碳霉素生物合成基因簇中细胞色素P450酶基因cbm2813的表征

Characterization of cbm2813 encoding the cytochrome P450 enzyme in the biosynthetic gene cluster of carbomycin

摘要:

碳霉素(carbomycin)是在耐热链霉菌中产生的16元大环内酯类抗生素，包括碳霉素A (carbomycin A, CA)和碳霉素B (carbomycin B, CB)这2种成分，CB在C12-C13位环氧化后形成CA。在carbomycin生物合成基因簇中，cbm2813基因编码细胞色素P450酶，推测其负责催化C12-C13位双键的环氧化。本研究旨在通过体内基因阻断和体外酶促反应鉴定carbomycin生物合成基因簇中细胞色素P450酶Cbm2813的功能和酶学特征。利用CRISPR-Cas9基因编辑系统构建cbm2813基因敲除菌株Δcbm2813，在Δcbm2813的发酵产物中只发现CB，而无CA；而cbm2813回复菌株又重新产生CA。在大肠杆菌中表达并纯化出Cbm2813，体外酶反应证实Cbm2813具有一定的底物特异性，能催化CB的C12-C13位进行环氧化生成CA，而不能催化交沙霉素、麦迪霉素、异戊酰螺旋霉素Ⅰ等其他结构类似的16元大环内酯类抗生素。酶学性质研究发现Cbm2813的最适pH为5.5，最适温度为36 ℃，米氏动力学研究表明Cbm2813的催化效率k_cat/K_m为4.39×10³L/(mol·s)。通过Cbm2813与CB的分子对接结果推测C9位羰基能与酶反应中心的Fe形成配位键从而固定底物，使其具有较强的底物选择性。本研究鉴定的细胞色素P450酶Cbm2813对于丰富P450氧化酶库具有重要意义，且能为深入解析carbomycin的生物合成过程奠定基础。

Abstract:

Carbomycin, a 16-membered macrolide antibiotic produced in Streptomyces thermotolerans, comprises two components, carbomycin A (CA) and carbomycin B (CB). CB is converted into CA through epoxidation of the C12-C13 double bond. The gene cbm2813, located in the biosynthetic gene cluster of carbomycin, encodes a cytochrome P450 enzyme considered to catalyze this epoxidation. In this study, the functional and enzymatic properties of the cytochrome P450 enzyme Cbm2813 in the carbomycin biosynthesis gene cluster were characterized by in vivo gene inactivation and in vitro enzymatic reactions. We employed the CRISPR-Cas9 system to delete cbm2813 and obtained the mutant Δcbm2813. The fermentation products of the mutant contained CB but not CA. Complementation of Δcbm2813 restored CA production. Cbm2813 was successfully expressed in Escherichia coli and then purified. In vitro enzyme assays confirmed that Cbm2813 specifically recognized CB but not structurally similar 16-membered macrolide antibiotics, such as josamycin, midecamycin, and isovalerylspiramycin I. Cbm2813 exhibited the maximal activity at pH 5.5 and 36 ℃, with the catalytic efficiency k_cat/K_m of 4.39×10³L/(mol·s). Molecular docking suggested that the C9 carbonyl group of CB coordinated with the heme iron in the active site of the enzyme, ensuring strict substrate specificity. This study expands the toolbox of characterized P450 enzymes and advances the understanding of carbomycin biosynthesis.