Abstract:

Myxobacteria are an important source for natural products. Sorangium cellulosum So0157-2 produces anticancer epothilone, and its genome of 14.78 Mb is the largest prokaryotic genome sequenced to date. Bioinformatic analysis indicated that the genome harbors 35 biosynthetic gene clusters (BGCs). In addition to the known epothilone BGC and another two terpene BGCs with 100% similarity to their predicted BGCs, there are 17 BGCs for polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), and PKS-NRPS hybrids, which mean this strain has great potential to produce novel natural products. However, limits on slow growth, difficult culture, and the absence of efficient genetic manipulation tools for So0157-2 impede the deep mining of its metabolic potential. Therefore, transferring its BGCs to a simple heterologous host and using heterologous expression strategy to achieve genome mining would be an effective way for discovering novel natural products produced by this strain. In this work, a PKS-NRPS hybrid BGC (BGC18) was directly cloned into an Escherichia coli expression vector and transferred into the heterologous host Schlegelella brevitalea DSM 7029. Fractionation was conducted by using repeated column chromatography (HPLC-MS) over a RP-C₁₈ column packing with silica gel, and three pure products were obtained, which were identified by extensive NMR analysis and the Marfey's method as new compounds: (1) cyclo(N-Me-L-Leu-L-Val), (2) cyclo(N-Me-L-Leu-L-Leu), and (3) cyclo(N-Me-L-Leu-L-Ile). Analysis of BGC18 reveals that the lack of thiolation domain in the PKS module might lead to the skipping of this PKS module, and only three cyclodipeptides were synthesized by the two NRPS modules, which might be a strategy for structural diversity in the bacterial NRPS-PKS BGCs. A NRPS-PKS hybrid BGC in S. cellulosum So0157-2 was successfully cloned, and expressed by using direct cloning and heterologous expression strategy with three corresponding products isolated and identified. This study lays a solid foundation for subsequent discovery of more active natural products in S. cellulosum So0157-2, and also provides theoretical guidance for mining secondary metabolites from other microbes which are difficult for culture.

Key words: heterologous expression, Sorangium cellulosum, genome mining, non-ribosomal peptide, diketopiperazines

摘要：

黏细菌是天然产物的重要来源。纤维堆囊菌So0157-2是抗癌药物埃博霉素的产生菌，并且是已知基因组最大的原核生物。生物信息学分析发现该菌一共含有35个次级代谢产物生物合成基因簇，除了已知的埃博霉素基因簇和其他两个萜类化合物生物合成基因簇与已知基因簇的相似度为100%之外，其他基因簇与已知化合物基因簇相似度均较低，其中包括17个聚酮合酶（polyketide synthase，PKS）、非核糖体肽合成酶（nonribosomal peptide synthetase，NRPS）及其杂合的基因簇。由于纤维堆囊菌So0157-2生长缓慢、培养困难且难以在本源菌中进行遗传改造。因此，将其生物合成基因簇转移到简单宿主中，利用异源表达策略是挖掘该菌中新颖天然产物的一个有效途径。本文利用直接克隆技术从纤维堆囊菌So0157-2基因组DNA中克隆了1个包含NRPS和PKS结构域的基因簇BGC18，将其转移至伯克氏菌DSM 7029中进行异源表达。通过液质联用分析，色谱柱靶向分离纯化，进而通过NMR结构鉴定和Marfey反应确定了3个化合物分别为Cyclo（N-Me-L-Leu-L-Val）（1）、Cyclo（N-Me-L-Leu-L-Leu）（2）、Cyclo（N-Me-L-Leu-L-Ile）（3）。化合物结构的多样性来源于第1个腺苷化结构域对底物识别的宽泛性（Val/Leu/Ile）。生物合成途径分析推测由于缺少硫醇化结构域导致PKS模块被跳过，从而只获得了NRPS指导合成的环二肽产物1～3，这可能是细菌中一种实现化合物多样性的方式。本论文以基因簇直接克隆与异源表达相结合的策略，成功实现了一个来源于纤维堆囊菌So0157-2中的NRPS-PKS杂合基因簇的异源表达，分离并鉴定了3个该基因簇对应的表达产物。本研究为后续从该菌株中挖掘更多活性天然产物奠定了技术基础，也为其他难培养菌株的次级代谢产物的挖掘提供了思路。

关键词: 异源表达, 纤维堆囊菌, 基因组挖掘, 非核糖体肽, 二酮哌嗪