Coding

Part:BBa_K5313021:Design

Designed by: Jinyang Gu   Group: iGEM24_NEFU-China   (2024-09-28)


ndm B Q289A


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

Key considerations in the design of this section include ensuring proper expression and function of ndmB Q289A in heterogeneous hosts. Codon optimization may be necessary to ensure effective translation in systems such as E. coli. In addition, unlike wild-type ndmB, ndmB Q289A does not need to work with ndmA, and it can complete the two-step conversion of caffeine to 7-MX on its own.

To demethylate the position 1 and position 3 methyl groups of caffeine (1,3,7-trimethylxanthine) and convert it into the high-value compound 7-MX, which has potential applications, we designed a strategy to mutate the enzymes NdmA and NdmB. Literature indicates that the NdmA enzyme specifically recognizes caffeine and removes the position 1 methyl group, generating 3,7-dimethylxanthine, while the NdmB enzyme further removes the position 3 methyl group, producing 7-MX. However, since NdmA cannot directly generate 7-MX and NdmB cannot recognize caffeine, it is necessary to engineer the functions of these enzymes to achieve a one-step conversion of caffeine to 7-MX, thereby reducing reaction steps and the metabolic burden on the strain.

Figure 1 The overall process of mutation screening

Source

It is a mutant of the ndmB gene derived from Pseudomonas sp.CBB5, a bacterium isolated from soil and known for its ability to use caffeine as its sole source of carbon and nitrogen. This gene is part of the N-demethylase system and performs sequential demethylation along with other enzymes such as NdmC and NdmD. The N-demethylase pathway in Pseudomonas CBB5 is one of the few naturally occurring mechanisms of caffeine metabolism.

References

[1] Kim, J. H., Kim, B. H., Brooks, S., Kang, S. Y., Summers, R. M., & Song, H. K. (2019). Structural and Mechanistic Insights into Caffeine Degradation by the Bacterial N-Demethylase Complex. Journal of molecular biology, 431(19), 3647–3661. https://doi.org/10.1016/j.jmb.2019.08.004 [2] Liu J, Tian J, Perry C, Lukowski AL, Doukov TI, Narayan ARH, Bridwell-Rabb J. Design principles for site-selective hydroxylation by a Rieske oxygenase. Nat Commun. 2022 Jan 11;13(1):255. doi: 10.1038/s41467-021-27822-3 [3] Tian J, Liu J, Knapp M, Donnan PH, Boggs DG, Bridwell-Rabb J. Custom tuning of Rieske oxygenase reactivity. Nat Commun. 2023 Sep 20;14(1):5858. doi: 10.1038/s41467-023-41428-x