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editorial
. 2015 Dec 2;14:196. doi: 10.1186/s12934-015-0387-1

Methanol regulated yeast promoters: production vehicles and toolbox for synthetic biology

Brigitte Gasser 1,2, Matthias G Steiger 1,2, Diethard Mattanovich 1,2,
PMCID: PMC4667464  PMID: 26627685

Abstract

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.

Keywords: Pichia pastoris, Komagataella, Promoter, Induction, Synthetic biology, Protein production

Background

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 [3]. Promoters are indispensable parts for synthetic biology approaches [4] and are needed for different expression strength in order to balance the expression levels in a synthetic pathway [5]. There are a plethora of studies which characterize, e.g. constitutive promoters of different strength for Escherichia coli [6], Aspergillus niger [7] or Pichia pastoris [8]. 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 [9].

Methanol regulated promoters

Methylotrophic yeasts such as P. pastoris (syn. Komagataella sp.) have gained great interest as production hosts for recombinant proteins [10] and more recently also as platform for metabolite production [2]. 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 [11], or by random mutagenesis [8]. Strong constitutive and regulated promoters were identified by transcriptomics studies [12, 13]. Delic et al. [14] 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].

A specific feature of methylotrophic yeasts is the carbon source dependent regulation of the genes involved in methanol metabolism. Recently we have redefined the methanol assimilation pathway of P. pastoris [16], a finding that was initially based on the identification of all genes that are upregulated on methanol as a substrate. These include hitherto unknown genes, controlled by promoters of a wide range of expression strength on methanol (Table 1). Beside different expression levels upon induction by methanol, these promoters feature a wide variety of induction degrees, defined as the ratio of expression levels in the induced state (presence of methanol) vs. the non-induced state (cells grown on glucose or glycerol). Some of these promoters are even deregulated on substrate limit without addition of methanol, illustrating a variety of regulation patterns which can be summarized by correlating the genes according to the similarity of their regulatory behavior in a plethora of different growth conditions, such as different carbon sources [17] or different growth rates, featuring different degrees of substrate limitation [18]. Thus they are allowing controllable expression of genes depending on the needs or growth conditions of the host cells.

Table 1.

Methanol regulated genes of P. pastoris as a source of regulated promoters

Ranked expression level (methanol)a Short name ORF nameb Co-regulation: 1 = with A/D/F; 2 = with A; 3 = with D/F; 4 = up at glucose limitc Methanol inductiond
1 DAS1 PP7435_Chr3-0352 1;4 Strong
2 AOX2 PP7435_Chr4-0863 2;4 Strong
3 AOX1 PP7435_Chr4-0130 1;4 Strong
4 DAS2 PP7435_Chr3-0350 3;4 Strong
5 FDH1 PP7435_Chr3-0238 1;4 Strong
6 PMP20 PP7435_Chr1-1351 Strong
7 THI11 PP7435_Chr4-0952 Weak
8 FLD PP7435_Chr3-0140 3 Intermediate
9 FBA1-2 PP7435_Chr1-0639 1 Strong
10 SHB17 PP7435_Chr2-0185 3 Intermediate
11 FGH1 PP7435_Chr3-0312 1 Intermediate
12 DAK2 PP7435_Chr3-0343 3 Intermediate
13 CTA1 PP7435_Chr2-0137 3 Weak
14 PMP47 PP7435_Chr3-1139 1 Strong
15 MPP1 PP7435_Chr3-0349 3 Weak
16 FBP1 PP7435_Chr3-0309 3 Weak
17 PIM1-2 PP7435_Chr1-0484 2 Weak
18 PAS_chr1-1_0037 PP7435_Chr1-0336 1 Strong
19 PAS_chr3_1071 PP7435_Chr3-0094 1 Strong
20 PEX11 PP7435_Chr2-0790 3;4 Intermediate
21 PEX13 PP7435_Chr2-0217 1 Weak
22 PAS_chr1-1_0343 PAS_Chr1-1_0343 4 Intermediate
23 PEX12 PP7435_Chr4-0200 1 Weak
24 INP1 PP7435_Chr4-0597 3 Weak
25 PEX6 PP7435_Chr1-0900 1 Weak
26 PEX17 PP7435_Chr4-0347 1 Weak
27 ATG37 PP7435_Chr4-0369 1 Weak
28 TAL1-2 PP7435_Chr2-0358 1 Intermediate
29 PEX5 PP7435_Chr2-0195 3 Intermediate
30 PEX2 PP7435_Chr3-1201 3 Weak
31 PAS_chr3_1020 PP7435_Chr3-0149 3 Strong
32 PEX1 PP7435_Chr3-0122 1 Weak
33 PEX26 PP7435_Chr4-0482 1 Weak
34 PEX10 PP7435_Chr1-1379 3 Weak
35 PEX14 PP7435_Chr4-0157 3 Weak
36 PAS_chr3_0408 PP7435_Chr3-0805 Intermediate
37 ARO7 PP7435_Chr4-0965 3 Weak
38 PEX8 PP7435_Chr1-1134 1 Weak
39 PAS_chr1-4_0459 PP7435_Chr1-1255 1 Intermediate
40 FAD1 PP7435_Chr1-0246 Intermediate
41 YLR177 W PP7435_Chr1-0659 3 Intermediate
42 PEX11C PP7435_Chr1-1331 3 Weak
43 ACS2 PP7435_Chr3-0810 Weak
44 PAS_chr3_0439 PAS_chr3_0439 2 Intermediate
45 RKI1-2 PP7435_Chr4-0797 3 Intermediate

aRelative gene expression levels were derived from signal intensities on DNA microarrays at methanol induction [16, 17] and ordered from highest to lowest

bORF names derived from published P. pastoris genome sequences [19, 20]

cThe gene correlation was calculated using transcriptomic datasets comprising 29 different conditions. The log2 fold change data was used to look for co-regulations in this data set. The data was processed via the DeGNServer to calculate Spearman´s rank correlation using a CLR-based Network and an association cut-off value of 3.8 [21]. Co-regulation was analyzed with three genes involved in methanol utilization: AOX1 (A), DAS1 (D), FBA1-2 (F). Up at glucose limit means that expression is deregulated in glucose limited culture conditions without methanol (data from [12])

dInduction on methanol was classified based on the transcriptional regulation patterns obtained by [16, 17] by comparing expression levels of cells grown on methanol to cells grown on glucose or glycerol

Conclusions

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.

Authors’ contributions

All authors contributed equally to this commentary. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Contributor Information

Brigitte Gasser, Email: brigitte.gasser@boku.ac.at.

Matthias G. Steiger, Email: matthias.steiger@boku.ac.at

Diethard Mattanovich, Phone: +43-1-47654-6569, Email: diethard.mattanovich@boku.ac.at.

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