PhiReX: a Programmable and Red Light-regulated Protein Expression Switch for Yeast

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2017 Sep 16

PMID 28911120

Citations 12

Authors

Lena Hochrein

Fabian Machens

Katrin Messerschmidt

Bernd Mueller-Roeber

Affiliations

Soon will be listed here.

Abstract

Highly regulated induction systems enabling dose-dependent and reversible fine-tuning of protein expression output are beneficial for engineering complex biosynthetic pathways. To address this, we developed PhiReX, a novel red/far-red light-regulated protein expression system for use in Saccharomyces cerevisiae. PhiReX is based on the combination of a customizable synTALE DNA-binding domain, the VP64 activation domain and the light-sensitive dimerization of the photoreceptor PhyB and its interacting partner PIF3 from Arabidopsis thaliana. Robust gene expression and high protein levels are achieved by combining genome integrated red light-sensing components with an episomal high-copy reporter construct. The gene of interest as well as the synTALE DNA-binding domain can be easily exchanged, allowing the flexible regulation of any desired gene by targeting endogenous or heterologous promoter regions. To allow low-cost induction of gene expression for industrial fermentation processes, we engineered yeast to endogenously produce the chromophore required for the effective dimerization of PhyB and PIF3. Time course experiments demonstrate high-level induction over a period of at least 48 h.

Citing Articles

Construction and Characterization of Light-Responsive Transcriptional Systems.

Gligorovski V, Rahi S Methods Mol Biol. 2024; 2844:261-275.

PMID: 39068346 DOI: 10.1007/978-1-0716-4063-0_18.

Dynamic Metabolic Control: From the Perspective of Regulation Logic.

Jiang T, Li C, Teng Y, Zhang J, Logan D, Yan Y Synth Biol Eng. 2024; 1(2).

PMID: 38572077 PMC: 10986841. DOI: 10.35534/sbe.2023.10012.

A roadmap to establish a comprehensive platform for sustainable manufacturing of natural products in yeast.

Naseri G Nat Commun. 2023; 14(1):1916.

PMID: 37024483 PMC: 10079933. DOI: 10.1038/s41467-023-37627-1.

PhiReX 2.0: A Programmable and Red Light-Regulated CRISPR-dCas9 System for the Activation of Endogenous Genes in .

Machens F, Ran G, Ruehmkorff C, Meyer Auf der Heyde J, Mueller-Roeber B, Hochrein L ACS Synth Biol. 2023; 12(4):1046-1057.

PMID: 37014634 PMC: 10127447. DOI: 10.1021/acssynbio.2c00517.

Use of red, far-red, and near-infrared light in imaging of yeasts and filamentous fungi.

Pocsi I, Szigeti Z, Emri T, Boczonadi I, Vereb G, Szollosi J Appl Microbiol Biotechnol. 2022; 106(11):3895-3912.

PMID: 35599256 PMC: 9200671. DOI: 10.1007/s00253-022-11967-2.

References

Rodriguez-Lopez M, Cotobal C, Fernandez-Sanchez O, Borbaran Bravo N, Oktriani R, Abendroth H . A CRISPR/Cas9-based method and primer design tool for seamless genome editing in fission yeast. Wellcome Open Res. 2017; 1:19. PMC: 5445975. DOI: 10.12688/wellcomeopenres.10038.3. View

Li L, Lagarias J . Phytochrome assembly in living cells of the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1994; 91(26):12535-9. PMC: 45473. DOI: 10.1073/pnas.91.26.12535. View

Si T, Luo Y, Xiao H, Zhao H . Utilizing an endogenous pathway for 1-butanol production in Saccharomyces cerevisiae. Metab Eng. 2014; 22:60-8. DOI: 10.1016/j.ymben.2014.01.002. View

Muller K, Engesser R, Timmer J, Nagy F, Zurbriggen M, Weber W . Synthesis of phycocyanobilin in mammalian cells. Chem Commun (Camb). 2013; 49(79):8970-2. DOI: 10.1039/c3cc45065a. View

Cherest H, Kerjan P . The Saccharomyces cerevisiae MET3 gene: nucleotide sequence and relationship of the 5' non-coding region to that of MET25. Mol Gen Genet. 1987; 210(2):307-13. DOI: 10.1007/BF00325699. View

Kennedy M, Hughes R, Peteya L, Schwartz J, Ehlers M, Tucker C . Rapid blue-light-mediated induction of protein interactions in living cells. Nat Methods. 2010; 7(12):973-5. PMC: 3059133. DOI: 10.1038/nmeth.1524. View

Moscou M, Bogdanove A . A simple cipher governs DNA recognition by TAL effectors. Science. 2009; 326(5959):1501. DOI: 10.1126/science.1178817. View

Brat D, Weber C, Lorenzen W, Bode H, Boles E . Cytosolic re-localization and optimization of valine synthesis and catabolism enables inseased isobutanol production with the yeast Saccharomyces cerevisiae. Biotechnol Biofuels. 2012; 5(1):65. PMC: 3476451. DOI: 10.1186/1754-6834-5-65. View

Khalil A, Lu T, Bashor C, Ramirez C, Pyenson N, Joung J . A synthetic biology framework for programming eukaryotic transcription functions. Cell. 2012; 150(3):647-58. PMC: 3653585. DOI: 10.1016/j.cell.2012.05.045. View

10.

Shimizu-Sato S, Huq E, Tepperman J, Quail P . A light-switchable gene promoter system. Nat Biotechnol. 2002; 20(10):1041-4. DOI: 10.1038/nbt734. View

11.

Pfeiffer A, Nagel M, Popp C, Wust F, Bindics J, Viczian A . Interaction with plant transcription factors can mediate nuclear import of phytochrome B. Proc Natl Acad Sci U S A. 2012; 109(15):5892-7. PMC: 3326511. DOI: 10.1073/pnas.1120764109. View

12.

Macreadie I, Horaitis O, Verkuylen A, Savin K . Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast. Gene. 1991; 104(1):107-11. DOI: 10.1016/0378-1119(91)90474-p. View

13.

Farzadfard F, Perli S, Lu T . Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. ACS Synth Biol. 2013; 2(10):604-13. PMC: 3805333. DOI: 10.1021/sb400081r. View

14.

Szurek B, Marois E, Bonas U, Van Den Ackerveken G . Eukaryotic features of the Xanthomonas type III effector AvrBs3: protein domains involved in transcriptional activation and the interaction with nuclear import receptors from pepper. Plant J. 2001; 26(5):523-34. DOI: 10.1046/j.0960-7412.2001.01046.x. View

15.

Ellis T, Wang X, Collins J . Diversity-based, model-guided construction of synthetic gene networks with predicted functions. Nat Biotechnol. 2009; 27(5):465-71. PMC: 2680460. DOI: 10.1038/nbt.1536. View

16.

Belli G, Gari E, Piedrafita L, Aldea M, Herrero E . An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. Nucleic Acids Res. 1998; 26(4):942-7. PMC: 147371. DOI: 10.1093/nar/26.4.942. View

17.

Zhang Y, Werling U, Edelmann W . SLiCE: a novel bacterial cell extract-based DNA cloning method. Nucleic Acids Res. 2012; 40(8):e55. PMC: 3333860. DOI: 10.1093/nar/gkr1288. View

18.

Galanie S, Thodey K, Trenchard I, Filsinger Interrante M, Smolke C . Complete biosynthesis of opioids in yeast. Science. 2015; 349(6252):1095-100. PMC: 4924617. DOI: 10.1126/science.aac9373. View

19.

Gietz R, Schiestl R . High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007; 2(1):31-4. DOI: 10.1038/nprot.2007.13. View

20.

Hochrein L, Machens F, Gremmels J, Schulz K, Messerschmidt K, Mueller-Roeber B . AssemblX: a user-friendly toolkit for rapid and reliable multi-gene assemblies. Nucleic Acids Res. 2017; 45(10):e80. PMC: 5449548. DOI: 10.1093/nar/gkx034. View