Skip to main content
SpringerLink
Log in

Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

With the increasing demand for biopharmaceutical proteins and industrial enzymes, it is necessary to optimize the production by microbial fermentation or cell cultures. Yeasts are well established for the production of a wide range of recombinant proteins, but there are also some limitations; e.g., metabolic and cellular stresses have a strong impact on recombinant protein production. In this work, we investigated the effect of the specific growth rate on the production of two different recombinant proteins. Our results show that human insulin precursor is produced in a growth-associated manner, whereas α-amylase tends to have a higher yield on substrate at low specific growth rates. Based on transcriptional analysis, we found that the difference in the production of the two proteins as function of the specific growth rate is mainly due to differences in endoplasmic reticulum processing, protein turnover, cell cycle, and global stress response. We also found that there is a shift at a specific growth rate of 0.1 h−1 that influences protein production. Thus, for lower specific growth rates, the α-amylase and insulin precursor-producing strains present similar cell responses and phenotypes, whereas for higher specific growth rates, the two strains respond differently to changes in the specific growth rate.

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

Similar content being viewed by others

References

  • Andersen DC, Krummen L (2002) Recombinant protein expression for therapeutic applications. Curr Opin Biotechnol 13(2):117–123

    Article  PubMed  CAS  Google Scholar 

  • Arvas M, Pakula T, Smit B, Rautio J, Koivistoinen H, Jouhten P, Lindfors E, Wiebe M, Penttilä M, Saloheimo M (2011) Correlation of gene expression and protein production rate—a system wide study. BMC Genomics 12(1):616

    Article  PubMed  CAS  Google Scholar 

  • Bernales S, Papa FR, Walter P (2006) Intracellular signaling by the unfolded protein response. Annu Rev Cell Dev Biol 22:487

    Article  PubMed  CAS  Google Scholar 

  • Buchetics M, Dragosits M, Maurer M, Rebnegger C, Porro D, Sauer M, Gasser B, Mattanovich D (2011) Reverse engineering of protein secretion by uncoupling of cell cycle phases from growth. Biotechnol Bioeng 108(10):2403–2412

    Article  CAS  Google Scholar 

  • Castrillo JI, Zeef LA, Hoyle DC, Zhang N, Hayes A, Gardner DCJ, Cornell MJ, Petty J, Hakes L, Wardleworth L (2007) Growth control of the eukaryote cell: a systems biology study in yeast. J Biol 6(2):4

    Article  PubMed  Google Scholar 

  • 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):323–337

    Article  PubMed  CAS  Google Scholar 

  • Dürrschmid K, Reischer H, Schmidt-Heck W, Hrebicek T, Guthke R, Rizzi A, Bayer K (2008) Monitoring of transcriptome and proteome profiles to investigate the cellular response of E. coli towards recombinant protein expression under defined chemostat conditions. J Biotechnol 135(1):34–44

    Article  PubMed  Google Scholar 

  • Fazio A, Jewett M, Daran-Lapujade P, Mustacchi R, Usaite R, Pronk J, Workman C, Nielsen J (2008) Transcription factor control of growth rate dependent genes in Saccharomyces cerevisiae: a three factor design. BMC Genomics 9(1):341

    Article  PubMed  Google Scholar 

  • Ferreira B, Calado C, Van Keulen F, Fonseca L, Cabral J, da Fonseca M (2003) Towards a cost effective strategy for cutinase production by a recombinant Saccharomyces cerevisiae: strain physiological aspects. Appl Microbiol Biotechnol 61(1):69–76

    PubMed  CAS  Google Scholar 

  • Freigassner M, Pichler H, Glieder A (2009) Tuning microbial hosts for membrane protein production. Microb Cell Fact 8(1):69

    Article  PubMed  Google Scholar 

  • Fu J, Wilson DB, Shuler ML (1993) Continuous, high level production and excretion of a plasmid-encoded protein by Escherichia coli in a two-stage chemostat. Biotechnol Bioeng 41(10):937–946

    Article  PubMed  CAS  Google Scholar 

  • Gill R, DeLisa M, Valdes J, Bentley W (2001) Genomic analysis of high-cell-density recombinant Escherichia coli fermentation and “cell conditioning” for improved recombinant protein yield. Biotechnol Bioeng 72(1):85–95

    Article  PubMed  CAS  Google Scholar 

  • Hackel BJ, Huang D, Bubolz JC, Wang XX, Shusta EV (2006) Production of soluble and active transferrin receptor-targeting single-chain antibody using Saccharomyces cerevisiae. Pharm Res 23(4):790–797

    Article  PubMed  CAS  Google Scholar 

  • Hardjito L, Greenfield PF, Lee PL (1993) Recombinant protein production via fed-batch culture of the yeast Saccharomyces cerevisiae. Enzyme Microb Technol 15(2):120–126

    Article  PubMed  CAS  Google Scholar 

  • Hatahet F, Ruddock LW (2009) Modulating proteostasis: peptidomimetic inhibitors and activators of protein folding. Curr Pharm Des 15(21):2488–2507

    Article  PubMed  CAS  Google Scholar 

  • Haynes CM, Titus EA, Cooper AA (2004) Degradation of misfolded proteins prevents ER-derived oxidative stress and cell death. Mol Cell 15(5):767–776

    Article  PubMed  CAS  Google Scholar 

  • Hoffmann F, Rinas U (2000) Kinetics of heat-shock response and inclusion body formation during temperature-induced production of basic fibroblast growth factor in high-cell-density cultures of recombinant Escherichia coli. Biotechnol Prog 16(6):1000–1007

    Article  PubMed  CAS  Google Scholar 

  • Hou J, Tyo K, Liu Z, Petranovic D, Nielsen J (2012a) Engineering of vesicle trafficking improves heterologous protein secretion in Saccharomyces cerevisiae. Metab Eng 14(2):120–127

    Article  PubMed  CAS  Google Scholar 

  • Hou J, Tyo K, Liu Z, Petranovic D, Nielsen J (2012b) Metabolic engineering of recombinant protein production by Saccharomyces cerevisiae. FEMS Yeast Res 12(5):491–510

    Article  PubMed  CAS  Google Scholar 

  • Idiris A, Tohda H, Kumagai H, Takegawa K (2010) Engineering of protein secretion in yeast: strategies and impact on protein production. Appl Microbiol Biotechnol 86(2):403–417

    Article  PubMed  CAS  Google Scholar 

  • Kim IK, Roldão A, Siewers V, Nielsen J (2012) A systems-level approach for metabolic engineering of yeast cell factories. FEMS Yeast Res 12(2):228–248

    Article  PubMed  CAS  Google Scholar 

  • Langer ES (2012) Biomanufacturing outsourcing outlook. BioPharm International 25(2):15–16

    Google Scholar 

  • Liu Z, Tyo K, Martínez J, Petranovic D, Nielsen J (2012) Different expression systems for production of recombinant proteins in Saccharomyces cerevisiae. Biotechnol Bioeng 109(5):1259–1268

    Article  PubMed  CAS  Google Scholar 

  • Lunter G, Goodson M (2011) Stampy: A statistical algorithm for sensitive and fast mapping of Illumina sequence reads. Genome Res 21(6):936–939

    Article  PubMed  CAS  Google Scholar 

  • Moye-Rowley WS (2002) Transcription factors regulating the response to oxidative stress in yeast. Antioxid Redox Sign 4(1):123–140

    Article  CAS  Google Scholar 

  • Nancib N, Boudrant J (1992) Effect of growth rate on stability and gene expression of a recombinant plasmid during continuous culture of Escherichia coli in a non-selective medium. Biotechnol Lett 14(8):643–648

    Article  CAS  Google Scholar 

  • Nurse P (2003) Systems biology: understanding cells. Nature 424(6951):883–883

    Article  PubMed  CAS  Google Scholar 

  • Oliveira AP, Patil KR, Nielsen J (2008) Architecture of transcriptional regulatory circuits is knitted over the topology of bio-molecular interaction networks. BMC Syst Biol 2(1):17

    Article  PubMed  Google Scholar 

  • Oliveira C, Teixeira JA, Lima N, Da Silva NA, Domingues L (2007) Development of stable flocculent Saccharomyces cerevisiae strain for continuous Aspergillus niger beta-galactosidase production. J Biosci Bioeng 103(4):318–324. doi:10.1263/jbb.103.318

    Article  PubMed  CAS  Google Scholar 

  • Patil KR, Nielsen J (2005) Uncovering transcriptional regulation of metabolism by using metabolic network topology. Proc Nati Acad Sci USA 102(8):2685–2689

    Article  CAS  Google Scholar 

  • Pincus D, Chevalier MW, Aragón T, Van Anken E, Vidal SE, El-Samad H, Walter P (2010) BiP binding to the ER-stress sensor Ire1 tunes the homeostatic behavior of the unfolded protein response. PLoS Biol 8(7):e1000415. doi:10.1371/journal.pbio.1000415

    Article  PubMed  Google Scholar 

  • Regenberg B, Grotkjær T, Winther O, Fausbøll A, Åkesson M, Bro C, Hansen LK, Brunak S, Nielsen J (2006) Growth-rate regulated genes have profound impact on interpretation of transcriptome profiling in Saccharomyces cerevisiae. Genome Biol 7(11):R107

    Article  PubMed  Google Scholar 

  • Schröder M (2008) Engineering eukaryotic protein factories. Biotechnol Lett 30(2):187–196

    Article  PubMed  Google Scholar 

  • Seresht AK, Palmqvist EA, Olsson L (2011) The impact of phosphate scarcity on pharmaceutical protein production in S. cerevisiae: linking transcriptomic insights to phenotypic responses. Microb Cell Fact 10(1):104

    Article  CAS  Google Scholar 

  • Thomas JG, Baneyx F (2000) ClpB and HtpG facilitate de novo protein folding in stressed Escherichia coli cells. Mol Microbiol 36(6):1360–1370

    Article  PubMed  CAS  Google Scholar 

  • Tyo K, Liu Z, Petranovic D, Nielsen J (2012) Imbalance of heterologous protein folding and disulfide bond formation rates yields runaway oxidative stress. BMC Biol 10:16

    Article  PubMed  CAS  Google Scholar 

  • Verripsab T, Duboc P, Visser C, Sagt C (2000) From gene to product in yeast: production of fungal cutinase. Enzyme Microb Technol 26(9–10):812–818

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Intawat Nookaew and Ph.D. student Leif Väremo from Chalmers University of Technology for kindly providing the PIANO software for microarray analysis. This work is financially supported by the European Research Council ERC project INSYSBIO (grant no. 247013), the Novo Nordisk Foundation, the Chalmers Foundation, and the Knut and Alice Wallenberg Foundation.

Author information

Authors and Affiliations

  1. Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, 41296, Göteborg, Sweden

    Zihe Liu, Jin Hou, José L. Martínez, Dina Petranovic & Jens Nielsen

  2. Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Fremtidsvej 3, 2970, Hørsholm, Denmark

    Jens Nielsen

  3. Metabolic Engineering Research Laboratory, Institute of Chemical and Engineering Sciences (ICES), Agency for Science, Technology and Research (A*STAR), 31 Biopolis Way #01-01 Nanos, Singapore, 138669, Singapore

    Zihe Liu

  4. State Key Laboratory of Microbial Technology, Shandong University, 27 Shanda Nan Road, Jinan, Shandong, 250100, China

    Jin Hou

Authors
  1. Zihe Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jin Hou
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. José L. Martínez
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Dina Petranovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jens Nielsen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jens Nielsen.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM 1

(PDF 104 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Liu, Z., Hou, J., Martínez, J.L. et al. Correlation of cell growth and heterologous protein production by Saccharomyces cerevisiae . Appl Microbiol Biotechnol 97, 8955–8962 (2013). https://doi.org/10.1007/s00253-013-4715-2

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-4715-2

Keywords

Search

Navigation