felix EA, Liu XZ, Zhang NY, An ZG, Bills GF (2016) Engineering of new pneumocandin side-chain analogues from Glarea lozoyensis by mutasynthesis and evaluation of their antifungal activity. ACS Chem Biol 11(10):2724–2733CrossRefGoogle Scholar
  9. Connors N, Petersen L, Hughes R, Saini K, Olewinski R, Salmon P (2000) Residual fructose and osmolality affect the levels of pneumocandins B0 and C0 produced by Glarea lozoyensis. Appl Microbiol Biotechnol 54(6):814–818CrossRefGoogle Scholar
  10. Culham DE, Vernikovska Y, Tschowri N, Keates RA, Wood JM, Boggs JM (2008) Periplasmic loops of osmosensory transporter ProP in Escherichia coli are sensitive to osmolality. Biochemistry 47(51):13584–13593CrossRefGoogle Scholar
  11. Denning DW (2003) Echinocandin antifungal drugs. Lancet 362(9390):1142–1151CrossRefGoogle Scholar
  12. Gasch AP, Spellman PT, Kao CM, Carmelharel O, Eisen MB, Storz G, Botstein D, Brown PO (2000) Genomic expression programs in the response of yeast cells to environmental changes. Mol Biol Cell 11(12):4241–4257CrossRefGoogle Scholar
  13. Görner W, Durchschlag E, Martinezpastor MT, Estruch F, Ammerer G, Hamilton B, Ruis H, Schüller C (1998) Nuclear localization of the C2H2 zinc finger protein Msn2p is regulated by stress and protein kinase A activity. Genes Dev 12(4):586–597CrossRefGoogle Scholar
  14. Grintzalis K, Vernardis SI, Klapa MI, Georgiou CD (2014) Role of oxidative stress in Sclerotial differentiation and aflatoxin B1 biosynthesis in Aspergillus flavus. Appl Environ Microbiol 80(18):5561–5571CrossRefGoogle Scholar
  15. Guedes SF, Leitao AL (2012) Effect of phenolic compounds and osmotic stress on the expression of penicillin biosynthetic genes from Penicillium chrysogenum var. halophenolicum strain. J Xenobiotics 2(2):7–12Google Scholar
  16. Hernandez-Onate MA, Herrera-Estrella A (2015) Damage response involves mechanisms conserved across plants, animals and fungi. Curr Genet 61(3):359–372CrossRefGoogle Scholar
  17. Houwaart S, Youssar L, Huttel W (2014) Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase. Chembiochem 15(16):2365–2369CrossRefGoogle Scholar
  18. Hu LB, Zhou W, Yang JD, Chen J, Yin YF, Shi ZQ (2011) Cinnamaldehyde induces PCD-like death of Microcystis aeruginosa via reactive oxygen species. Water Air Soil Pollut 217(1–4):105–113CrossRefGoogle Scholar
  19. Hu X, Ma X, Tang P, Yuan Q (2013) Improved β-carotene production by oxidative stress in Blakeslea trispora induced by liquid paraffin. Biotechnol Lett 35(4):559–563CrossRefGoogle Scholar
  20. Hu XC, Ren LJ, Chen SL, Zhang L, Ji XJ, Huang H (2015) The roles of different salts and a novel osmotic pressure control strategy for improvement of DHA production by Schizochytrium sp. Bioprocess Biosyst Eng 38(11):2129–2136CrossRefGoogle Scholar
  21. Jia Y, Zhong JJ (2011) Enhanced production of ansamitocin P-3 by addition of Mg2+ in fermentation of Actinosynnema pretiosum. Bioresour Technol 102(21):10147–10150CrossRefGoogle Scholar
  22. Kenne GJ, Gummadidala PM, Omebeyinje MH (2018) Activation of aflatoxin biosynthesis alleviates Total ROS in Aspergillus parasiticus. Toxins 10(2):57CrossRefGoogle Scholar
  23. Kogej T, Stein M, Volkmann M, Gorbushina AA, Galinski EA, Gundecimerman N (2007) Osmotic adaptation of the halophilic fungus Hortaea werneckii: role of osmolytes and melanization. Microbiology 153(12):4261–4273CrossRefGoogle Scholar
  24. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with bowtie 2. Nat Methods 9(4):357–359CrossRefGoogle Scholar
  25. Li B (2011) RSEM: accurate transcript quantification from RNA-Seq data with or without a reference genome. Bmc Bioinformatics 12(1):323–323CrossRefGoogle Scholar
  26. Li Y, Chen L, Yue Q, Liu XZ, An ZQ, Bills GF (2015) Genetic manipulation of the pneumocandin biosynthetic pathway for generation of analogues and evaluation of their antifungal activity. ACS Chem Biol 10(7):1702–1710CrossRefGoogle Scholar
  27. Liu HJ, Liu DH, Zhong JJ (2006) Quantitative response of trehalose and glycerol syntheses by Candida krusei to osmotic stress of the medium. Process Biochem 41(2):473–476CrossRefGoogle Scholar
  28. Macarisin D, Droby S, Bauchan G, Wisniewski M (2010) Superoxide anion and hydrogen peroxide in the yeast antagonist–fruit interaction: a new role for reactive oxygen species in postharvest biocontrol? Postharvest Biol Technol 58(3):194–202CrossRefGoogle Scholar
  29. Mignolet-Spruyt L, Xu E, Idänheimo N, Hoeberichts FA, Mühlenbock P, Brosché M, Van Breusegem F, Kangasjärvi J (2016) Spreading the news: subcellular and organellar reactive oxygen species production and signalling. J Exp Bot 67(13):3831–3844CrossRefGoogle Scholar
  30. Montibus M, Ducos C, Bonninverdal MN, Bormann J, Ponts N, Richardforget F, Barreau C (2013) The bZIP transcription factor Fgap1 mediates oxidative stress response and trichothecene biosynthesis but not virulence in Fusarium graminearum. PLoS One 8(12):83377CrossRefGoogle Scholar
  31. Nanou K, Roukas T, Papadakis E (2011) Oxidative stress and morphological changes in Blakeslea trispora induced by enhanced aeration during carotene production in a bubble column reactor. Biochem Eng J 54(3):172–177CrossRefGoogle Scholar
  32. Narasaiah KV, Sashidhar RB, Subramanyam C (2006) Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates. Mycopathologia 162(3):179–189CrossRefGoogle Scholar
  33. Nguyen AN, Lee A, Place W, Shiozaki K (2000) Multistep phosphorelay proteins transmit oxidative stress signals to the fission yeast stress-activated protein kinase. Mol Biol Cell 11(4):1169–1181CrossRefGoogle Scholar
  34. Nicholls S, Straffon M, Enjalbert B, Nantel A, Macaskill S, Whiteway M, Brown AJ (2004) Msn2- and Msn4-like transcription factors play no obvious roles in the stress responses of the fungal pathogen Candida albicans. Eukaryot Cell 3(5):1111–1123CrossRefGoogle Scholar
  35. Papoudou-Bai A, Hatzimichael E, Barbouti A, Kanavaros P (2017) Expression patterns of the activator protein-1 (AP-1) family members in lymphoid neoplasms. Clin Exper Med 17(3):291–304CrossRefGoogle Scholar
  36. Petersen J, Förster K, Turina P, Gräber P (2012) Comparison of the H+/ATP ratios of the H+-ATP synthases from yeast and from chloroplast. Proc Natl Acad Sci USA 109(28):11150–11155CrossRefGoogle Scholar
  37. Qin TT, Song P, Wang XT, Ji XJ, Ren LJ, Huang H (2016) Protoplast mutant selection of Glarea lozoyensis and statistical optimization of medium for pneumocandin B0 yield-up. Biosci Biotechnol Biochem 80(11):2241–2246CrossRefGoogle Scholar
  38. Reczek CR, Chandel NS (2015) ROS-dependent signal transduction. Curr Opin Cell Biol 33:8–13CrossRefGoogle Scholar
  39. Ren LJ, Hu XC, Zhao XY, Chen SL, Wu Y, Li D, Yu YD, Geng LJ, Ji XJ, Huang H (2017) Transcriptomic analysis of the regulation of lipid fraction migration and fatty acid biosynthesis in Schizochytrium sp. Sci Rep 7:3562CrossRefGoogle Scholar
  40. Reverberi M, Zjalic S, Ricelli A, Fabbri AA, Fanelli C (2006) Oxidant/antioxidant balance in Aspergillus parasiticus affects aflatoxin biosynthesis. Mycotoxin Res 22(1):39–47CrossRefGoogle Scholar
  41. Reverberi M, Ricelli A, Zjalic S, Fabbri AA, Fanelli C (2010) Natural functions of mycotoxins and control of their biosynthesis in fungi. Appl Microbiol Biotechnol 87(3):899–911CrossRefGoogle Scholar
  42. Reverberi M, Punelli M, Smith CA, Zjalic S, Scarpari M, Scala V, Cardinali G, Aspite N, Pinzari F, Payne GA (2012) How peroxisomes affect aflatoxin biosynthesis in Aspergillus frlavus. PLoS One 7(10):48097CrossRefGoogle Scholar
  43. Roetzer A, Gregori C, Jennings AM, Quintin J, Ferrandon D, Butler G, Kuchler K, Ammerer G, Schüller C (2008) Candida glabrata environmental stress response involves Saccharomyces cerevisiae Msn2/4 orthologous transcription factors. Mol Microbiol 69(3):603–620CrossRefGoogle Scholar
  44. Sheih IC, Wu TK, Fang TJ (2009) Antioxidant properties of a new antioxidative peptide from algae protein waste hydrolysate in different oxidation systems. Bioresour Technol 100(13):3419–3425CrossRefGoogle Scholar
  45. Shi K, Gao Z, Shi TQ, Song P, Ren LJ, Huang H, Ji XJ (2017) Reactive oxygen species-mediated cellular stress response and lipid accumulation in oleaginous microorganisms: the state of the art and future perspectives. Front Microbiol 8:793CrossRefGoogle Scholar
  46. Song P, Yuan K, Qin T, Zhang K, Ji XJ, Ren L, Guan R, Wen J, Huang H (2018) Metabolomics profiling reveals the mechanism of increased pneumocandin B0 production by comparing mutant and parent strains. J Ind Microbiol Biotechnol 45(9):767–780CrossRefGoogle Scholar
  47. Toledano MB, Delaunay A, Monceau L, Tacnet F (2004) Microbial H2O2 sensors as archetypical redox signaling modules. Trends Biochem Sci 29(7):351–357CrossRefGoogle Scholar
  48. Vreeland RH, Mierau BD, Litchfield CD, Martin EL (2011) Relationship of the internal solute composition to the salt tolerance. Can J Microbiol 29(29):407–414Google Scholar
  49. Wang Y, Lu ZY, Sun KL, Zhu WM (2011) Effects of high salt stress on secondary metabolite production in the marine-derived fungus Spicaria elegans. Mar Drugs 9(4):535–542CrossRefGoogle Scholar
  50. Wang R, Diao PP, Chen Q, Wu H, Xu N, Duan SS (2017) Identification of novel pathways for biodegradation of bisphenol A by the green alga Desmodesmus sp.WR1, combined with mechanistic analysis at the transcriptome level. Chem Eng J 321:424–431CrossRefGoogle Scholar
  51. Wucherpfennig T, Hestler T, Krull R (2011) Morphology engineering - osmolality and its effect on Aspergillus niger morphology and productivity. Microb Cell Factories 10(1):1CrossRefGoogle Scholar
  52. Xia ML, Huang D, Li SS, Wen JP, Jia XQ, Chen YL (2013) Enhanced FK506 production in Streptomyces tsukubaensis by rational feeding strategies based on comparative metabolic profiling analysis. Biotechnol Bioeng 110(10):2717–2730CrossRefGoogle Scholar
  53. Xu YN, Xia XX, Zhong JJ (2013) Induced effect of Na(+) on ganoderic acid biosynthesis in static liquid culture of Ganoderma lucidum via calcineurin signal transduction. Biotechnol Bioeng 110(7):1913–1923CrossRefGoogle Scholar
  54. Yang LB, Zhan XB, Zheng ZY, Wu JR, Gao MJ, Lin CC (2014) A novel osmotic pressure control fed-batch fermentation strategy for improvement of erythritol production by Yarrowia lipolytica from glycerol. Bioresour Technol 151:120–127CrossRefGoogle Scholar
  55. Yin WB, Reinke AW, Szilágyi M, Emri T, Chiang YM, Keating AE, Pócsi I, Wang CCC, Keller NP (2013) bZIP transcription factors affecting secondary metabolism, sexual development and stress responses in Aspergillus nidulans. Microbiology 159:77–88CrossRefGoogle Scholar
  56. Yoshimi A, Kojima K, Takano Y, Tanaka C (2005) Group III histidine kinase is a positive regulator of Hog1-type mitogen-activated protein kinase in filamentous fungi. Eukaryot Cell 4(11):1820–1828.  https://doi.org/10.1128/Ec.4.11.1820-1828.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhike Z, Wolfram L, Edda K (2010) A quantitative study of the Hog1 MAPK response to fluctuating osmotic stress in Saccharomyces cerevisiae. PLoS One 5(3):9522CrossRefGoogle Scholar
  58. Zhou X, Ferraris JD, Burg MB (2006) Mitochondrial reactive oxygen species contribute to high NaCl-induced activation of the transcription factor TonEBP/OREBP. Am J Physiol Renal Physiol 290(5):F1169–F1176CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.SynBio Research Platform, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), School of Chemical Engineering and TechnologyTianjin UniversityTianjinChina
  2. 2.Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture, College of Biotechnology and Pharmaceutical EngineeringNanjing Tech UniversityNanjingChina
  3. 3.Jiangsu Collaboration Innovation Center of Chinese Medical Resources Industrialization, College of PharmacyNanjing University of Chinese MedicineNanjingChina
  4. 4.School of Pharmaceutical SciencesNanjing Tech UniversityNanjingChina

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