RESUMEN
Herein, we report a short semisynthesis of the potent transient receptor potential canonical (TRPC) channel agonist englerin A (EA) and the related guaianes oxyphyllol and orientalol E. The guaia-6,10(14)-diene starting material was systematically engineered in Escherichia coli and Saccharomyces cerevisiae using the CRISPR/Cas9 system and was produced with high titers. The potentially scalable approach combines the advantages of synthetic biology and chemical synthesis providing an efficient and economical method for producing EA and analogues.
Asunto(s)
Ingeniería Metabólica , Plantas/química , Sesquiterpenos de Guayano/química , Sistemas CRISPR-Cas , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Sesquiterpenos de Guayano/síntesis químicaRESUMEN
Darwinolide, a recently identified marine natural product from the Antarctic sponge Dendrilla membranosa, was previously shown to exhibit promising activity against the biofilm phase of methicillin-resistant Staphylococcus aureus. Its challenging tetracyclic rearranged spongian diterpenoid structure links a trimethylcyclohexyl subunit to a seven-membered core with two fused tetrahydrofuran units. Herein, we describe the first synthesis of (+)-darwinolide, which features a convergent aldol fragment coupling, an Ireland-Claisen rearrangement, and an organocatalytic desymmetrization as the key steps. Our results provide a foundation for the development of novel antibiofilm-specific antibiotics.
Asunto(s)
Antibacterianos/síntesis química , Biopelículas/efectos de los fármacos , Diterpenos/síntesis química , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Diterpenos/química , Diterpenos/farmacología , Staphylococcus aureus Resistente a Meticilina/fisiología , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
Two sesquiterpene cyclases from Fusarium fujikuroi were expressed in Escherichia coli and purified. The first enzyme was inactive because of a critical mutation, but activity was restored by sequence correction through site-directed mutagenesis. The mutated enzyme and two naturally functional homologues from other fusaria converted farnesyl diphosphate into guaia-6,10(14)-diene. The second enzyme produced eremophilene. The absolute configuration of guaia-6,10(14)-diene was elucidated by enantioselective synthesis, while that of eremophilene was evident from the sign of its optical rotation and is opposite to that in plants but the same as in Sorangium cellulosum. The mechanisms of both terpene cyclases were studied with various (13) C- and (2) H-labelled FPP isotopomers.