Part:BBa_K4365000

FIG1 inducible promoter

The Factor-Induced Gene 1 (FIG1) is a pheromone-induced promoter in yeast that is activated by the alpha mating factor. The FIG1 promoter is a great device for synthetic biology applications aiming to engineer productive stationary-phase systems in S. cerevisiae. This is because the induction of the FIG1 promoter by the alpha mating factor, in addition to activating expression, leads to the arrest of growth and maintenance of active metabolism in S. cerevisiae. As a result, the synthesis of a product of interest is decoupled from population growth, and cellular resources, such as carbon and nitrogen, can be redirected from biomass production to the synthesis of the desired bioproduct. Moreover, the FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids cross-activation of other pathways and has enabled the construction and fine-tuning of a multitude of synthetic regulatory circuits.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 172
  
• 1000
  COMPATIBLE WITH RFC[1000]

Biology and Usage

Factor-Induced Gene 1 and pheromone induced mating pathway

Factor-Induced Gene 1 (FIG1) is a pheromone-responsive gene whose expression is activated by a transcription factor Ste12 [1]. Ste12 is one of transcriptional factors regulated by pheromone induced mating pathway. The pathway is activated when the mating pheromone (alpha mating factor in Saccharomyces cerevisiae) binds to its receptor (Ste2) which leads to signal transduction via MAP kinase cascade. This results in phosphorylation of Ste12 transcription factor and allows it to bind to PRE motif present in pheromone-responsive genes promoter [2].

FIG1 promoter and its advantages for synthethic biology in yeast

FIG1 promoter is a pheromone-induced promoter that is activated by the alpha mating factor. This promoter can be applied for engineering productive stationary-phase systems in S. cerevisiae and has been used to improve heterologous protein yield [3] or to control cell-cell communication in yeast cultures [4]. FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids the cross-activation of other pathways [3].

Figure 1. Induction of turboRFP expression driven by the FIG1 promoter by addition of 500 nM alpha mating factor pheromone into the growth medium. The image was taken under a UV lamp.

The induction of the FIG1 promoter by the alpha mating factor not only activates the expression of a gene of interest, but also stops the growth and maintenance of active metabolism in S. cerevisiae. As a result, the synthesis of a product of interest becomes independent of population growth. At the same time, cellular resources, such as carbon and nitrogen, can be redirected from biomass production to the synthesis of the desired bioproduct [3].

References

• [1] Pincus D, Ryan CJ, Smith RD, Brent R, Resnekov O. Assigning quantitative function to post-translational modifications reveals multiple sites of phosphorylation that tune yeast pheromone signaling output [published correction appears in PLoS One. 2013;8(6). doi: 10.1371/annotation/06dfa4e4-30f5-4d37-8559-0f2a9d11f0de]. PLoS One. 2013;8(3):e56544. doi:10.1371/journal.pone.0056544
• [2] Wong Sak Hoi J, Dumas B. Ste12 and Ste12-like proteins, fungal transcription factors regulating development and pathogenicity. Eukaryot Cell. 2010 Apr;9(4):480-5. doi: 10.1128/EC.00333-09. Epub 2010 Feb 5. PMID: 20139240; PMCID: PMC2863410.
• [3] Thomas C. Williams, Bingyin Peng, Claudia E. Vickers, Lars K. Nielsen, The Saccharomyces cerevisiae pheromone-response is a metabolically active stationary phase for bio-production, Metabolic Engineering Communications, Volume 3, 2016, Pages 142-152, ISSN 2214-0301, https://doi.org/10.1016/j.meteno.2016.05.001.
• [4] Hennig, S., Rödel, G. & Ostermann, K. Artificial cell-cell communication as an emerging tool in synthetic biology applications. J Biol Eng 9, 13 (2015). https://doi.org/10.1186/s13036-015-0011-2.

Characterization of FIG1 promoter

Pheromone response induction and activation of the FIG1 promoter in Δfar1 Δbar1 strain

Figure 2. Growth curves of Δfar1Δbar1 S. cerevisiae cells after induction with the 500 nM of alpha mating factor pheromone.

To characterize the *FIG1* promoter and the pheromone response it induces we conducted a series of tests. We first verified the effect of the alpha mating factor pheromone on growth by culturing yeast cells in two conditions in flasks. BY4741 yeast strain with Δfar1 and Δbar1 deletions (Euroscarf, Acc. No. Y00000). These deletions prevent the degradation of the pheromone [12] and the cell cycle arrest [13]. The far1 deletion is beneficial to sensing systems as it avoids complete arrest of the cell cycle so that the strain does not get lost.

The two yeast cultures were grown overnight in MV medium at 30°C in a shaking incubator. The following day, a 20 mL culture was prepared by adjusting the overnight cultures to 1 OD and 500 nM alpha mating factor pheromone were added into the medium to activate the pheromone response.

The growth of the cultures was monitored over the course of several hours, starting with 12 hours after induction, by measuring their optical density using a spectrophotometer (Figure 5). Due to the size of the yeast cells, it was always necessary to perform a 1:10 dilution of the yeast culture before measuring the OD. The data was plotted using R-studio.

The growth curves show that the pheromone is still able to slow down growth despite the deletion of the FAR1 gene.

Characterization of the FIG1 promoter-driven expression

Figure 3. Experiment of visualization of turboRFP expression driven by the FIG1 promoter in induced (500 nM alpha mating factor pheromone) and uninduced state. The images were taken in bright field mode and with the TexasRed emission channel using the Keyence Biozero fluorescence microscope.