An in vitro platform for engineering and harnessing modular polyketide synthases
To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates...
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| Publicado no: | Nat Commun |
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| Main Authors: | , , , |
| Formato: | Artigo |
| Idioma: | Inglês |
| Publicado em: |
Nature Publishing Group UK
2020
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| Assuntos: | |
| Acesso em linha: | https://ncbi.nlm.nih.gov/pmc/articles/PMC6941969/ https://ncbi.nlm.nih.gov/pubmed/31900404 https://ncbi.nlm.nih.govhttp://dx.doi.org/10.1038/s41467-019-13811-0 |
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| Resumo: | To harness the synthetic power of modular polyketide synthases (PKSs), many aspects of their biochemistry must be elucidated. A robust platform to study these megadalton assembly lines has not yet been described. Here, we in vitro reconstitute the venemycin PKS, a short assembly line that generates an aromatic product. Incubating its polypeptides, VemG and VemH, with 3,5-dihydroxybenzoic acid, ATP, malonate, coenzyme A, and the malonyl-CoA ligase MatB, venemycin production can be monitored by HPLC and NMR. Multi-milligram quantities of venemycin are isolable from dialysis-based reactors without chromatography, and the enzymes can be recycled. Assembly line engineering is performed using pikromycin modules, with synthases designed using the updated module boundaries outperforming those using the traditional module boundaries by over an order of magnitude. Using combinations of VemG, VemH, and their engineered derivatives, as well as the alternate starter unit 3-hydroxybenzoic acid, a combinatorial library of six polyketide products is readily accessed. |
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