Difference between revisions of "Part:BBa K4805008"

 
 
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<partinfo>BBa_K4805008 short</partinfo>
  
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==description==
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ERG20 catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). ERG20_F96W, as a F96W mutant can help increase the supply of FPP. 
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BmeTC_Y167A, D373C refers to a variant of the Tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658) that contains two specific mutations: Y167A and D373C. In a cell-free system, the synergistic effect of BmeTC_Y167A, D373C and BmeTC can reach 53.26% higher activity than BmeTC_Y167A, D373C. This inspired us to attempt to co-expressed BmeTC and BmeTC_Y167A, D373C in S.cerevisiae to produce ambrein (Tsutomu S. et al, 2020). 
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It belongs to the terpenoids producing collection we create for the production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012. This collection can provide inspiration and efficient methods to utilize the MVA pathway in S.cerevisiae for other teams.
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==usage and biology==
 +
ERG20 catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). ERG20_F96W, as a F96W mutant can help increase the supply of FPP. 
 +
BmeTC_Y167A, D373C refers to a variant of the Tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658) that contains two specific mutations: Y167A and D373C. Although BmeTC can only catalyze the bicyclic structure at the end of squalene, its variant BmeTC_Y167A, D373C possess the ability of catalyzing both monocyclic and bicyclic structure, which can produce ambrein. Besides, it has a higher affinity for bicyclic substances(8α-hydroxypolypoda-13,17,21-triene) compared to squalene. In a cell-free system, the synergistic effect of BmeTC_Y167A, D373C and BmeTC can reach 53.26% higher activity than BmeTC_Y167A, D373C. This inspired us to attempt to co-expressed BmeTC and BmeTC_Y167A, D373C in S.cerevisiae to produce ambrein (Tsutomu S. et al, 2020). 
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==source==
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composite
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==characterization==
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We tried to insert BmeTC_Y167A, D373C and BmeTC into 106 site to construct our strain Lv2a-1(Figure 1A), and the successful integration can be seen in strain as it is shown in Figure 1C.
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<html>
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  <img src="https://static.igem.wiki/teams/4805/wiki/engineering-success/es9.png" style="width: 50vw;">
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  <p style="font-size: smaller; margin-top: 10px;">Figure 1. (A) Schematic strategy of BmeTC_Y167A, D373C and BmeTC integration into the site 106. (B) Schematic strategy of BmeTC_Y167A, D373C-Flexible Linker-BmeTC integration into the site 106. (C) These two genes were confirmed to be inserted into site 106 in the strain 6 of Lv2a-1.(D) These two genes were confirmed to be inserted into site 106 in the strain 2-8 of Lv2a-2.</p>
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</html>
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Unfortunately, we did not observe any peaks suspected of ambrein. But the reaction substrate, squalene, can be detected at 9.70-9.83 min. Compared with strain Lv1, the yield of squalene significantly decreased in the further modified strains. To be specific, the yield of squalene decrease up to 77.40% in strain Lv2a-1. Although there's no ambrein can be detected, the decrease of squalene might indicate the synthesis of some potential intermediates. We will optimize our testing method and the specificity of cyclase to reach the goal of ambrein production.
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<html>
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  <img src="https://static.igem.wiki/teams/4805/wiki/engineering-success/es10.png" style="width: 50vw;">
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  <p style="font-size: smaller; margin-top: 10px;">Figure 2. (A) Analysis of ambrein and squalene, accumulated in strain Lv1, Lv2a-1 and Lv2a-2 by GC-MS. (B) The yield comparison of squalene in different strains based on GC-MS. </p>
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</html>
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==sequencing and features==
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<partinfo>BBa_K4805008 SequenceAndFeatures</partinfo>
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==reference==
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Yamabe, Y., Kawagoe, Y., Okuno, K., Inoue, M., Chikaoka, K., Ueda, D., Tajima, Y., Yamada, T. K., Kakihara, Y., Hara, T., & Sato, T. (2020). Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein. Scientific Reports, 10(1), 19643. https://doi.org/10.1038/s41598-020-76624-y

Latest revision as of 14:00, 12 October 2023

pTDH3-BmeTC_Y167A,D373C-tTDH1-pTEF2-ERG20_F96W-tSSA1-pPGK1-BmeTC-tPGK1

description

ERG20 catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). ERG20_F96W, as a F96W mutant can help increase the supply of FPP. BmeTC_Y167A, D373C refers to a variant of the Tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658) that contains two specific mutations: Y167A and D373C. In a cell-free system, the synergistic effect of BmeTC_Y167A, D373C and BmeTC can reach 53.26% higher activity than BmeTC_Y167A, D373C. This inspired us to attempt to co-expressed BmeTC and BmeTC_Y167A, D373C in S.cerevisiae to produce ambrein (Tsutomu S. et al, 2020). It belongs to the terpenoids producing collection we create for the production of santalol and ambrein in S. cerevisiae, which includes BBa_K4805000-BBa_K4805012. This collection can provide inspiration and efficient methods to utilize the MVA pathway in S.cerevisiae for other teams.

usage and biology

ERG20 catalyzes the sequential formation of geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). ERG20_F96W, as a F96W mutant can help increase the supply of FPP. BmeTC_Y167A, D373C refers to a variant of the Tetraprenyl-beta-curcumene Cyclase from Bacillus megaterium (Genbank Accession: CP001982.1, 2130781–2132658) that contains two specific mutations: Y167A and D373C. Although BmeTC can only catalyze the bicyclic structure at the end of squalene, its variant BmeTC_Y167A, D373C possess the ability of catalyzing both monocyclic and bicyclic structure, which can produce ambrein. Besides, it has a higher affinity for bicyclic substances(8α-hydroxypolypoda-13,17,21-triene) compared to squalene. In a cell-free system, the synergistic effect of BmeTC_Y167A, D373C and BmeTC can reach 53.26% higher activity than BmeTC_Y167A, D373C. This inspired us to attempt to co-expressed BmeTC and BmeTC_Y167A, D373C in S.cerevisiae to produce ambrein (Tsutomu S. et al, 2020).

source

composite

characterization

We tried to insert BmeTC_Y167A, D373C and BmeTC into 106 site to construct our strain Lv2a-1(Figure 1A), and the successful integration can be seen in strain as it is shown in Figure 1C.

Figure 1. (A) Schematic strategy of BmeTC_Y167A, D373C and BmeTC integration into the site 106. (B) Schematic strategy of BmeTC_Y167A, D373C-Flexible Linker-BmeTC integration into the site 106. (C) These two genes were confirmed to be inserted into site 106 in the strain 6 of Lv2a-1.(D) These two genes were confirmed to be inserted into site 106 in the strain 2-8 of Lv2a-2.

Unfortunately, we did not observe any peaks suspected of ambrein. But the reaction substrate, squalene, can be detected at 9.70-9.83 min. Compared with strain Lv1, the yield of squalene significantly decreased in the further modified strains. To be specific, the yield of squalene decrease up to 77.40% in strain Lv2a-1. Although there's no ambrein can be detected, the decrease of squalene might indicate the synthesis of some potential intermediates. We will optimize our testing method and the specificity of cyclase to reach the goal of ambrein production.

Figure 2. (A) Analysis of ambrein and squalene, accumulated in strain Lv1, Lv2a-1 and Lv2a-2 by GC-MS. (B) The yield comparison of squalene in different strains based on GC-MS.

sequencing and features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1357
    Illegal NheI site found at 6331
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 4112
    Illegal BglII site found at 4173
    Illegal BglII site found at 5638
    Illegal BamHI site found at 760
    Illegal BamHI site found at 5734
    Illegal XhoI site found at 2567
    Illegal XhoI site found at 4289
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 3828
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 4275
    Illegal BsaI site found at 4536
    Illegal BsaI.rc site found at 4525
    Illegal BsaI.rc site found at 5648

reference

Yamabe, Y., Kawagoe, Y., Okuno, K., Inoue, M., Chikaoka, K., Ueda, D., Tajima, Y., Yamada, T. K., Kakihara, Y., Hara, T., & Sato, T. (2020). Construction of an artificial system for ambrein biosynthesis and investigation of some biological activities of ambrein. Scientific Reports, 10(1), 19643. https://doi.org/10.1038/s41598-020-76624-y