RESUMEN
Biosynthesis is an environmentally benign and renewable approach that can be used to produce a broad range of natural and, in some cases, new-to-nature products. However, biology lacks many of the reactions that are available to synthetic chemists, resulting in a narrower scope of accessible products when using biosynthesis rather than synthetic chemistry. A prime example of such chemistry is carbene-transfer reactions1. Although it was recently shown that carbene-transfer reactions can be performed in a cell and used for biosynthesis2,3, carbene donors and unnatural cofactors needed to be added exogenously and transported into cells to effect the desired reactions, precluding cost-effective scale-up of the biosynthesis process with these reactions. Here we report the access to a diazo ester carbene precursor by cellular metabolism and a microbial platform for introducing unnatural carbene-transfer reactions into biosynthesis. The α-diazoester azaserine was produced by expressing a biosynthetic gene cluster in Streptomyces albus. The intracellularly produced azaserine was used as a carbene donor to cyclopropanate another intracellularly produced molecule-styrene. The reaction was catalysed by engineered P450 mutants containing a native cofactor with excellent diastereoselectivity and a moderate yield. Our study establishes a scalable, microbial platform for conducting intracellular abiological carbene-transfer reactions to functionalize a range of natural and new-to-nature products and expands the scope of organic products that can be produced by cellular metabolism.
Asunto(s)
Azaserina , Azaserina/biosíntesis , Azaserina/química , Productos Biológicos/química , Productos Biológicos/metabolismo , Familia de Multigenes/genética , Estireno/química , Ciclopropanos/química , Coenzimas/química , Coenzimas/metabolismo , Biocatálisis , Sistema Enzimático del Citocromo P-450/genética , Sistema Enzimático del Citocromo P-450/metabolismoRESUMEN
The LL-D05139 complex, containing LL-D05139 beta and azaserine, was recovered from the fermentation filtrate of Glycomyces harbinensis (NRRL 15337). A chemically defined medium was developed which favored the production of LL-D05139 beta. Antibiotic LL-D05139 beta was isolated from the fermentation filtrate by adsorption on granular carbon and further purified by chromatography on microcrystalline cellulose. Acid hydrolysis of LL-D05139 beta gave one molar equivalent each of alanine and serine. Both amino acids were found to have the L-configuration by GC analysis on a chiral column and alanine was assigned to be the N-terminal amino acid by Edman degradation. This information coupled with IR, UV, 1H NMR, 13C NMR and MS spectral data allowed us to assign the structure of LL-D05139 beta as alanylazaserine. LL-D05139 beta demonstrated greater antibacterial and biochemical induction assay activities than azaserine. The two drugs showed similar antitumor activities.
