Difference between revisions of "Part:BBa K4273020"

 
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<partinfo>BBa_K4273020 short</partinfo>
 
<partinfo>BBa_K4273020 short</partinfo>
  
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Gene from cyanobacteria N. punctiforme and Nostoc linkia NIES-25 involved in biosynthesis of shinorine.
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Np5598 is a gene found in cyanobacteria Nostoc punctiforme that encodes for AGL whereas NlmysD encodes for AlaL in Nostoc linckia. AGL-AlaL allows 4-DG to be converted into the MAAs shinorine or porphyra-334. In our experiment, we found that AlaL encoded by NlmysD has a strong preference toward the amino acid threonine. Therefore, this part could efficiently produce the MAA porphyra-334 primarily, making it the first successful case of producing pure samples of porphyra-334. This part is within our part collection that allows efficiently production of MAAs in S. cerevisiae.
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Our part collection contains necessary genes to produce gadusol and the MAAs shinorine, porphyra-334, and palythine at a high rate. Xyl1, Xyl2, and Xyl3 are genes that allow S. cerevisiae to utilize xylose to produce S7P. DDGS and OMT converts S7P to the precursor of MAA, 4-deoxygadusol (4-DG). AGL converts 4-DG into M-glycine (MG). AlaL, by adding either serine or threonine, produces shinorine and porphyra-334, respectively. MysH could be added to the circuit of shinorine to produce palythine. S7P could also be converted into gadusol under the catalyzation of EEVS and M-Tox. In this part collection, we included multiple pathways and methods to increase the production of the upstream S7P and downstream MAAs.
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encoding  ATP-grasp ligase(AG-L)  that converts 4-deoxygadusol (4-DG) to mycosporine-glycine(MG) and D-Ala-D-Ala ligase(ALA-L)  that converts mycosporine-glycine(MG) to Shinorine.
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This part collection can provide inspiration and efficient methods to utilize the penta phosphate pathway or to produce other types of MAAs in S. cerevisiae for other teams.
  
 
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===Usage and Biology===
 
===Usage and Biology===
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We selected promoters pTDH3, pPGK1, and pTEF2 due to their stability expression in S. cerevisiae (Apel et. al., 2016). These promoters are shown to have stable and strong expression in YPD culture mediums. Among the three, pTDH3 has highest stability and strength, followed by pPGK1. For expression of AGL and AlaL enzymes, we used pTDH3 for AGL and pPGK1 for AlaL. In order to optimize our production, we inserted this part into the yeast’s genome at position 106, chromosome I (Apel et. al., 2016).
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===Experiment===
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Design and Absorbance of Nine AGL-AlaL Combinations
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We expressed AGL-AlaL genes first using plasmid vector and then through genome insertion. Due to the existence of multiple different types of AGL and AlaL with different efficiency and amino acid preference in nature, we selected ligases from three different marine organisms: Nostoc punctiform (Np5598 and Np5597), Nostoc linckia (NlmysC and NImysD) and Actinosynnema mirum (Am4257 and Am4256), and expected to create nine combinations of AGL-AlaL. We used Lee's yeast toolkit to produce Level1 plasmids containing only AGL or AlaL, and used Golden Gate assembly on the basis of Level1 to construct Level2 plasmid containing the 9 combinations of AGL and AlaL. Finally, we transformed these Level2 plasmids into SC.L3 strains, which are S. cerevisiae which could produce 4-DG efficiently from the addition of DDGS and OMT, to form strain SC.L5.
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[[t-links-china-figure8.png|thumb|right|300px|'''Figure 1.
  
 
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Revision as of 10:54, 12 October 2022


pTDH3-Np5598-tTDH1-pPGK1-NlmysD-tPGK1

Np5598 is a gene found in cyanobacteria Nostoc punctiforme that encodes for AGL whereas NlmysD encodes for AlaL in Nostoc linckia. AGL-AlaL allows 4-DG to be converted into the MAAs shinorine or porphyra-334. In our experiment, we found that AlaL encoded by NlmysD has a strong preference toward the amino acid threonine. Therefore, this part could efficiently produce the MAA porphyra-334 primarily, making it the first successful case of producing pure samples of porphyra-334. This part is within our part collection that allows efficiently production of MAAs in S. cerevisiae. Our part collection contains necessary genes to produce gadusol and the MAAs shinorine, porphyra-334, and palythine at a high rate. Xyl1, Xyl2, and Xyl3 are genes that allow S. cerevisiae to utilize xylose to produce S7P. DDGS and OMT converts S7P to the precursor of MAA, 4-deoxygadusol (4-DG). AGL converts 4-DG into M-glycine (MG). AlaL, by adding either serine or threonine, produces shinorine and porphyra-334, respectively. MysH could be added to the circuit of shinorine to produce palythine. S7P could also be converted into gadusol under the catalyzation of EEVS and M-Tox. In this part collection, we included multiple pathways and methods to increase the production of the upstream S7P and downstream MAAs. This part collection can provide inspiration and efficient methods to utilize the penta phosphate pathway or to produce other types of MAAs in S. cerevisiae for other teams.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 3423
    Illegal XhoI site found at 2075
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2321
    Illegal BsaI.rc site found at 3433