Part:BBa_K4701306

Synthetic tpaK-tphII operon (High transcription)

A synthetic operon designed to enable the use of terephthalic acid (TPA) as a carbon source by Pseudomonas putida. The design includes a TPA transporter from Rhodococcus jostii encoded by tpaK, followed by four genes tphA2_II, tphA3_II, tphB_II, tphA1_II from the catalytic tph operon of Comamonas sp. E6 [1]. The rough design is inspired by the wild-type operon in Comamonas sp E6, previously published research on TPA utilization by P. putida [2], and biophysical models [3-5].

TPA assimilation pathway

The catabolic tph operon found in Comamonas sp. E6 contains genes that are sufficient to enable TPA utilization. Briefly, TPA is metabolized into 1,2-dihydroxy-3,5-cyclohexadiene-1,4-dicarboxylic acid (DCD), by TPA 1,2-dioxygenase (TPADO) [1], which is a protein complex encoded by tphA1_IIA2_IIA3_II. DCD is further metabolized into protocatechuic acid (PCA) by a DCD dehydrogenase encoded by tphB_II. PCA is a common intermediate utilized by various organisms, and in P. putida, the pathway continues towards central metabolism via the PCA-3,4-dioxygenase pathway [6].

Figure 1: Sketch of the assimilation pathway. Structures for tpaK, tphB, and tphA1 are predicted by AlphaFold. The oxygenase subunit of TPADO formed by tphA2 and tphA3 is shown as a heterohexamer as determined from a crystal structure [7].

Cloning strategy

As the length of the synthetic operon is 5392 bp, it is too long to be synthesized in one go. For this reason, our team decided to attempt cloning using Gibson assembly. The operon is broken down into four fragments with 40 bp overlaps between them. The overall structure and fragments are shown in figure 2. For more information refer to the design page or our wiki.

Figure 2: Overview of the construct and correspondence to Gibson fragments used in assembly. See the design page for a description of each part and our wiki for the design process.

The operon can be cloned into various backbones as it acts as a standalone expression cassette. Our team aimed to clone the part into pSEVA231 linearized with PacI and SpeI.

Figure 3: Geneious plasmid map after simulating the Gibson assembly of BBa_K4701306 into pSEVA231 linearized by PacI and SpeI.

References

[1] Sasoh, M. et al. (2006) Characterization of the Terephthalate Degradation Genes of Comamonas sp. Strain E6. Applied and Environmental Microbiology. 72(3), 1825–1832. https://doi.org/10.1128/AEM.72.3.1825-1832.2006

[2] Werner, AZ. et al. (2021) Tandem chemical deconstruction and biological upcycling of poly(ethylene terephthalate) to β-ketoadipic acid by Pseudomonas putida KT2440. Metabolic Engineering. 67, 250–261. https://doi.org/10.1016/j.ymben.2021.07.005

[3] Cetnar, DP. Salis, HM. et al. (2021) Systematic Quantification of Sequence and Structural Determinants Controlling mRNA stability in Bacterial Operons. ACS Synthetic Biology. 10(2), 318–332. https://pubs.acs.org/doi/10.1021/acssynbio.0c00471

[4] Salis, HM. et al. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nature Biotechnology. 27(10), 946–950. https://doi.org/10.1038/nbt.1568

[5] Tian T, Salis HM. (2015) A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons. Nucleic Acids Research. 43(14), 7137–7151. https://doi.org/10.1093/nar/gkv635

[6] Salvador, M. et al. (2019) Microbial Genes for a Circular and Sustainable Bio-PET Economy. Genes. 10(5), 373. https://doi.org/10.3390/genes10050373

[7] Kincannon, WM. et al. (2022) Biochemical and structural characterization of an aromatic ring–hydroxylating dioxygenase for terephthalic acid catabolism. Proceedings of the National Academy of Sciences. 119(13). https://doi.org/10.1073/pnas.2121426119

Figures on this page were created with BioRender.com.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 5244
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 5343
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 5334
  Illegal SapI site found at 5291