Difference between revisions of "Part:BBa K4365000"

(Biology and Usage)
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===Biology and Usage===
 
===Biology and Usage===
 
====Factor-Induced Gene 1 and pheromone induced mating pathway====
 
====Factor-Induced Gene 1 and pheromone induced mating pathway====
 
<b>Factor-Induced Gene 1 (FIG1)</b> is a pheromone-responsive gene whose expression is activated by a transcription factor Ste12 <html><a href="https://doi.org/10.1371/journal.pone.0056544">[1]</a></html>. Ste12 is one of transcriptional factors regulated by pheromone induced mating pathway. The pathway is activated when the mating pheromone (alpha mating factor in <i>Saccharomyces cerevisiae</i>) 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 <html><a href="https://doi.org/10.1128/EC.00333-09">[2]</a></html>.
 
<b>Factor-Induced Gene 1 (FIG1)</b> is a pheromone-responsive gene whose expression is activated by a transcription factor Ste12 <html><a href="https://doi.org/10.1371/journal.pone.0056544">[1]</a></html>. Ste12 is one of transcriptional factors regulated by pheromone induced mating pathway. The pathway is activated when the mating pheromone (alpha mating factor in <i>Saccharomyces cerevisiae</i>) 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 <html><a href="https://doi.org/10.1128/EC.00333-09">[2]</a></html>.
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====FIG1 promoter and its advantages for synthethic biology in yeast====
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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 <i>S. cerevisiae</i> and has been used to improve heterologous protein yield <html><a href="https://doi.org/10.1016/j.meteno.2016.05.001">[3]</a> or to control cell-cell communication in yeast cultures <a href = "https://doi.org/10.1186/s13036-015-0011-2">[4]</a>. FIG1 promoter is strictly regulated by a well-understood signaling cascade, which avoids the cross-activation of other pathways <a href="https://doi.org/10.1016/j.meteno.2016.05.001">[3]</a></html>.
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[[File:BBa_K4365000_Fig1_fig1.png|thumb|<b>Figure 1.</b> 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.]]
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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 <i>S. cerevisiae</i>. 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 <html><a href="https://doi.org/10.1016/j.meteno.2016.05.001">[3]</a></html>.
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====Use of yeast pheromone response for protein production====
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*[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  
 
*[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  
  
====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. The FIG1 promoter is a great device for synthetic biology applications. It can be used to engineer productive stationary-phase systems in <i>S. cerevisiae</i> and has been used to improve heterologous protein yield [3] or for the controlled cell-cell communication in yeast cultures [4].
 
  
[[File:BBa_K4365000_Fig1_fig1.png|thumb|<b>Figure 1.</b> 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, in addition to activating the expression of a gene of interest, 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 [3].
 
 
 
Moreover, the *FIG1* promoter is strictly regulated by a well-understood signaling cascade, which avoids the cross-activation of other pathways and has enabled the construction and fine-tuning of a multitude of synthetic regulatory circuits [3].
 
 
====Use of yeast pheromone response for protein production====
 
 
The *S. cerevisiae* mating system has become a cornerstone of eukaryotic synthetic biology [7]. The key regulator of the mating system is the alpha mating factor pheromone, functioning as an inductor of the pheromone response.
 
The *S. cerevisiae* mating system has become a cornerstone of eukaryotic synthetic biology [7]. The key regulator of the mating system is the alpha mating factor pheromone, functioning as an inductor of the pheromone response.
  

Revision as of 19:23, 10 October 2022


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].

Use of yeast pheromone response for protein production

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


The *S. cerevisiae* mating system has become a cornerstone of eukaryotic synthetic biology [7]. The key regulator of the mating system is the alpha mating factor pheromone, functioning as an inductor of the pheromone response.


When large-scale production by means of engineered organisms is conducted in parallel with biomass growth, the organism uses carbon and nitrogen for both growth and synthesis of the desired bioproduct. Biomass accumulation is essential to achieve the productivity required for commercial processes; yet, excess biomass limits yields because of the aforementioned competition for resource allocation and the accumulation of toxic intermediates.


  • S. cerevisiae* undergoes an exponential growth phase where carbon and nitrogen resources are consumed until they limit further biomass accumulation. During this phase, 90% of the energy is directed toward ribosome biogenesis [8]. After the growth phase, the cells enter a ‘stationary phase’, which is characterized by the induction of survival mechanisms and a drastic reduction in the rate of protein synthesis.


The alpha mating factor pheromone induces the pheromone-response in *S. cerevisiae*, which leads to the arrest of growth. Moreover, the pheromone response triggered by the alpha mating factor has been shown to maintain an active metabolism in *S. cerevisiae*. This stationary phase phenotype induced by the pheromone response has the potential to improve heterologous protein yield [4].


This means that, after a rapid growth phase where biomass accumulates to a sufficient level to enable high productivity, the alpha mating factor can be easily added by the operator to switch to a metabolically active stationary phase. This phase can be maintained in the presence of high concentrations of resources such as carbon and nitrogen. With cells metabolically active but not growing and dividing, a greater proportion of resources can therefore be directed towards target metabolites.


In addition to bioprocess engineering, the pheromone response has been successfully adopted into synthetic biology applications in the field of artificial cell-cell communication systems to directly activate and finetune a desired function in yeast cells [6].


For example, a quorum sensing circuit was developed in yeast by utilizing the alpha factor pheromone [9]. In this system, yeast populations were engineered to produce and respond to extracellular alpha mating factor pheromone by expressing GFP in a population-density-dependent manner.


Moreover, the pheromone response is a well characterized pathway that can engineered to allow couple positive or negative regulators to of the pheromone response cascade to modify sensitivity or timing of the cellular response of yeast-based sensing systems [10].


A further example of the potential of the pheromone response and the induction by the alpha mating factor pheromone is illustrated by the development of synthetic inter-species communication systems between *S. cerevisiae* and *S. pombe* (Figure 4) [11]. *S. cerevisiae* cells were engineered to secrete the P-factor pheromone of *S. pombe*, whereas *S. pombe* cells were engineered to secrete the alpha mating factor pheromone of *S. cerevisiae*. Co-cultures of the two engineered species were shown to be able to communicate using their respective pheromones.

Characterization of FIG1 promoter