- Open Access
Methanol regulated yeast promoters: production vehicles and toolbox for synthetic biology
© Gasser et al. 2015
- Received: 19 November 2015
- Accepted: 25 November 2015
- Published: 2 December 2015
Promoters are indispensable elements of a standardized parts collection for synthetic biology. Regulated promoters of a wide variety of well-defined induction ratios and expression strengths are highly interesting for many applications. Exemplarily, we discuss the application of published genome scale transcriptomics data for the primary selection of methanol inducible promoters of the yeast Pichia pastoris (Komagataella sp.). Such a promoter collection can serve as an excellent toolbox for cell and metabolic engineering, and for gene expression to produce heterologous proteins.
- Pichia pastoris
- Synthetic biology
- Protein production
A major task of synthetic biology is the provision of standardized elements for rapid assembly of predictable recombinant gene expression cassettes [1, 2]. These elements include vectors, selection markers, and most importantly collections of regulatory elements like promoters, transcription terminators, secretory leaders and other signal sequences. Ideally, collections of these parts are cataloged in standardized, easy to assemble formats like BioBrick . Promoters are indispensable parts for synthetic biology approaches  and are needed for different expression strength in order to balance the expression levels in a synthetic pathway . There are a plethora of studies which characterize, e.g. constitutive promoters of different strength for Escherichia coli , Aspergillus niger  or Pichia pastoris . Depending on the application it might be necessary to tightly control the promoter activity. Especially regulated promoters are often strictly host specific, so that they need to be identified, characterized and standardized for the host species of interest, as shown e.g. for E. coli .
Methanol regulated promoters
Methylotrophic yeasts such as P. pastoris (syn. Komagataella sp.) have gained great interest as production hosts for recombinant proteins  and more recently also as platform for metabolite production . Both applications require promoter collections of different strength for metabolic and cell engineering to enable and enhance productivity. Promoter libraries were developed based on mutating transcription factor binding sites , or by random mutagenesis . Strong constitutive and regulated promoters were identified by transcriptomics studies [12, 13]. Delic et al.  described a collection of native regulated promoters of different strength with the main aim of providing repressible promoters for gene knockdown studies. Synthetic core promoters represent a source for transcriptional initiators at different strength, however with the loss of regulatory features [1, 15].
Methanol regulated genes of P. pastoris as a source of regulated promoters
Ranked expression level (methanol)a
Co-regulation: 1 = with A/D/F; 2 = with A; 3 = with D/F; 4 = up at glucose limitc
Genome scale transcriptomic studies are a valuable source of information on native promoters and have been successfully used to identify promoters of different strength and desired regulatory behavior. Well defined promoters are core elements of synthetic biology part collections. The collection of P. pastoris promoters presented here, and others analyzed in the cited references can serve as a basis for setting up a P. pastoris promoter collection. Promoters with different regulatory strength are crucial elements of toolboxes for cell and metabolic engineering. In addition, they can be directly employed for gene expression to produce heterologous proteins or metabolites in yeasts.
All authors contributed equally to this commentary. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Redden H, Alper HS. The development and characterization of synthetic minimal yeast promoters. Nat Commun. 2015;6:7810.View ArticleGoogle Scholar
- Zhang X, Liu J, Yu X, Wang F, Yi L, Li Z, Liu Y, Ma L. High-level expression of human arginase I in Pichia pastoris and its immobilization on chitosan to produce L-ornithine. BMC Biotechnol. 2015;15:66.View ArticleGoogle Scholar
- Røkke G, Korvald E, Pahr J, Oyås O, Lale R. BioBrick assembly standards and techniques and associated software tools. Methods Mol Biol. 2014;1116:1–24.View ArticleGoogle Scholar
- Yadav VG, De Mey M, Lim CG, Ajikumar PK, Stephanopoulos G. The future of metabolic engineering and synthetic biology: towards a systematic practice. Metab Eng. 2012;14:233–41.View ArticleGoogle Scholar
- Keasling JD. Manufacturing molecules through metabolic engineering. Science. 2010;330:1355–8.View ArticleGoogle Scholar
- Kelly JR, Rubin AJ, Davis JH, Ajo-Franklin CM, Cumbers J, Czar MJ, de Mora K, Glieberman AL, Monie DD, Endy D. Measuring the activity of BioBrick promoters using an in vivo reference standard. J Biol Eng. 2009;3:4.View ArticleGoogle Scholar
- Blumhoff M, Steiger MG, Marx H, Mattanovich D, Sauer M. Six novel constitutive promoters for metabolic engineering of Aspergillus niger. Appl Microbiol Biotechnol. 2013;97:259–67.View ArticleGoogle Scholar
- Qin X, Qian J, Yao G, Zhuang Y, Zhang S, Chu J. GAP promoter library for fine-tuning of gene expression in Pichia pastoris. Appl Environ Microbiol. 2011;77:3600–8.View ArticleGoogle Scholar
- Balzer S, Kucharova V, Megerle J, Lale R, Brautaset T, Valla S. A comparative analysis of the properties of regulated promoter systems commonly used for recombinant gene expression in Escherichia coli. Microb Cell Fact. 2013;12:26.View ArticleGoogle Scholar
- Gasser B, Prielhofer R, Marx H, Maurer M, Nocon J, Steiger M, Puxbaum V, Sauer M, Mattanovich D. Pichia pastoris: protein production host and model organism for biomedical research. Future Microbiol. 2013;8:191–208.View ArticleGoogle Scholar
- Hartner F, Ruth C, Langenegger D, Johnson S, Hyka P, Lin-Cereghino G, Lin-Cereghino J, Kovar K, Cregg J, Glieder A. Promoter library designed for fine-tuned gene expression in Pichia pastoris. Nucleic Acids Res. 2008;36:e76.View ArticleGoogle Scholar
- Prielhofer R, Maurer M, Klein J, Wenger J, Kiziak C, Gasser B, Mattanovich D. Induction without methanol: novel regulated promoters enable high-level expression in Pichia pastoris. Microb Cell Fact. 2013;12:5.View ArticleGoogle Scholar
- Stadlmayr G, Mecklenbräuker A, Rothmüller M, Maurer M, Sauer M, Mattanovich D, Gasser B. Identification and characterisation of novel Pichia pastoris promoters for heterologous protein production. J Biotechnol. 2010;150:519–29.View ArticleGoogle Scholar
- Delic M, Mattanovich D, Gasser B. Repressible promoters—a novel tool to generate conditional mutants in Pichia pastoris. Microb Cell Fact. 2013;12:6.View ArticleGoogle Scholar
- Vogl T, Ruth C, Pitzer J, Kickenweiz T, Glieder A. Synthetic core promoters for Pichia pastoris. ACS Synth Biol. 2014;3:188–91.View ArticleGoogle Scholar
- Russmayer H, Buchetics M, Gruber C, Valli M, Grillitsch K, Modarres G, Guerrasio R, Klavins K, Neubauer S, Drexler H, et al. Systems-level organization of yeast methylotrophic lifestyle. BMC Biol. 2015;13:80.View ArticleGoogle Scholar
- Prielhofer R, Cartwright SP, Graf AB, Valli M, Bill RM, Mattanovich D, Gasser B. Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level. BMC Genomics. 2015;16:167.View ArticleGoogle Scholar
- Rebnegger C, Graf AB, Valli M, Steiger MG, Gasser B, Maurer M, Mattanovich D. In Pichia pastoris, growth rate regulates protein synthesis and secretion, mating and stress response. Biotechnol J. 2014;9:511–25.View ArticleGoogle Scholar
- Küberl A, Schneider J, Thallinger GG, Anderl I, Wibberg D, Hajek T, Jaenicke S, Brinkrolf K, Goesmann A, Szczepanowski R, et al. High-quality genome sequence of Pichia pastoris CBS7435. J Biotechnol. 2011;154:312–20.View ArticleGoogle Scholar
- Mattanovich D, Graf A, Stadlmann J, Dragosits M, Redl A, Maurer M, Kleinheinz M, Sauer M, Altmann F, Gasser B. Genome, secretome and glucose transport highlight unique features of the protein production host Pichia pastoris. Microb Cell Fact. 2009;8:29.View ArticleGoogle Scholar
- Li J, Wei H, Zhao PX. DeGNServer: deciphering genome-scale gene networks through high performance reverse engineering analysis. Biomed Res Int. 2013;2013:856325.Google Scholar