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Development of a dual temperature control system for isoprene biosynthesis in Saccharomyces cerevisiae

Abstract

Conflict between cell growth and product accumulation is frequently encountered in the biosynthesis of secondary metabolites. To address the growth-production conflict in yeast strains harboring the isoprene synthetic pathway in the mitochondria, the dynamic control of isoprene biosynthesis was explored. A dual temperature regulation system was developed through engineering and expression regulation of the transcriptional activator Gal4p. A cold-sensitive mutant, Gal4ep19, was created by directed evolution of Gal4p based on an internally developed growth-based high-throughput screening method and expressed under the heat-shock promoter PSSA4 to control the expression of PGAL-driven pathway genes in the mitochondria. Compared to the control strain with constitutively expressed wild-type Gal4p, the dual temperature regulation strategy led to 34.5% and 72% improvements in cell growth and isoprene production, respectively. This study reports the creation of the first cold-sensitive variants of Gal4p by directed evolution and provides a dual temperature control system for yeast engineering that may also be conducive to the biosynthesis of other high-value natural products.

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References

  1. 1.

    Vickers C E, Suriana S. Isoprene. Advances in Biochemical Engineering/Biotechnology, 2015, 148(9): 289–317

    CAS  Article  Google Scholar 

  2. 2.

    Lv X M, Wang F, Zhou P P, Ye L D, Xie W P, Xu H M, Yu H W. Dual regulation of cytoplasmic and mitochondrial acetyl-CoA utilization for improved isoprene production in Saccharomyces cerevisiae. Nature Communications, 2016, 7(1): 12851

    CAS  Article  Google Scholar 

  3. 3.

    Yao Z, Zhou P P, Su B M, Su S S, Ye L D, Yu H W. Enhanced isoprene production by reconstruction of metabolic balance between strengthened precursor supply and improved isoprene synthase in Saccharomyces cerevisiae. ACS Synthetic Biology, 2018, 7(9): 2308–2316

    CAS  Article  Google Scholar 

  4. 4.

    Wang F, Lv X M, Xie W P, Zhou P P, Zhu Y Q, Yao Z, Yang C C, Yang X H, Ye L D, Yu H W. Combining Gal4p-mediated expression enhancement and directed evolution of isoprene synthase to improve isoprene production in Saccharomyces cerevisiae. Metabolic Engineering, 2017, 39: 257–266

    Article  Google Scholar 

  5. 5.

    Cao X, Yang S, Cao C, Zhou Y J. Harnessing sub-organelle metabolism for biosynthesis of isoprenoids in yeast. Synthetic and Systems Biotechnology, 2020, 5(3): 179–186

    Article  Google Scholar 

  6. 6.

    Traven A, Jelicic B, Sopta M. Yeast Gal4: a transcriptional paradigm revisited. EMBO Reports, 2006, 7(5): 496–499

    CAS  Article  Google Scholar 

  7. 7.

    Da Silva N A, Srikrishnan S. Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae. FEMS Yeast Research, 2012, 12(2): 197–214

    CAS  Article  Google Scholar 

  8. 8.

    Xie W P, Liu M, Lv X M, Lu W Q, Gu J L, Yu H W. Construction of a controllable β-carotene biosynthetic pathway by decentralized assembly strategy in Saccharomyces cerevisiae. Biotechnology and Bioengineering, 2014, 111(1): 125–133

    CAS  Article  Google Scholar 

  9. 9.

    Tan G H, Chen M, Foote C, Tan C. Temperature-sensitive mutations made easy: generating conditional mutations by using temperature-sensitive inteins that function within different temperature ranges. Genetics, 2009, 183(1): 13–22

    CAS  Article  Google Scholar 

  10. 10.

    Zeidler M P, Tan C, Bellaiche Y, Cherry S, Häder S, Gayko U, Perrimon N. Temperature-sensitive control of protein activity by conditionally splicing inteins. Nature Biotechnology, 2004, 22(7): 871–876

    CAS  Article  Google Scholar 

  11. 11.

    Chakshusmathi G, Mondal K, Lakshmi G S, Singh G, Roy A, Babu Ch R, Madhusudhanan S, Varadarajan R. Design of temperature-sensitive mutants solely from amino acid sequence. Proceedings of the National Academy of Sciences of the United States of America, 2004, 101(21): 7925–7930

    CAS  Article  Google Scholar 

  12. 12.

    Mondal K, Dastidar A G, Singh G, Madhusudhanan S, Gande S L, VijayRaghavan K, Varadarajan R. Design and isolation of temperature-sensitive mutants of Gal4 in yeast and Drosophila. Journal of Molecular Biology, 2007, 370(5): 939–950

    CAS  Article  Google Scholar 

  13. 13.

    Zhou P P, Xie W P, Yao Z, Zhu Y Q, Ye L D, Yu H W. Development of a temperature-responsive yeast cell factory using engineered Gal4 as a protein switch. Biotechnology and Bioengineering, 2018, 115(5): 1321–1330

    CAS  Article  Google Scholar 

  14. 14.

    Mondal K, VijayRaghavan K, Varadarajan R. Design and utility of temperature-sensitive Gal4 mutants for conditional gene expression in Drosophila. Fly, 2007, 1(5): 282–286

    Article  Google Scholar 

  15. 15.

    Boeke J D, LaCroute F, Fink G R. A positive selection for mutants lacking orotidine-5′-phosphate decarboxylase activity in yeast 5-fluoro-orotic acid resistance. Molecular Genetics and Genomics, 1984, 197(2): 345–346

    CAS  Article  Google Scholar 

  16. 16.

    Sikorski R, Boeke J D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods in Enzymology, 1991, 194: 302–318

    CAS  Article  Google Scholar 

  17. 17.

    Meng X D, Smith R M, Giesecke A V, Joung J K, Wolfe S A. Counter-selectable marker for bacterial-based interaction trap systems. BioTechniques, 2006, 40(2): 179–184

    CAS  Article  Google Scholar 

  18. 18.

    Baliga C, Majhi S, Mondal K, Bhattacharjee A, VijayRaghavan K, Varadarajan R. Rational elicitation of cold-sensitive phenotypes. Proceedings of the National Academy of Sciences of the United States of America, 2016, 113(18): E2506–E2515

    CAS  Article  Google Scholar 

  19. 19.

    Brachmann C B, Davies A, Cost G J, Caputo E, Li J, Hieter P, Boeke J D. Designer deletion strains derived from Saccharomyces cerevisiae S288C: a useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast (Chichester, England), 1998, 14(2): 115–132

    CAS  Article  Google Scholar 

  20. 20.

    Gietz R D, Schiestl R H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols, 2007, 2(1): 31–34

    CAS  Article  Google Scholar 

  21. 21.

    Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods (San Diego, Calif.), 2001, 25(4): 402–408

    CAS  Article  Google Scholar 

  22. 22.

    van Hoek P, de Hulster E, van Dijken J P, Pronk J T. Fermentative capacity in high-cell-density fed-batch cultures of baker’s yeast. Biotechnology and Bioengineering, 2000, 68(5): 517–523

    CAS  Article  Google Scholar 

  23. 23.

    Meng X, Smith R M, Giesecke A V, Keith Joung J, Wolfe S A. Counter-selectable marker for bacterial-based interaction trap systems. BioTechniques, 2006, 40(2): 179–184

    CAS  Article  Google Scholar 

  24. 24.

    Hong M Q, Fitzgerald M X, Harper S, Luo C, Speicher D W, Marmorstein R. Structural basis for dimerization in DNA recognition by Gal4. Structure (London, England), 2008, 16(7): 1019–1026

    CAS  Article  Google Scholar 

  25. 25.

    Schjerling P, Holmberg S. Comparative amino acid sequence analysis of the C6 zinc cluster family of transcriptional regulators. Nucleic Acids Research, 1996, 24(23): 4599–4607

    CAS  Article  Google Scholar 

  26. 26.

    Ma J, Ptashne M. Deletion analysis of GAL4 defines two transcriptional activating segments. Cell, 1987, 48(5): 847–853

    CAS  Article  Google Scholar 

  27. 27.

    Johnston M, Dover J. Mutational analysis of the Gal4-encoded transcriptional activator protein of Saccharomyces cerevisiae. Genetics, 1988, 120(1): 63–74

    CAS  Article  Google Scholar 

  28. 28.

    Zhou P P, Xu N N, Yang Z F, Du Y, Yue C L, Xu N, Ye L D. Directed evolution of the transcription factor Gal4 for development of an improved transcriptional regulation system in Saccharomyces cerevisiae. Enzyme and Microbial Technology, 2020, 142: 109675

    CAS  Article  Google Scholar 

  29. 29.

    Boorstein W R, Craig E A. Structure and regulation of the SSA4 HSP70 gene of Saccharomyces cerevisiae. Journal of Biological Chemistry, 1990, 265(31): 18912–18921

    CAS  Article  Google Scholar 

  30. 30.

    Chen Y, Daviet L, Schalk M, Siewers V, Nielsen J. Establishing a platform cell factory through engineering of yeast acetyl-CoA metabolism. Metabolic Engineering, 2013, 15: 48–54

    CAS  Article  Google Scholar 

  31. 31.

    Gill G, Ptashne M. Negative effect of the transcriptional activator GAL4. Nature, 1988, 334(6184): 721–724

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This work was financially supported by the National Key Research and Development Program of China (Grant Nos. 2018YFA0901800 and 2020YFA0908400), the National Natural Science Foundation of China (Grant No. 21776244), and Zhejiang Provincial Natural Science Foundation of China (Grant No. LZ20B060002).

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Correspondence to Lidan Ye or Hongwei Yu.

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Lin, J., Yao, Z., Lyu, X. et al. Development of a dual temperature control system for isoprene biosynthesis in Saccharomyces cerevisiae. Front. Chem. Sci. Eng. (2021). https://doi.org/10.1007/s11705-021-2088-0

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Keywords

  • transcriptional activator
  • directed evolution
  • dynamic control
  • heat-shock
  • isoprene