犹他游动放线菌SW1311 中阿库来菌素A 酰基酶在棘白菌素类抗真菌药物合成中的应用(英文).

棘白菌素类抗真菌药物米卡芬净和阿尼芬净的合成工艺中包括一个关键步骤：水解除去FR901379分子和棘白菌素B（echinocandin B, ECB）分子的脂肪酸侧链，形成环状的6元多肽核心。FR901379和ECB的水解可以分别被FR901379酰基酶和阿库来菌素A酰基酶（aculeacin A acylase, AAC）催化，因此，FR901379酰基酶和AAC的发掘、表征和生产在米卡芬净和阿尼芬净的工业生产上具有重要的应用价值。本研究首先筛选到了犹他游动放线菌SW1311，发现该菌株发酵液具有酰基酶活性，并摸索了不同发酵条件对酰基酶活性的影响。然后将犹他游动放线菌SW1311中的AAC 基因克隆到改造过的质粒载体pIJ8660 中，并将该质粒转化到天蓝色链霉菌（Streptomyces coelicolor）A3(2)中对AAC 基因进行高表达，得到重组菌株sSCO-AAC。最后将sSCO-AAC生产的AAC活性和所需培养时间与犹他游动放线菌SW1311进行比较，表征了sSCO-AAC的发酵液水解FR901379的反应。结果表明，犹他游动放线菌SW1311中的酰基酶具有水解FR901379和青霉素V的酰基活性。用重组菌株sSCO-AAC生产的AAC活性比犹他游动放线菌SW1311的高4.6倍，且该重组菌株所需的培养时间比犹他游动放线菌SW1311缩短了30%。该结果不仅将ACC的应用范围从阿尼芬净合成拓宽到了米卡芬净合成，而且还揭示出天蓝色链霉菌A3(2)可以作为一个良好的AAC表达菌株。本研究对阿尼芬净和米卡芬净的工业化生产具有潜在的应用价值。 通讯作者: JIANG Hui(http://orcid.org/0000-0002-4534-1322);ZHANG Xiaosheng (http://orcid.org/0000-0002-4379-6497) E-mail: jianghuisioc@zju.edu.cn;sid@zju.edu.cn
Abstract: Echinocandins are the first class of antifungals to target fungal cell wall. Two antifungals, including micafungin and anidulafungin, have been widely used in clinic. The synthesis of them includes an essential process: hydrolytic removal of fatty acyl side chains from FR901379 and echinocandin B (ECB) to generate cyclic hexapeptide nuclei. In this study, Actinoplanes utahensis SW1311 with acylase activity was isolated for both FR901379 and penicillin V. Then a recombinant Streptomyces coelicolor A3(2) harboring aculeacin A acylase (AAC) gene from A. utahensis SW1311 was constructed. The result showed that the AAC activity produced by the recombinant strain was 4.6-fold higher than that of A. utahensis SW1311. Additionally, the fermentation time of the recombinant strain was 30% shorter than that of A. utahensis SW1311. The results not only provide a new application of AAC for micafungin synthesis but also identify a new suitable host for AAC gene.