Part:BBa_K5477006
pPDC1 - Lex6Op - pENO1 bidirectional synthetic promoter with Lex6Op
The pPDC1 core promoter drives the expression of the PDC1 gene, one of the three pyruvate decarboxylase isozymes in Saccharomyces cerevisiae. PDC1 plays a critical role in alcoholic fermentation, catalyzing the conversion of pyruvate to acetaldehyde, a key step in ethanol production. This enzyme is also involved in amino acid catabolism and is regulated by glucose, ethanol, and autoregulatory mechanisms (1) (3) (5).
The pENO1 core promoter drives the expression of ENO1, which encodes Enolase I, a key enzyme in glycolysis that catalyzes the conversion of 2-phosphoglycerate to phosphoenolpyruvate (2) (4).
In our system, we used the core region of the pPDC1 promoter with an emphasis on glucose-dependent regulation and the core region of pENO1. In our Lex6Op bidirectional system, pPDC1 serves as a promoter, working in tandem with another promoter (pENO1) to drive the expression of two different genes in opposite orientations (6). The Lex6Op operator cassette, composed of six tandem LexA operator sites, regulates the bidirectional activation of both promoters (6). When a LexA fusion protein (such as LexA-ERα, LexA-mERα or LexA-ERRγ) binds to the Lex6Op sequence, it activates transcription through both pENO1 and pPDC1, enabling the coordinated expression of two genes simultaneously.
In our system, we combined our receptor module and reporter module. On top of that, this composite part is a detoxification module which was integrated. These three modules comprise our SUPERMOM. Upon presence of BPA, the contaminant will bind to the chimeric activator LexA-ERα. The chimeric activator will bind to the Lex6Op in the reporter module triggering the transcription of NanoLuc and also the detoxification module, since it contains the Lex6Op between the two promoters of UGT2B15 and UDPD.
| Constructs | Module | Composite Part Name |
|---|---|---|
| LexA-ERα | Receptor module | Composite part Number |
| Lex6Op-pLEU2-NanoLuc | Reporter Module | Composite part Number |
| UDPD-pPDC1-Lex6Op-pENO1-UGT2B15 | Detoxification Module | Composite part Number |
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
References
1. Boer VM, de Winde JH, Pronk JT, Piper MD. The genome-wide transcriptional responses of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. J Biol Chem. 2003 Jan 31;278(5):3265-74. doi: 10.1074/jbc.M209759200. Epub 2002 Oct 31. PMID: 12414795.
2. Entian KD, Meurer B, Köhler H, Mann KH, Mecke D. Studies on the regulation of enolases and compartmentation of cytosolic enzymes in Saccharomyces cerevisiae. Biochim Biophys Acta. 1987 Feb 20;923(2):214-21. doi: 10.1016/0304-4165(87)90006-7. PMID: 3545298.
3. Kellermann E, Seeboth PG, Hollenberg CP. Analysis of the primary structure and promoter function of a pyruvate decarboxylase gene (PDC1) from Saccharomyces cerevisiae. Nucleic Acids Res. 1986 Nov 25;14(22):8963-77. doi: 10.1093/nar/14.22.8963. PMID: 3537965; PMCID: PMC311923.
4. Klein CJL, Olsson L, Nielsen J. Glucose control in Saccharomyces cerevisiae: the role of Mig1 in metabolic functions. Microbiology (Reading). 1998 Jan;144 ( Pt 1):13-24. doi: 10.1099/00221287-144-1-13. PMID: 9467897.
5. Pronk JT, Yde Steensma H, Van Dijken JP. Pyruvate metabolism in Saccharomyces cerevisiae. Yeast. 1996 Dec;12(16):1607-33. doi: 10.1002/(sici)1097-0061(199612)12:16<1607::aid-yea70>3.0.co;2-4. PMID: 9123965.
6. Rantasalo A, Czeizler E, Virtanen R, et al. Synthetic Transcription Amplifier System for Orthogonal Control of Gene Expression in Saccharomyces cerevisiae. PLoS One. 2016;11(2):e0148320. Published 2016 Feb 22. doi:10.1371/journal.pone.0148320
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