RhlABC + tags + scaffold
This part encodes rhlA,B,C as well as tags for each enzyme and scaffold to connect these three enzymes together, and it is the most essential plasmid in our protein scaffold project. We place the ORF of specific tags in front of each enzyme's ORF. These tags could bind with the corresponding domain on the scaffold and form a protein complex that can carry out the three needed reactions to synthesize rhamnolipid. For the detailed function of each enzyme, see BBa_K3745030.
The ratio of each domain on the scaffold was adopted from "Synthetic protein scaffolds provide modular control over metabolic flux", and we plan to fine-tune the ratio of the domain in the future.
Usage and Biology
In our project, the protein scaffold is used to increase the rhamnolipid production rate and reduce the content of toxic intermediates. To be specific, di-rhamnolipid is not catalyzed from its precursor R-3-hydroxydecanoyl-CoA in a simple addition reaction. By using a protein scaffold, however, a stepwise reaction that involves multiple enzymes, including RhlA, RhlB, RhlC, and its corresponding substrates, can happen. Originally, all these reactants are distributed separately throughout the cytoplasm, but after applying protein scaffolds, we can close the distance between a series of subsequent enzymes. In other words, an enzyme complex instead of 3 individual enzymes can be used now. Therefore, although the overall concentration remains the same, the enzyme complexes formed through protein scaffolds limit the area where the reaction takes place, concentrating the substrates, and thereby increasing the rate of successful collisions. Plus, since the enzyme complex is concentrated in one place after the modification, the shunt products, especially some of them are toxic to the organisms, produced during each step can be reduced. In this way, an efficient and safe approach of increasing rhamnolipids’ production can be applied.
Due to experimental issues, there is no current characterization of this part.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 2281
Illegal BamHI site found at 6640
Illegal XhoI site found at 6649
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 2149
Illegal NgoMIV site found at 2554
Illegal NgoMIV site found at 4123
Illegal NgoMIV site found at 5446
Illegal NgoMIV site found at 5761
Illegal NgoMIV site found at 6076
Illegal NgoMIV site found at 6391
Illegal AgeI site found at 3166
- 1000Illegal BsaI site found at 5676
Illegal BsaI site found at 5991
Illegal BsaI site found at 6306
Illegal BsaI site found at 6621
Illegal SapI.rc site found at 4262
Illegal SapI.rc site found at 5305