Open Access

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

Microbial Cell Factories201514:196

https://doi.org/10.1186/s12934-015-0387-1

Received: 19 November 2015

Accepted: 25 November 2015

Published: 2 December 2015

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.

Declarations

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.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Department of Biotechnology, BOKU-University of Natural Resources and Life Sciences Vienna
(2)
Austrian Centre of Industrial Biotechnology (ACIB GmbH)

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Copyright

© Gasser et al. 2015

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