Computation-driven redesign of an NRPS-like carboxylic acid reductase improves activity and selectivity
Engineering nonribosomal peptide synthetases (NRPSs) has been a "holy grail" in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the "gate-keeper" adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, <i>Mab</i>CAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub>) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.
Authors
- Zhi‐Jun Zhang (ORCID: https://orcid.org/0000-0001-9836-0204)
- Ju‐Mou Li (ORCID: https://orcid.org/0000-0002-3023-0283)
- Frank Hollmann (ORCID: https://orcid.org/0000-0003-4821-756X)
- Y. J. Zhang
- Hui‐Lei Yu (ORCID: https://orcid.org/0000-0002-1925-3679)
- Qi Chen (ORCID: https://orcid.org/0000-0002-1287-2557)
- Kun Shi (ORCID: https://orcid.org/0000-0002-1830-9167)
- Jian‐He Xu (ORCID: https://orcid.org/0000-0003-4301-7630)
- Mu-Qiang Wang
Institutions
- East China University of Science and Technology (CN)
- Delft University of Technology (NL)
Publication Details
- Journal
- Science Advances
- Published
- 2024-11-29
- DOI
- https://doi.org/10.1126/sciadv.adp6775
- Citations
- 15
- Primary Topic
- Chemical Synthesis and Analysis
- Type
- article
- Field-Weighted Citation Impact
- 3.51
Funders
- East China University of Science and Technology
- National Key Research and Development Program of China