Composite

Part:BBa_K5175037:Design

Designed by: Xihong Zeng   Group: iGEM24_HUST-China   (2024-10-01)


T7 promoter- tphA2-tphA3 -tphA1-T7 terminator- T7 promoter - tphB-tpaK-T7 terminator


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal XbaI site found at 912
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 1951
    Illegal PstI site found at 4853
    Illegal PstI site found at 5142
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 2826
    Illegal NheI site found at 2891
    Illegal NheI site found at 2947
    Illegal NheI site found at 5409
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 1951
    Illegal PstI site found at 4853
    Illegal PstI site found at 5142
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 426
    Illegal BglII site found at 3189
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 912
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 1951
    Illegal PstI site found at 4853
    Illegal PstI site found at 5142
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 912
    Illegal PstI site found at 949
    Illegal PstI site found at 982
    Illegal PstI site found at 1951
    Illegal PstI site found at 4853
    Illegal PstI site found at 5142
    Illegal NgoMIV site found at 477
    Illegal NgoMIV site found at 1516
    Illegal NgoMIV site found at 2266
    Illegal NgoMIV site found at 3236
    Illegal NgoMIV site found at 3911
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphA2, tphA3, tphA1, tphB, tpaK. It is responsible for converting TPA to 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD), oxidising the diol moiety (two hydroxyl groups) of DCD to a keto group to result in the production of PCA and transporting TPA. TphA2, TphA3 constitute the large and small subunits of the TPADO oxidase component responsible for binding to the TPA substrate and catalysing the oxygenation reaction in the active site. TphA1 contains a [2Fe -2S] iron-sulfur cluster and a flavin adenine dinucleotide (FAD) binding site that transfers electrons from an electron donor (e.g., NADPH) to the oxidised component of TPADO. TphB is a dehydrogenase that oxidises the diol moiety (two hydroxyl groups) of DCD to a keto group, resulting in the production of PCA. TpaK is the TPA transporter protein.



Source

tphA2, tphA1, tphA3, tphB are from Comamonas sp.E6. tpaK is from Rhodococcus erythropolis

References

[1] NIKEL P I, DE LORENZO V. Pseudomonas putida as a functional chassis for industrial biocatalysis: From native biochemistry to trans-metabolism [J]. Metab Eng, 2018, 50: 142-55.

[2] HARWOOD C S, PARALES R E. THE β-KETOADIPATE PATHWAY AND THE BIOLOGY OF SELF-IDENTITY [J]. Annual Review of Microbiology, 1996, 50(Volume 50, 1996): 553-90.

[3] SASOH M, MASAI E, ISHIBASHI S, et al. Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6 [J]. Applied and Environmental Microbiology, 2006, 72(3): 1825-32.

[4] KINCANNON W M, ZAHN M, CLARE R, et al. Biochemical and structural characterization of an aromatic ring-hydroxylating dioxygenase for terephthalic acid catabolism [J]. Proc Natl Acad Sci U S A, 2022, 119(13): e2121426119.

[5] HOSAKA M, KAMIMURA N, TORIBAMI S, et al. Novel tripartite aromatic acid transporter essential for terephthalate uptake in Comamonas sp. strain E6 [J]. Appl Environ Microbiol, 2013, 79(19): 6148-55.