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Development of conditional cell lysis mutants of Saccharomyces cerevisiae as production hosts by modulating OCH1 and CHS3 expression

  • Van-Trinh Luu
  • Hye Yun Moon
  • Su Jin Yoo
  • Jin Ho Choo
  • Eun Jung Thak
  • Hyun Ah KangEmail author
Applied genetics and molecular biotechnology
  • 54 Downloads

Abstract

The traditional yeast Saccharomyces cerevisiae has been widely used as a host for the production of recombinant proteins and metabolites with industrial potential. However, its thick and rigid cell wall presents problems for the effective recovery of products. In this study, we modulated the expression of ScOCH1, encoding the α-1,6-mannosyltransferase responsible for outer chain biosynthesis of N-glycans, and ScCHS3, encoding the chitin synthase III required for synthesis of the majority of cell wall chitin, by exploiting the repressible ScMET3 promoter. The conditional single mutants PMET3-OCH1 and PMET3-CHS3 and the double mutant PMET3-OCH1/PMET3-CHS3 showed comparable growth to the wild-type strain under normal conditions but exhibited increased sensitivity to temperature and cell wall-disturbing agents in the presence of methionine. Such conditional growth defects were fully recovered by supplementation with 1 M sorbitol. The osmotic lysis of the conditional mutants cultivated with methionine was sufficient to release the intracellularly expressed recombinant protein, nodavirus capsid protein, with up to 60% efficiency, compared to lysis by glass bead breakage. These mutant strains also showed approximately three-fold-enhanced secretion of a recombinant extracellular glycoprotein, Saccharomycopsis fibuligera β-glucosidase, with markedly reduced hypermannosylation, particularly in the PMET3-OCH1 mutants. Furthermore, a substantial increase of extracellular glutathione production, up to four-fold, was achieved with the conditional mutant yeast cells. Together, our data support that the conditional cell wall lysis mutants constructed based on the modulation of ScOCH1 and ScCHS3 expression would likely be useful hosts for the improved recovery of proteins and metabolites with industrial application.

Keywords

Saccharomyces cerevisiae Conditional mutant α-1,6-Mannosyltransferase Chitin synthase III MET3 promoter 

Notes

Funding

This work was supported by grants from the National Research Foundation of Korea (NRF), NRF-2013M3A6A8073554 (Global Frontier Program for the Intelligent Synthetic Biology), NRF-2017M3C1B5019295 (STEAM Research Project), and NRF-2018R1025077 (Advanced Research Center Program). Van-Trinh Luu is a recipient of CASSY fellowship from Chung-Ang University.

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_2019_9614_MOESM1_ESM.pdf (971 kb)
ESM 1 The online version of this article (10.1007/....) contains supplementary material, which is available to all users. (PDF 970 kb)

References

  1. Alvarez P, Sampedro M, Molina M, Nombela C (1994) A new system for the release of heterologous proteins from yeast based on mutant strains deficient in cell integrity. J Biotechnol 38:81–88.  https://doi.org/10.1016/0168-1656(94)90149-X CrossRefGoogle Scholar
  2. Alvarez P, Sánchez M, Molina M, Nombela C (1995) Release of virus-like particles by osmotic shock from a mutant strain of yeast deficient in cell integrity. Biotechnol Tech 9:441–444.  https://doi.org/10.1007/bf00160833 CrossRefGoogle Scholar
  3. Amberg D, Burke D, Strathern J (2005) Methods in yeast genetics. In: Cold Spring Harbor laboratory course manual, vol 230. Cold Spring Harbor Laboratory Press, Cold Spring HarborGoogle Scholar
  4. Andrews B, Huang R, Asenjo J (1995) Purification of virus like particles from yeast cells using aqueous two-phase systems. Bioseparation 5:105–112 https://www.ncbi.nlm.nih.gov/pubmed/7772946 Google Scholar
  5. Asenjo J, Ventom A, Huang R-B, Andrews B (1993) Selective release of recombinant protein particles (VLPs) from yeast using a pure lytic glucanase enzyme. Nat Biotechol 11:214–217.  https://doi.org/10.1038/nbt0293-214 CrossRefGoogle Scholar
  6. Balasundaram B, Harrison S, Bracewell DG (2009) Advances in product release strategies and impact on bioprocess design. Trends Biotechnol 27:477–485.  https://doi.org/10.1016/j.tibtech.2009.04.004 CrossRefGoogle Scholar
  7. Bellí G, Garí E, Piedrafita L, Aldea M, Herrero E (1998) An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Res 26:942–947.  https://doi.org/10.1093/nar/26.4.942 CrossRefGoogle Scholar
  8. Berghem LE, Pettersson LG (1974) The mechanism of enzymatic cellulose degradation. FEBS J 46:295–305.  https://doi.org/10.1111/j.1432-1033.1974.tb03621.x Google Scholar
  9. Bujdoso R, Williamson M, Roy D, Hunt P, Blacklaws B, Sargan D, McConnell I (1995) Molecular cloning and expression of DNA encoding ovine interleukin 2. Cytokine 7:223–231.  https://doi.org/10.1006/cyto.1995.0025 CrossRefGoogle Scholar
  10. Choi E-S, Sohn J-H, Rhee S-K (1994) Optimization of the expression system using galactose-inducible promoter for the production of anticoagulant hirudin in Saccharomyces cerevisiae. Appl Microbiol Biotechnol 42:587–594.  https://doi.org/10.1007/BF00173925 CrossRefGoogle Scholar
  11. Choi YR, Kim HJ, Lee JY, Kang HA, Kim H-J (2013) Chromatographically-purified capsid proteins of red-spotted grouper nervous necrosis virus expressed in Saccharomyces cerevisiae form virus-like particles. Protein Expr Purif 89:162–168.  https://doi.org/10.1016/j.pep.2013.03.007 CrossRefGoogle Scholar
  12. Choo JH, Hong CP, Lim JY, Seo J-A, Kim Y-S, Lee DW, Park S-G, Lee GW, Carroll E, Lee Y-W (2016) Whole-genome de novo sequencing, combined with RNA-Seq analysis, reveals unique genome and physiological features of the amylolytic yeast Saccharomycopsis fibuligera and its interspecies hybrid. Biotechnol Biofuels 9:246.  https://doi.org/10.1186/s13068-016-0653-4 CrossRefGoogle Scholar
  13. De Nobel JG, Klis FM, Priem J, Munnik T, Van Den Ende H (1990) The glucanase-soluble mannoproteins limit cell wall porosity in Saccharomyces cerevisiae. Yeast 6:491–499.  https://doi.org/10.1002/yea.320060606 CrossRefGoogle Scholar
  14. Dean N (1995) Yeast glycosylation mutants are sensitive to aminoglycosides. Proc Natl Acad Sci U S A 92:1287–1291.  https://doi.org/10.1073/pnas.92.5.1287 CrossRefGoogle Scholar
  15. Elbaz-Alon Y, Morgan B, Clancy A, Amoako TN, Zalckvar E, Dick TP, Schwappach B, Schuldiner M (2014) The yeast oligopeptide transporter Opt2 is localized to peroxisomes and affects glutathione redox homeostasis. FEMS Yeast Res 14:1055–1067.  https://doi.org/10.1111/1567-1364.12196 Google Scholar
  16. Fernández E, Toledo JR, Mansur M, Sánchez O, Gil DF, González-González Y, Lamazares E, Fernández Y, Parra F, Farnós O (2015) Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor. Appl Microbiol Biotechnol 99:3875–3886.  https://doi.org/10.1007/s00253-014-6171-z CrossRefGoogle Scholar
  17. Forman HJ, Zhang H, Rinna A (2009) Glutathione: overview of its protective roles, measurement, and biosynthesis. Mol Asp Med 30:1–12.  https://doi.org/10.1016/j.mam.2008.08.006 CrossRefGoogle Scholar
  18. Ganeva V, Galutzov B, Angelova B, Suckow M (2018) Electroinduced extraction of human ferritin heavy chain expressed in Hansenula polymorpha. Appl Biochem Biotechnol 184:1286–1307.  https://doi.org/10.1007/s12010-017-2627-9 CrossRefGoogle Scholar
  19. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96.  https://doi.org/10.1016/S0076-6879(02)50957-5 CrossRefGoogle Scholar
  20. Gmeiner C, Saadati A, Maresch D, Krasteva S, Frank M, Altmann F, Herwig C, Spadiut O (2015) Development of a fed-batch process for a recombinant Pichia pastoris Δoch1 strain expressing a plant peroxidase. Microb Cell Factories 14(1):1.  https://doi.org/10.1186/s12934-014-0183-3 CrossRefGoogle Scholar
  21. Grabek-Lejko D, Kurylenko OO, Sibirny VA, Ubiyvovk VM, Penninckx M, Sibirny AA (2011) Alcoholic fermentation by wild-type Hansenula polymorpha and Saccharomyces cerevisiae versus recombinant strains with an elevated level of intracellular glutathione. J Ind Microbiol Biotechnol 38:1853–1859.  https://doi.org/10.1007/s10295-011-0974-z CrossRefGoogle Scholar
  22. Gurgu L, Lafraya Á, Polaina J, Marín-Navarro J (2011) Fermentation of cellobiose to ethanol by industrial Saccharomyces strains carrying the β-glucosidase gene (BGL1) from Saccharomycopsis fibuligera. Bioresour Technol 102:5229–5236.  https://doi.org/10.1016/j.biortech.2011.01.062 CrossRefGoogle Scholar
  23. Kang H, Choi E-S, Hong W-K, Kim J-Y, Ko S-M, Sohn J-H, Rhee S (2000) Proteolytic stability of recombinant human serum albumin secreted in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 53:575–582.  https://doi.org/10.1007/s002530051659 CrossRefGoogle Scholar
  24. Kim H, Lee J, Kang H, Lee Y, Park EJ, Kim HJ (2014) Oral immunization with whole yeast producing viral capsid antigen provokes a stronger humoral immune response than purified viral capsid antigen. Lett Appl Microbiol 58:285–291.  https://doi.org/10.1111/lam.12188 CrossRefGoogle Scholar
  25. Kim H, Yoo SJ, Kang HA (2015) Yeast synthetic biology for the production of recombinant therapeutic proteins. FEMS Yeast Res 15:1–16.  https://doi.org/10.1111/1567-1364.12195 CrossRefGoogle Scholar
  26. Klis FM, Boorsma A, De Groot PW (2006) Cell wall construction in Saccharomyces cerevisiae. Yeast 23:185–202.  https://doi.org/10.1002/yea.1349 CrossRefGoogle Scholar
  27. Lambou K, Perkhofer S, Fontaine T, Latge JP (2010) Comparative functional analysis of the OCH1 mannosyltransferase families in Aspergillus fumigatus and Saccharomyces cerevisiae. Yeast 27:625–636.  https://doi.org/10.1002/yea.1798 CrossRefGoogle Scholar
  28. Lesage G, Bussey H (2006) Cell wall assembly in Saccharomyces cerevisiae. Microbiol Mol Biol Rev 70:317–343.  https://doi.org/10.1128/MMBR.00038-05 CrossRefGoogle Scholar
  29. Levin DE (2011) Regulation of cell wall biogenesis in Saccharomyces cerevisiae: the cell wall integrity signaling pathway. Genetics 189:1145–1175.  https://doi.org/10.1534/genetics.111.128264 CrossRefGoogle Scholar
  30. Lipke PN, Ovalle R (1998) Cell wall architecture in yeast: new structure and new challenges. J Bacteriol 180:3735–3740 https://www.ncbi.nlm.nih.gov/pubmed/9683465 Google Scholar
  31. Liu Z, Ho S-H, Sasaki K, Den Haan R, Inokuma K, Ogino C, Van Zyl WH, Hasunuma T, Kondo A (2016) Engineering of a novel cellulose-adherent cellulolytic Saccharomyces cerevisiae for cellulosic biofuel production. Sci Rep 6:24550.  https://doi.org/10.1038/srep24550 CrossRefGoogle Scholar
  32. Lu SC (2013) Glutathione synthesis. Biochim Biophys Acta Gen Subj 1830:3143–3153.  https://doi.org/10.1016/j.bbagen.2012.09.008 CrossRefGoogle Scholar
  33. Luu V-T, Moon HY, Hwang JY, Kang B-K, Kang HA (2017) Development of recombinant Yarrowia lipolytica producing virus-like particles of a fish nervous necrosis virus. J Microbiol 55:655–664.  https://doi.org/10.1007/s12275-017-7218-5 CrossRefGoogle Scholar
  34. Mao X, Hu Y, Liang C, Lu C (2002) MET3 promoter: a tightly regulated promoter and its application in construction of conditional lethal strain. Curr Microbiol 45:37–40.  https://doi.org/10.1007/s00284-001-0046-0 CrossRefGoogle Scholar
  35. Marini NJ, Meldrum E, Buehrer B, Hubberstey AV, Stone DE, Traynor Kaplan A, Reed SI (1996) A pathway in the yeast cell division cycle linking protein kinase C (Pkc1) to activation of Cdc28 at START. EMBO J 15:3040–3052 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC450245 CrossRefGoogle Scholar
  36. Nagasu T, Shimma YI, Nakanishi Y, Kuromitsu J, Iwama K, Nakayama KI, Suzuki K, Jigami Y (1992) Isolation of new temperature-sensitive mutants of Saccharomyces cerevisiae deficient in mannose outer chain elongation. Yeast 8:535–547.  https://doi.org/10.1002/yea.320080705 CrossRefGoogle Scholar
  37. Omara WA, Rash BM, Hayes A, Wickham MS, Oliver SG, Stateva LI (2010) Conditional cell-wall mutants of Saccharomyces cerevisiae as delivery vehicles for therapeutic agents in vivo to the GI tract. J Biotechnol 147:136–143.  https://doi.org/10.1016/j.jbiotec.2010.03.010 CrossRefGoogle Scholar
  38. Orlean P (2012) Architecture and biosynthesis of the Saccharomyces cerevisiae cell wall. Genetics 192:775–818.  https://doi.org/10.1534/genetics.112.144485 CrossRefGoogle Scholar
  39. Ram AF, Wolters A, Hoopen RT, Klis FM (1994) A new approach for isolating cell wall mutants in Saccharomyces cerevisiae by screening for hypersensitivity to calcofluor white. Yeast 10:1019–1030.  https://doi.org/10.1002/yea.320100804 CrossRefGoogle Scholar
  40. Rollini M, Musatti A, Manzoni M (2010) Production of glutathione in extracellular form by Saccharomyces cerevisiae. Process Biochem 45:441–445.  https://doi.org/10.1016/j.procbio.2009.10.016 CrossRefGoogle Scholar
  41. Romanos MA, Scorer CA, Clare JJ (1992) Foreign gene expression in yeast: a review. Yeast 8:423–488.  https://doi.org/10.1002/yea.320080602 CrossRefGoogle Scholar
  42. Rozkov A, Enfors S-O (1999) Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing. Biotechnol Bioeng 62:730–738.  https://doi.org/10.1002/(SICI)1097-0290(19990320)62:6%3C730::AID-BIT12%3E3.0.CO;2-Q CrossRefGoogle Scholar
  43. Shedlovskiy D, Shcherbik N, Pestov DG (2017) One-step hot formamide extraction of RNA from Saccharomyces cerevisiae. RNA Biol 14:1722–1726.  https://doi.org/10.1080/15476286.2017.1345417 CrossRefGoogle Scholar
  44. Stateva L, Oliver S, Trueman L, Venkov P (1991) Cloning and characterization of a gene which determines osmotic stability in Saccharomyces cerevisiae. Mol Cell Biol 11:4235–4243.  https://doi.org/10.1128/MCB.11.8.4235 CrossRefGoogle Scholar
  45. Stowers CC, Boczko EM (2007) Reliable cell disruption in yeast. Yeast 24:533–541.  https://doi.org/10.1002/yea.1491 CrossRefGoogle Scholar
  46. Tang H, Hou J, Shen Y, Xu L, Yang H, Fang X, Bao X (2013) High β-glucosidase secretion in Saccharomyces cerevisiae improves the efficiency of cellulase hydrolysis and ethanol production in simultaneous saccharification and fermentation. J Microbiol Biotechnol 23:1577–1585.  https://doi.org/10.4014/jmb.1305.05011 CrossRefGoogle Scholar
  47. Tang H, Wang S, Wang J, Song M, Xu M, Zhang M, Shen Y, Hou J, Bao X (2016) N-hypermannose glycosylation disruption enhances recombinant protein production by regulating secretory pathway and cell wall integrity in Saccharomyces cerevisiae. Sci Rep 6.  https://doi.org/10.1038/srep25654
  48. Thiéry R, Cozien J, Cabon J, Lamour F, Baud M, Schneemann A (2006) Induction of a protective immune response against viral nervous necrosis in the European sea bass Dicentrarchus labrax by using betanodavirus virus-like particles. J Virol 80:10201–10207.  https://doi.org/10.1128/JVI.01098-06 CrossRefGoogle Scholar
  49. Toledano MB, Delaunay-Moisan A, Outten CE, Igbaria A (2013) Functions and cellular compartmentation of the thioredoxin and glutathione pathways in yeast. Antioxid Redox Signal 18:1699–1711.  https://doi.org/10.1089/ars.2012.5033 CrossRefGoogle Scholar
  50. Vallejo-Illarramendi A, Marciano DK, Reichardt LF (2013) A novel method that improves sensitivity of protein detection in PAGE and Western blot. Electrophoresis 34:1148–1150.  https://doi.org/10.1002/elps.201200534 CrossRefGoogle Scholar
  51. Ye J, Ly J, Watts K, Hsu A, Walker A, McLaughlin K, Berdichevsky M, Prinz B, Sean Kersey D, d'Anjou M (2011) Optimization of a glycoengineered Pichia pastoris cultivation process for commercial antibody production. Biotechnol Prog 27:1744–1750.  https://doi.org/10.1002/btpr.695 CrossRefGoogle Scholar
  52. Yoo SJ, Chung SY, Lee D-j, Kim H, Cheon SA, Kang HA (2015) Use of the cysteine-repressible HpMET3 promoter as a novel tool to regulate gene expression in Hansenula polymorpha. Biotechnol Lett 37:2237–2245.  https://doi.org/10.1007/s10529-015-1902-5 CrossRefGoogle Scholar
  53. Yurkiv M, Kurylenko O, Vasylyshyn R, Dmytruk K, Fickers P, Sibirny A (2018) Gene of the transcriptional activator MET4 is involved in regulation of glutathione biosynthesis in the methylotrophic yeast Ogataea (Hansenula) polymorpha. FEMS Yeast Res 18:foy004.  https://doi.org/10.1093/femsyr/foy004 CrossRefGoogle Scholar
  54. Zhang N, Gardner DC, Oliver SG, Stateva LI (1999a) Down-regulation of the expression of PKC1 and SRB1/PSA1/VIG9, two genes involved in cell wall integrity in Saccharomyces cerevisiae, causes flocculation. Microbiology 145:309–316.  https://doi.org/10.1099/13500872-145-2-309 CrossRefGoogle Scholar
  55. Zhang N, Gardner DC, Oliver SG, Stateva LI (1999b) Genetically controlled cell lysis in the yeast Saccharomyces cerevisiae. Biotechnol Bioeng 64:607–615.  https://doi.org/10.1002/(SICI)1097-0290(19990905)64:5%3C607::AID-BIT11%3E3.0.CO;2-0 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Van-Trinh Luu
    • 1
  • Hye Yun Moon
    • 1
  • Su Jin Yoo
    • 1
  • Jin Ho Choo
    • 1
  • Eun Jung Thak
    • 1
  • Hyun Ah Kang
    • 1
    Email author
  1. 1.Department of Life Science, College of Natural ScienceChung-Ang UniversitySeoulSouth Korea

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