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 P_SSA4 to control the expression of P_GAL-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.

Graphical abstract

Keywords

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

References

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

[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

[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

[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

[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

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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

[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

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

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

[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

[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

[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

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

RIGHTS & PERMISSIONS

Higher Education Press