Part:BBa_K5175040:Design
T7 promoter-tphA2-tphA3-tphB-tphA1-T7 terminator-T7 promoter-tpaK-T7 terminator
- 10INCOMPATIBLE 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 2938
Illegal PstI site found at 4853
Illegal PstI site found at 5142 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3813
Illegal NheI site found at 3878
Illegal NheI site found at 3934
Illegal NheI site found at 5409
Illegal PstI site found at 949
Illegal PstI site found at 982
Illegal PstI site found at 2938
Illegal PstI site found at 4853
Illegal PstI site found at 5142 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 426
Illegal BglII site found at 2027 - 23INCOMPATIBLE 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 2938
Illegal PstI site found at 4853
Illegal PstI site found at 5142 - 25INCOMPATIBLE 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 2938
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 2074
Illegal NgoMIV site found at 2749
Illegal NgoMIV site found at 3253 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
It is a composite component consisting of the T7 promoter, T7 terminator, target genes tphA2, tphA3, tphB, tphA1, 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, tphA3, tphB, tphA1 are fromComamonas sp.E6. tpaK is from Rhodococcus erythropolis RHA1.
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.