Engineering of Synthetic, Stress-responsive Yeast Promoters

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 2016 Jun 22

PMID 27325743

Citations 39

Authors

Arun S Rajkumar

Guodong Liu

David Bergenholm

Dushica Arsovska

Mette Kristensen

Jens Nielsen

Michael K Jensen

Jay D Keasling

Affiliations

Soon will be listed here.

Abstract

Advances in synthetic biology and our understanding of the rules of promoter architecture have led to the development of diverse synthetic constitutive and inducible promoters in eukaryotes and prokaryotes. However, the design of promoters inducible by specific endogenous or environmental conditions is still rarely undertaken. In this study, we engineered and characterized a set of strong, synthetic promoters for budding yeast Saccharomyces cerevisiae that are inducible under acidic conditions (pH ≤ 3). Using available expression and transcription factor binding data, literature on transcriptional regulation, and known rules of promoter architecture we improved the low-pH performance of the YGP1 promoter by modifying transcription factor binding sites in its upstream activation sequence. The engineering strategy outlined for the YGP1 promoter was subsequently applied to create a response to low pH in the unrelated CCW14 promoter. We applied our best promoter variants to low-pH fermentations, enabling ten-fold increased production of lactic acid compared to titres obtained with the commonly used, native TEF1 promoter. Our findings outline and validate a general strategy to iteratively design and engineer synthetic yeast promoters inducible to environmental conditions or stresses of interest.

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References

Van Driessche B, Tafforeau L, Hentges P, Carr A, Vandenhaute J . Additional vectors for PCR-based gene tagging in Saccharomyces cerevisiae and Schizosaccharomyces pombe using nourseothricin resistance. Yeast. 2005; 22(13):1061-8. DOI: 10.1002/yea.1293. View

Destruelle M, Holzer H, Klionsky D . Identification and characterization of a novel yeast gene: the YGP1 gene product is a highly glycosylated secreted protein that is synthesized in response to nutrient limitation. Mol Cell Biol. 1994; 14(4):2740-54. PMC: 358640. DOI: 10.1128/mcb.14.4.2740-2754.1994. View

Lawrence C, Botting C, Antrobus R, Coote P . Evidence of a new role for the high-osmolarity glycerol mitogen-activated protein kinase pathway in yeast: regulating adaptation to citric acid stress. Mol Cell Biol. 2004; 24(8):3307-23. PMC: 381676. DOI: 10.1128/MCB.24.8.3307-3323.2004. View

Jensen N, Strucko T, Kildegaard K, David F, Maury J, Mortensen U . EasyClone: method for iterative chromosomal integration of multiple genes in Saccharomyces cerevisiae. FEMS Yeast Res. 2013; 14(2):238-48. PMC: 4282123. DOI: 10.1111/1567-1364.12118. View

Muller P, Janovjak H, Miserez A, Dobbie Z . Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques. 2002; 32(6):1372-4, 1376, 1378-9. View

Livak K, Schmittgen T . Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2002; 25(4):402-8. DOI: 10.1006/meth.2001.1262. View

Fernandes A, Mira N, Vargas R, Canelhas I, Sa-Correia I . Saccharomyces cerevisiae adaptation to weak acids involves the transcription factor Haa1p and Haa1p-regulated genes. Biochem Biophys Res Commun. 2005; 337(1):95-103. DOI: 10.1016/j.bbrc.2005.09.010. View

Paddon C, Westfall P, Pitera D, Benjamin K, Fisher K, McPhee D . High-level semi-synthetic production of the potent antimalarial artemisinin. Nature. 2013; 496(7446):528-32. DOI: 10.1038/nature12051. View

de Boer C, Hughes T . YeTFaSCo: a database of evaluated yeast transcription factor sequence specificities. Nucleic Acids Res. 2011; 40(Database issue):D169-79. PMC: 3245003. DOI: 10.1093/nar/gkr993. View

10.

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

11.

Haase S, Wittenberg C . Topology and control of the cell-cycle-regulated transcriptional circuitry. Genetics. 2014; 196(1):65-90. PMC: 3872199. DOI: 10.1534/genetics.113.152595. View

12.

Bitter G, Chang K, Egan K . A multi-component upstream activation sequence of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene promoter. Mol Gen Genet. 1991; 231(1):22-32. DOI: 10.1007/BF00293817. View

13.

Jakociunas T, Rajkumar A, Zhang J, Arsovska D, Rodriguez A, Jendresen C . CasEMBLR: Cas9-Facilitated Multiloci Genomic Integration of in Vivo Assembled DNA Parts in Saccharomyces cerevisiae. ACS Synth Biol. 2015; 4(11):1226-34. DOI: 10.1021/acssynbio.5b00007. View

14.

Carmelo V, Sa-Correia I . HySP26 gene transcription is strongly induced during Saccharomyces cerevisiae growth at low pH. FEMS Microbiol Lett. 1997; 149(1):85-8. DOI: 10.1111/j.1574-6968.1997.tb10312.x. View

15.

Ishida N, Saitoh S, Onishi T, Tokuhiro K, Nagamori E, Kitamoto K . The effect of pyruvate decarboxylase gene knockout in Saccharomyces cerevisiae on L-lactic acid production. Biosci Biotechnol Biochem. 2006; 70(5):1148-53. DOI: 10.1271/bbb.70.1148. View

16.

Jung U, Levin D . Genome-wide analysis of gene expression regulated by the yeast cell wall integrity signalling pathway. Mol Microbiol. 1999; 34(5):1049-57. DOI: 10.1046/j.1365-2958.1999.01667.x. View

17.

MacIsaac K, Wang T, Gordon D, Gifford D, Stormo G, Fraenkel E . An improved map of conserved regulatory sites for Saccharomyces cerevisiae. BMC Bioinformatics. 2006; 7:113. PMC: 1435934. DOI: 10.1186/1471-2105-7-113. View

18.

Brown S, Clastre M, Courdavault V, OConnor S . De novo production of the plant-derived alkaloid strictosidine in yeast. Proc Natl Acad Sci U S A. 2015; 112(11):3205-10. PMC: 4371906. DOI: 10.1073/pnas.1423555112. View

19.

Raveh-Sadka T, Levo M, Shabi U, Shany B, Keren L, Lotan-Pompan M . Manipulating nucleosome disfavoring sequences allows fine-tune regulation of gene expression in yeast. Nat Genet. 2012; 44(7):743-50. DOI: 10.1038/ng.2305. View

20.

Mira N, Henriques S, Keller G, Teixeira M, Matos R, Arraiano C . Identification of a DNA-binding site for the transcription factor Haa1, required for Saccharomyces cerevisiae response to acetic acid stress. Nucleic Acids Res. 2011; 39(16):6896-907. PMC: 3167633. DOI: 10.1093/nar/gkr228. View