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Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica

  • Applied genetics and molecular biotechnology
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Both varied and strong promoters are essential for metabolic and pathway engineering applications in any host organism. To enable this capacity, here we demonstrate a generalizable method for the de novo construction of strong, synthetic hybrid promoter libraries. Specifically, we demonstrate how promoter truncation and fragment dissection analysis can be utilized to identify both novel upstream activating sequences (UAS) and core promoters—the two components required to generate hybrid promoters. As a base case, the native TEF promoter in Yarrowia lipolytica was examined to identify putative UAS elements that serve as modular synthetic transcriptional activators. Resulting synthetic promoters containing a core promoter region activated by between one and twelve tandem repeats of the newly isolated, 230 nucleotide UASTEF#2 element showed promoter strengths 3- to 4.5-fold times the native TEF promoter. Further analysis through transcription factor binding site abrogation revealed the GCR1p binding site to be necessary for complete UASTEF#2 function. These various promoters were tested for function in a variety of carbon sources. Finally, by combining disparate UAS elements (in this case, UASTEF and UAS1B), we developed a high-strength promoter with for Y. lipolytica with an expression level of nearly sevenfold higher than that of the strong, constitutive TEF promoter. Thus, the general strategy described here enables the efficient, de novo construction of synthetic promoters to both increase native expression capacity and to produce libraries for tunable gene expression.

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  • Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. Proc Natl Acad Sci USA 102:12678–12683

    Article  CAS  Google Scholar 

  • Barth G, Gaillardin C (1997) Physiology and genetics of the dimorphic fungus Yarrowia lipolytica. FEMS Microbiol Rev 19:219–237

    Article  CAS  Google Scholar 

  • Beopoulos A, Chardot T, Nicaud JM (2009) Yarrowia lipolytica: a model and a tool to understand the mechanisms implicated in lipid accumulation. Biochimie 91:692–696

    Article  CAS  Google Scholar 

  • Blanchin-roland S, Otero RRC, Gaillardin C (1994) Two upstream activation sequences control the expression of the XPR2 gene in the yeast Yarrowia lipolytica. Mol Cell Biol 14:327–338

    CAS  Google Scholar 

  • Blazeck J, Liu L, Redden H, Alper H (2011) Tuning gene expression in Yarrowia lipolytica by a hybrid promoter approach. Appl Environ Microbiol 77:7905–7914

    Article  CAS  Google Scholar 

  • Blazeck J, Garg R, Reed B, Alper H (2012) Controlling promoter strength and regulation in Saccharomyces cerevisiae using synthetic hybrid promoters. Biotechnol Bioeng. doi:10.1002/bit.24552

  • Chen DC, Yang BC, Kuo TT (1992) One step transformation of yeast in stationary phase. Curr Genet 21:83–84

    Article  CAS  Google Scholar 

  • Damude H.G.H., Gillies, Peter John, Macool, Daniel Joseph, Picataggio, Stephen K., Pollak, Dana Walters M., Ragghianti, James John, Xue, Zhixiong, Yadav, Narendra S., Zhang, Hongxiang, Zhu, Quinn Qun. (2006) High eicosapentaenoic acid producing strains of Yarrowia lipolytica, United States

  • Davidow LS, Apostolakos D, Odonnell MM, Proctor AR, Ogrydziak DM, Wing RA, Stasko I, Dezeeuw JR (1985) Integrative transformation of the yeast Yarrowia lipolytica. Curr Genet 10:39–48

    Article  CAS  Google Scholar 

  • Deboer HA, Comstock LJ, Vasser M (1983) The Tac promoter—a functional hybrid derived from the Trp and Lac promoters. Proc Natl Acad Sci USA 80:21–25

    Article  CAS  Google Scholar 

  • Drazinic CM, Smerage JB, Lopez MC, Baker HV (1996) Activation mechanism of the multifunctional transcription factor repressor-activator protein 1 (Rap1p). Mol Cell Biol 16:3187–3196

    CAS  Google Scholar 

  • Fournier P, Abbas A, Chasles M, Kudla B, Ogrydziak DM, Yaver D, Xuan JW, Peito A, Ribet AM, Feynerol C, He F, Gaillardin C (1993) Colocalization of centromeric and replicative functions on autonomously replicating sequences isolated from the yeast Yarrowia lipolytica. Proc Natl Acad Sci USA 90:4912–4916

    Article  CAS  Google Scholar 

  • Gaillardin C, Ribet AM (1987) LEU2 directed expression of beta-galactosidase activity and phleomycin resistance in Yarrowia lipolytica. Curr Genet 11:369–375

    Article  CAS  Google Scholar 

  • Gorgens JF, van Zyl WH, Knoetze JH, Hahn-Hagerdal B (2001) The metabolic burden of the PGK1 and ADH2 promoter systems for heterologous xylanase production by Saccharomyces cerevisiae in defined medium. Biotechnol Bioeng 73:238–245

    Article  CAS  Google Scholar 

  • Jensen PR, Hammer K (1998) The sequence of spacers between the consensus sequences modulates the strength of prokaryotic promoters. Appl Environ Microbiol 64:82–87

    CAS  Google Scholar 

  • Jeppsson M, Johansson B, Jensen PR, Hahn-Hagerdal B, Gorwa-Grauslund MF (2003) The level of glucose-6-phosphate dehydrogenase activity strongly influences xylose fermentation and inhibitor sensitivity in recombinant Saccharomyces cerevisiae strains. Yeast 20:1263–1272

    Article  CAS  Google Scholar 

  • Juretzek T, Le Dall MT, Mauersberger S, Gaillardin C, Barth G, Nicaud JM (2001) Vectors for gene expression and amplification in the yeast Yarrowia lipolytica. Yeast 18:97–113

    Article  CAS  Google Scholar 

  • Kalnins A, Otto K, Ruther U, Mullerhill B (1983) Sequence of the lacZ gene of Escherichia coli. EMBO J 2:593–597

    CAS  Google Scholar 

  • Ledall MT, Nicaud JM, Gaillardin C (1994) Multiple-copy integration in the yeast Yarrowia lipolytica. Curr Genet 26:38–44

    Article  CAS  Google Scholar 

  • Liu L, Reed B, Alper H (2011) From pathways to genomes and beyond: the metabolic engineering toolbox and its place in biofuels production. Green 1:81–95

    Article  Google Scholar 

  • Madzak C, Blanchin-Roland S, Otero RRC, Gaillardin C (1999) Functional analysis of upstream regulating regions from the Yarrowia lipolytica XPR2 promoter. Microbiology 145:75–87

    Article  CAS  Google Scholar 

  • Madzak C, Treton B, Blanchin-Roland S (2000) Strong hybrid promoters and integrative expression/secretion vectors for quasi-constitutive expression of heterologous proteins in the yeast Yarrowia lipolytica. J Mol Microbiol Biotechnol 2:207–216

    CAS  Google Scholar 

  • Madzak C, Gaillardin C, Beckerich JM (2004) Heterologous protein expression and secretion in the non-conventional yeast Yarrowia lipolytica: a review. J Biotechnol 109:63–81

    Article  CAS  Google Scholar 

  • Matsuoka M, Matsubara M, Daidoh H, Imanaka T, Uchida K, Aiba S (1993) Analysis of regions essential for the function of chromosomal replicator sequences from Yarrowia lipolytica. Mol Gen Genet 237:327–333

    CAS  Google Scholar 

  • Miller J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, [Cold Spring Harbor, N.Y.]

  • Mukai H., Horii H., Tsujikawa M., Kawabe H., Arimura H., Suyama T. (1992) Yeast promoter and process for preparing heterologous protein, GREEN CROSS CORPORATION (3-3, Imabashi 1-chome Chuo-ku, Osaka-shi, Osaka, JP), Europe

  • Muller S, Sandal T, Kamp-Hansen P, Dalboge H (1998) Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis. Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica. Yeast 14:1267–1283

    Article  CAS  Google Scholar 

  • Mumberg D, Muller R, Funk M (1995) Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds. Gene 156:119–122

    Article  CAS  Google Scholar 

  • Nevoigt E, Kohnke J, Fischer CR, Alper H, Stahl U, Stephanopoulos G (2006) Engineering of promoter replacement cassettes for fine-tuning of gene expression in Saccharomyces cerevisiae. Appl Environ Microbiol 72:5266–5273

    Article  CAS  Google Scholar 

  • Nicaud JM, Madzak C, van den Broek P, Gysler C, Duboc P, Niederberger P, Gaillardin C (2002) Protein expression and secretion in the yeast Yarrowia lipolytica. Fems Yeast Res 2:371–379

    CAS  Google Scholar 

  • Pfleger BF, Pitera DJD, Smolke C, Keasling JD (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat Biotechnol 24:1027–1032

    Article  CAS  Google Scholar 

  • Rosenberg S., Tekamp-olson P. (1992) Enhanced yeast transcription employing hybrid GAPDH promoter region constructs, Chiron Corporation (Emeryville, CA), United States

  • Rud I, Jensen PR, Naterstad K, Axelsson L (2006) A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiology 152:1011–1019

    Article  CAS  Google Scholar 

  • Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y

    Google Scholar 

  • Vanheerikhuizen H, Ykema A, Klootwijk J, Gaillardin C, Ballas C, Fournier P (1985) Heterogeneity in the ribosomal RNA genes of the yeast Yarrowia lipolytica—cloning and analysis of two size classes of repeats. Gene 39:213–222

    Article  CAS  Google Scholar 

  • Vernis L, Abbas A, Chasles M, Gaillardin CM, Brun C, Huberman JA, Fournier P (1997) An origin of replication and a centromere are both needed to establish a replicative plasmid in the yeast Yarrowia lipolytica. Mol Cell Biol 17:1995–2004

    CAS  Google Scholar 

  • Vernis L, Poljak L, Chasles M, Uchida K, Casaregola S, Kas E, Matsuoka M, Gaillardin C, Fournier P (2001) Only centromeres can supply the partition system required for ARS function in the yeast Yarrowia lipolytica. J Mol Biol 305:203–217

    Article  CAS  Google Scholar 

  • Yamane T, Sakai H, Nagahama K, Ogawa T, Matsuoka M (2008) Dissection of centromeric DNA from yeast Yarrowia lipolytica and identification of protein-binding site required for plasmid transmission. J Biosci Bioeng 105:571–578

    Article  CAS  Google Scholar 

  • Young E, Alper H (2010) Synthetic biology: tools to design, build, and optimize cellular processes. J Biomed Biotechnol 2010:130781

    Article  Google Scholar 

  • Zhu J, Zhang MQ (1999) SCPD: a promoter database of the yeast Saccharomyces cerevisiae. Bioinformatics 15:607–611

    Article  CAS  Google Scholar 

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This work was funded by the Office of Naval Research Young Investigator Program and DuPont Young Professor Grant for funding. We would like to thank Masayoshi Matsuoka for the generous gift of plasmid pSl16-Cen1-1(227).

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Correspondence to Hal S. Alper.

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Blazeck, J., Reed, B., Garg, R. et al. Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica . Appl Microbiol Biotechnol 97, 3037–3052 (2013).

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