Modulating transcription through development of semi-synthetic yeast core promoters
Autoři:
Thomas Decoene aff001; Sofie L. De Maeseneire aff002; Marjan De Mey aff001
Působiště autorů:
Centre for Synthetic Biology (CSB), Ghent University, Ghent, Belgium
aff001; Centre for Industrial Biotechnology and Biocatalysis (InBio.be), Ghent University, Coupure links, Ghent, Belgium
aff002
Vyšlo v časopise:
PLoS ONE 14(11)
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pone.0224476
Souhrn
Altering gene expression regulation by promoter engineering is a very effective way to fine-tune heterologous pathways in eukaryotic hosts. Typically, pathway building approaches in yeast still use a limited set of long, native promoters. With the today’s introduction of longer and more complex pathways, an expansion of this synthetic biology toolbox is necessary. In this study we elucidated the core promoter structure of the well-characterized yeast TEF1 promoter and determined the minimal length needed for sufficient protein expression. Furthermore, this minimal core promoter sequence was used for the creation of a promoter library covering different expression strengths. This resulted in a group of short, 69 bp promoters with an 8.0-fold expression range. One exemplar had a two and four times higher expression compared to the native CYC1 and ADH1 promoter, respectively. Additionally, as it was described that the protein expression range could be broadened by upstream activating sequences (UASs), we integrated earlier described single and multiple short, synthetic UASs in front of the strongest yeast core promoter. This approach resulted to further variation in protein expression and an overall promoter library spanning a 20-fold activity range and covering a length from 69 bp to maximally 129 bp. Furthermore, the robustness of this library was assessed on three alternative carbon sources besides glucose. As such, the suitability of short yeast core promoters for metabolic engineering applications on different media, either in an individual context or combined with UAS elements, was demonstrated.
Klíčová slova:
Gene expression – Engineering and technology – Yeast – Saccharomyces cerevisiae – DNA transcription – Protein expression – Transcriptional control – TATA box
Zdroje
1. Jullesson D, David F, Pfleger B, Nielsen J. Impact of synthetic biology and metabolic engineering on industrial production of fine chemicals. Biotechnol Adv. 2015;33: 1395–402. doi: 10.1016/j.biotechadv.2015.02.011 25728067
2. Nielsen J, Keasling JD. Engineering Cellular Metabolism. Cell. 2016;164: 1185–1197. doi: 10.1016/j.cell.2016.02.004 26967285
3. Curran K a., Karim AS, Gupta A, Alper HS. Use of expression-enhancing terminators in Saccharomyces cerevisiae to increase mRNA half-life and improve gene expression control for metabolic engineering applications. Metab Eng. 2013;19: 88–97. doi: 10.1016/j.ymben.2013.07.001 23856240
4. Curran K a., Morse NJ, Markham K a., Wagman AM, Gupta A, Alper HS. Short, Synthetic Terminators for Improved Heterologous Gene Expression in Yeast. ACS Synth Biol. 2015;4: 824–32. doi: 10.1021/sb5003357 25686303
5. MacPherson M, Saka Y. Short Synthetic Terminators for Assembly of Transcription Units in Vitro and Stable Chromosomal Integration in Yeast S. cerevisiae. ACS Synth Biol. 2016;6: 130–138. doi: 10.1021/acssynbio.6b00165 27529501
6. Redden H, Morse N, Alper HS. The synthetic biology toolbox for tuning gene expression in yeast. FEMS Yeast Res. 2015;15: 1–10. doi: 10.1111/1567-1364.12188 25047958
7. Sun J, Shao Z, Zhao H, Nair N, Wen F, Xu JH, et al. Cloning and characterization of a panel of constitutive promoters for applications in pathway engineering in Saccharomyces cerevisiae. Biotechnol Bioeng. 2012;109: 2082–2092. doi: 10.1002/bit.24481 22383307
8. Lee ME, DeLoache WC, Cervantes B, Dueber JE. A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. ACS Synth Biol. 2015;4: 975–986. doi: 10.1021/sb500366v 25871405
9. Da Silva N a., Srikrishnan S. Introduction and expression of genes for metabolic engineering applications in Saccharomyces cerevisiae. FEMS Yeast Res. 2012;12: 197–214. doi: 10.1111/j.1567-1364.2011.00769.x 22129153
10. Mumberg D, Müller R, Funk M. Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene. 1995;156: 119–122. doi: 10.1016/0378-1119(95)00037-7 7737504
11. Blazeck J, Alper HS. Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol J. 2013;8: 46–58. doi: 10.1002/biot.201200120 22890821
12. Decoene T, Peters G, De Maeseneire S, De Mey M. Toward predictable 5’UTRs in Saccharomyces cerevisiae: Development of a yUTR calculator. ACS Synth Biol. 2018. doi: 10.1021/acssynbio.7b00366 29366325
13. Petersen SD, Zhang J, Lee JS, JakočiJakoˇJakoči T, Grav LM, Kildegaard HF, et al. Modular 5-UTR hexamers for context-independent tuning of protein expression in eukaryotes. Nucleic Acids Res. 2018; 1–11. doi: 10.1093/nar/gkx1156
14. Cuperus J, Groves B, Kuchina A, Rosenberg AB, Jojic N, Fields S, et al. Deep learning of the regulatory grammar of yeast 5’ untranslated regions from 500,000 random sequences. Genome Res. 2017;27: 2015–2024. doi: 10.1101/gr.224964.117 29097404
15. Dvir S, Velten L, Sharon E, Zeevi D, Carey LB, Weinberger A, et al. Deciphering the rules by which 5 ′ -UTR sequences affect protein expression in yeast. PNAS. 2013;110: e2792–801. doi: 10.1073/pnas.1222534110 23832786
16. Lubliner S, Keren L, Segal E. Sequence features of yeast and human core promoters that are predictive of maximal promoter activity. Nucleic Acids Res. 2013;41: 5569–5581. doi: 10.1093/nar/gkt256 23599004
17. Lubliner S, Regev I, Lotan-Pompan M, Edelheit S, Weinberger A, Segal E. Core promoter sequence in yeast is a major determinant of expression level. Genome Res. 2015;25: 1008–1017. doi: 10.1101/gr.188193.114 25969468
18. Danino YM, Even D, Ideses D, Juven-Gershon T. The core promoter: at the heart of gene expression. Biochim Biophys Acta—Gene Regul Mech. 2015;1847: 1116–31. doi: 10.1016/j.bbagrm.2015.04.003 25934543
19. Ede C, Chen X, Lin M-Y, Chen YY. Quantitative Analyses of Core Promoters Enable Precise Engineering of Regulated Gene Expression in Mammalian Cells. ACS Synth Biol. 2016;5: 395–404. doi: 10.1021/acssynbio.5b00266 26883397
20. Portela RMC, Vogl T, Kniely C, Fischer JE, Oliveira R, Glieder A. Synthetic core promoters as universal parts for fine-tuning expression in different yeast species. ACS Synth Biol. 2017;6: 471–484. doi: 10.1021/acssynbio.6b00178 27973777
21. Basehoar AD, Zanton SJ, Pugh BF. Identification and distinct regulation of yeast TATA box-containing genes. Cell. 2004;116: 699–709. doi: 10.1016/s0092-8674(04)00205-3 15006352
22. Hampsey M. Molecular Genetics of the RNA Polymerase II General Transcriptional Machinery. Microbiol Mol Biol Rev. 1998;62: 465–503. 9618449
23. Butler JEF, Kadonaga JT. The RNA polymerase II core promoter: a key component in the regulation of gene expression. Genes Dev. 2002;16: 2583–2592. doi: 10.1101/gad.1026202 12381658
24. Mcmillan J, Lu Z, Rodriguez JS, Ahn T, Lin Z. YeasTSS : an integrative web database of yeast transcription start sites. Database. 2019;2019: 1–12. doi: 10.1093/database/baz048 31032841
25. Hahn S, Young ET. Transcriptional regulation in saccharomyces cerevisiae: Transcription factor regulation and function, mechanisms of initiation, and roles of activators and coactivators. Genetics. 2011;189: 705–736. doi: 10.1534/genetics.111.127019 22084422
26. Blazeck J, Garg R, Reed B, Alper HS. Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng. 2012;109: 2884–2895. doi: 10.1002/bit.24552 22565375
27. Alper H, Fischer C, Nevoigt E, Stephanopoulos G. Tuning genetic control through promoter engineering. Proc Natl Acad Sci U S A. 2005;102: 12678–12683. doi: 10.1073/pnas.0504604102 16123130
28. Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, Stephanopoulos G. Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol. 2006;72: 5266–5273. doi: 10.1128/AEM.00530-06 16885275
29. Blazeck J, Liu L, Redden H, Alper H. Tuning gene expression in yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol. 2011;77: 7905–7914. doi: 10.1128/AEM.05763-11 21926196
30. Curran K a Crook NC, Karim AS, Gupta A, Wagman AM, Alper HS. Design of synthetic yeast promoters via tuning of nucleosome architecture. Nat Commun. 2014;5: 4002. doi: 10.1038/ncomms5002 24862902
31. Ottoz DSM, Rudolf F, Stelling J. Inducible, tightly regulated and growth condition-independent transcription factor in Saccharomyces cerevisiae. Nucleic Acids Res. 2014;42: e130. doi: 10.1093/nar/gku616 25034689
32. Mcisaac RS, Gibney PA, Chandran SS, Benjamin KR, Botstein D. Synthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae. Nucleic Acids Res. 2014;42: e48. doi: 10.1093/nar/gkt1402 24445804
33. Mcisaac RS, Oakes BL, Wang X, Dummit KA, Botstein D, Noyes MB. Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast. Nucleic Acids Res. 2013;41: e57. doi: 10.1093/nar/gks1313 23275543
34. Machens F, Balazadeh S, Bernd M-R, Katrin M. Synthetic Promoters and Transcription Factors for Heterologous Protein Expression in Saccharomyces cerevisiae. Front Bioeng Biotechnol. 2017;5. doi: 10.3389/fbioe.2017.00063 29098147
35. Lian J, HamediRad M, Sumeng H, Huimin Z. Combinatorial metabolic engineering using an orthogonal tri-functional CRISPR system. Nat Commun. 2017;8: 1–9. doi: 10.1038/s41467-016-0009-6
36. Rantasalo A, Landowski CP, Kuivanen J, Korppoo A, Reuter L, Koivistoinen O, et al. A universal gene expression system for fungi. Nucleic Acids Res. 2018;46: e111. doi: 10.1093/nar/gky558 29924368
37. Rantasalo A, Kuivanen J, Penttilä M, Jäntti J, Mojzita D. A synthetic toolkit for complex genetic circuit engineering in S. cerevisiae. ACS Synth Biol. 2018. doi: 10.1021/acssynbio.8b00076 29750501
38. Lucks JB, Qi L, Whitaker WR, Arkin AP. Toward scalable parts families for predictable design of biological circuits. Curr Opin Microbiol. 2008;11: 567–573. doi: 10.1016/j.mib.2008.10.002 18983935
39. Decoene T, De Paepe B, Maertens J, Coussement P, Peters G, De Maeseneire SL, et al. Standardization in synthetic biology: an engineering discipline coming of age. Crit Rev Biotechnol. 2018;38: 647–656. doi: 10.1080/07388551.2017.1380600 28954542
40. Dower K, Rosbash M. T7 RNA polymerase-directed transcripts are processed in yeast and link 3′ end formation to mRNA nuclear export. Rna. 2002;8: 686–697. doi: 10.1017/s1355838202024068 12022234
41. Benton BM, Eng WK, Dunn JJ, Studier FW, Sternglanz R, Fisher PA. Signal-mediated import of bacteriophage T7 RNA polymerase into the Saccharomyces cerevisiae nucleus and specific transcription of target genes. Mol Cell Biol. 1990;10: 353–360. doi: 10.1128/mcb.10.1.353 2403641
42. Redden H, Alper HS. The development and characterization of synthetic minimal yeast promoters. Nat Commun. 2015;6: 7810. doi: 10.1038/ncomms8810 26183606
43. Quan J, Tian J. Circular polymerase extension cloning of complex gene libraries and pathways. PLoS One. 2009;4: e6441. doi: 10.1371/journal.pone.0006441 19649325
44. Brachmann CB, Davies A, Cost GJ, Caputo E, Li J, Hieter P, et al. Designer deletion strains derived from Saccharomyces cerevisiae S288C: A useful set of strains and plasmids for PCR-mediated gene disruption and other applications. Yeast. 1998;14: 115–132. doi: 10.1002/(SICI)1097-0061(19980130)14:2<115::AID-YEA204>3.0.CO;2-2 9483801
45. Sheff M a., Thorn KS. Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae. Yeast. 2004;21: 661–670. doi: 10.1002/yea.1130 15197731
46. Shaner NC, Steinbach P a, Tsien RY. A guide to choosing fluorescent proteins. Nat Methods. 2005;2: 905–909. doi: 10.1038/nmeth819 16299475
47. Gietz RD, Schiestl RH. Quick and easy yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc. 2007;2: 35–37. doi: 10.1038/nprot.2007.14 17401335
48. Salis HM, Mirsky E a, Voigt C a. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol. 2009;27: 946–50. doi: 10.1038/nbt.1568 19801975
49. Bonde MT, Pedersen M, Klausen MS, Jensen SI, Wulff T, Harrison S, et al. Predictable tuning of protein expression in bacteria. Nat Methods. 2016;13: 233–236. doi: 10.1038/nmeth.3727 26752768
50. Weenink T, Mckiernan RM, Ellis T. Rational Design of RNA Structures that Predictably Tune Eukaryotic Gene Expression. BioRxiv. 2017; http://dx.doi.org/10.1101/137877.
51. Li S, Si T, Wang M, Zhao H. Development of a Synthetic Malonyl-CoA Sensor in Saccharomyces cerevisiae for Intracellular Metabolite Monitoring and Genetic Screening. ACS Synth Biol. 2015;4: 1308–1315. doi: 10.1021/acssynbio.5b00069 26149896
52. Siddiqui MS, Thodey K, Trenchard I, Smolke CD. Advancing secondary metabolite biosynthesis in yeast with synthetic biology tools. FEMS Yeast Res. 2012;12: 144–170. doi: 10.1111/j.1567-1364.2011.00774.x 22136110
53. Jensen NB, Strucko T, Kildegaard KR, David F, Maury J, Mortensen UH, et al. EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 2014;14: 238–248. doi: 10.1111/1567-1364.12118 24151867
54. David F, Nielsen J, Siewers V. Flux control at the malonyl-CoA node through hierarchical dynamic pathway regulation in Saccharomyces cerevisiae. ACS Synth Biol. 2016;5: 224–233. doi: 10.1021/acssynbio.5b00161 26750662
55. Jendresen CB, Stahlhut SG, Li M, Gaspar P, Siedler S, Frster J, et al. Highly Active and Specific Tyrosine Ammonia-Lyases from Diverse Origins Enable Enhanced Production of Aromatic Compounds in Bacteria and Saccharomyces cerevisiae. Appl Environ Microbiol. 2015;81: 4458–4476. doi: 10.1128/AEM.00405-15 25911487
56. Nagalakshmi U, Wang Z, Waern K, Shou C, Raha D, Gerstein M, et al. The transcriptional landscape of the yeast genome defined by RNA sequencing. Science. 2008;320: 1344–1349. doi: 10.1126/science.1158441 18451266
57. Tuller T, Ruppin E, Kupiec M. Properties of untranslated regions of the S. cerevisiae genome. BMC Genomics. 2009;10: doi: 10.1186/1471-2164-10-391 19698117
58. Cramer P, Cramer P, Bushnell DA, Kornberg RD. Structural Basis of Transcription : RNA Polymerase II at 2. 8 Ångstrom Resolution. Science (80-). 2001;1863: 1863–1877. doi: 10.1126/science.1059493 11313498
59. Segal E, Widom J. Poly(dA:dT) tracts: major determinants of nucleosome organization. Curr Opin Struct Biol. 2009;19: 65–71. doi: 10.1016/j.sbi.2009.01.004 19208466
60. Raveh-Sadka T, Levo M, Shabi U, Shany B, Keren L, Lotan-Pompan M, et al. Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast. Nat Genet. 2012;44: 743–750. doi: 10.1038/ng.2305 22634752
61. Zeevi D, Sharon E, Lotan-pompan M, Lubling Y, Shipony Z, Raveh-sadka T, et al. Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters. Genome Res. 2011;21: 2114–2128. doi: 10.1101/gr.119669.110 22009988
62. Levo M, Segal E. In pursuit of design principles of regulatory sequences. Nat Rev Genet. 2014;15: 453–68. doi: 10.1038/nrg3684 24913666
63. Jansen A, Van Der Zande E, Meert W, Fink GR, Verstrepen KJ. Distal chromatin structure influences local nucleosome positions and gene expression. Nucleic Acids Res. 2012;40: 3870–3885. doi: 10.1093/nar/gkr1311 22241769
64. Portela R M. C., Vogl T, Ebner K, Oliveira R, Glieder A. Pichia pastoris alcohol oxidase 1 (AOX1) core promoter engineering by high resolution systematic mutagenesis. Biotechnol J. 2018;13.
65. Zhang Z, Dietrich FS. Mapping of transcription start sites in Saccharomyces cerevisiae using 5′ SAGE. Nucleic Acids Res. 2005;33: 2838–2851. doi: 10.1093/nar/gki583 15905473
66. Chen W, Struhl K. Yeast mRNA initiation sites are determined primarily by specific sequences, not by the distance from the TATA element. EMBO J. 1985;4: 3273–80. 3912167
67. Hahn S, Hoar ET, Guarente L. Each of three “TATA elements” specifies a subset of the transcription initiation sites at the CYC-1 promoter of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1985;82: 8562–8566. doi: 10.1073/pnas.82.24.8562 3001709
68. Li XY, Bhaumik SR, Zhu X, Li L, Shen WC, Dixit BL, et al. Selective recruitment of TAFs by yeast upstream activating sequences: Implications for eukaryotic promoter structure. Curr Biol. 2002;12: 1240–1244. doi: 10.1016/s0960-9822(02)00932-6 12176335
69. Partow S, Siewers V, Bjørn S, Nielsen J, Maury J. Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae. Yeast. 2010;27: 955–964. doi: 10.1002/yea.1806 20625983
70. Peng B, Williams TC, Henry M, Nielsen LK, Vickers CE. Controlling heterologous gene expression in yeast cell factories on different carbon substrates and across the diauxic shift: A comparison of yeast promoter activities. Microb Cell Fact. 2015;14: 1–11. doi: 10.1186/s12934-014-0183-3
71. Turcotte B, Liang XB, Robert F, Soontorngun N. Transcriptional regulation of nonfermentable carbon utilization in budding yeast. FEMS Yeast Res. 2016;10: 2–13. doi: 10.1111/j.1567-1364.2009.00555.x.Transcriptional
72. Mitchell L a., Chuang J, Agmon N, Khunsriraksakul C, Phillips N a., Cai Y, et al. Versatile genetic assembly system (VEGAS) to assemble pathways for expression in S. cerevisiae. Nucleic Acids Res. 2015;43: 6620–30. doi: 10.1093/nar/gkv466 25956652
Článok vyšiel v časopise
PLOS One
2019 Číslo 11
- Metamizol jako analgetikum první volby: kdy, pro koho, jak a proč?
- Nejasný stín na plicích – kazuistika
- Masturbační chování žen v ČR − dotazníková studie
- Úspěšná resuscitativní thorakotomie v přednemocniční neodkladné péči
- Dlouhodobá recidiva a komplikace spojené s elektivní operací břišní kýly
Najčítanejšie v tomto čísle
- A daily diary study on maladaptive daydreaming, mind wandering, and sleep disturbances: Examining within-person and between-persons relations
- A 3’ UTR SNP rs885863, a cis-eQTL for the circadian gene VIPR2 and lincRNA 689, is associated with opioid addiction
- A substitution mutation in a conserved domain of mammalian acetate-dependent acetyl CoA synthetase 2 results in destabilized protein and impaired HIF-2 signaling
- Molecular validation of clinical Pantoea isolates identified by MALDI-TOF