基因组指导海洋来源金黄拟诺卡氏菌活性次级代谢产物的挖掘

Marine-derived actinomycetes could form a special genetic, metabolic, physiological and biochemical mechanisms during environmental adaption and own the ability to produce various kinds of secondary metabolites. Thus, they are recognized as a critical source of lead compounds. Genome-based mining of bioactive secondary metabolites from marine-derived actinomycetes represents a new research trend. Nocardiopsis flavescens CGMCC 4.5723 is a marine-derived actinomycete isolated from the seashore of Lianyungang. Previous bioinformatics analyses revealed that there are 22 gene clusters for secondary metabolites in N. flavescens CGMCC 4.5723 genome. However, only the β-carboline alkaloids were identified from the conventional fermentation extracts, which means most of its gene clusters were still silent. In order to exploit the biosynthesis potential of N. flavescens CGMCC 4.5723, genome-based mining of bioactive secondary metabolites is employed in this project and the main methods and techniques are listed as follows: 1) gradient knockout of biosynthetic gene (clusters), genetic manipulation of transcriptional or regulatory genes and heterologous expression for the biosynthesis of secondary metabolites; 2) extensive media screening and orientation optimization of the culture as well as scale fermentation, extraction, isolation and purification; 3)various spectroscopic strategies for the structure elucidation of these isolated compounds and 4) biological activities evaluation including anti-pathogenic bacteria, anti-drug-resistant bacteria and anti-tumor activities towards the isolated compounds are screened to find those with significant activities. The project will provide a systematic technical platform for the genome mining of marine-derived actinomycetes and will supply significant lead compounds for the development of marine drugs with independent intellectual property rights.

海洋放线菌是先导化合物发现的重要源泉，在基因组信息指导下对其所蕴藏的活性次级代谢产物进行发掘是相关研究的新趋势。我们前期对一株海洋放线菌CGMCC 4.5723的序列进行分析，发现其具产22种次级代谢产物的潜能，但经菌株的常规发酵分离，仅获得1种生物碱类产物。这表明该菌株绝大多数次级代谢产物生物合成基因簇处于沉默状态。因此，本项目以深入发掘该菌株活性次级代谢产物的生产潜能为目标，综合运用1）次级代谢产物生物合成基因（簇）的梯度敲除、转录、调控基因的遗传操作和异源表达等技术；2）培养基的广泛筛选和定向优化以及规模化发酵、提取、分离纯化等手段；3）目标单体化合物结构鉴定的多种波谱学策略以及4）抗病原菌、抗耐药菌、抗肿瘤等多方面的生物学活性评价，从而针对性发掘菌株蕴藏的活性次级代谢产物。本项目的开展可为海洋放线菌产物的发掘建立系统的技术平台，也可为开发具我国自主知识产权的海洋药物提供先导化合物。

海洋放线菌蕴含丰富的药用先导化合物，在基因组信息指导下对其所藏活性次级代谢产物进行发掘是当前研究的新趋势。本项目系统利用了异源表达、辅因子工程、核糖体工程等多种基因组挖掘技术，激活并鉴定了海洋放线菌金黄拟诺卡氏菌（Nocardiopsis flavescens CGMCC 4.5723，CGMCC4.5723）中的10个活性代谢产物，其中包括3个新的β-咔啉生物碱。相关初步生物合成研究发现在不影响marinacarboline A相关生物合成基因簇nfmcb表达的情况下，倍增内源核黄素生物合成关键基因nfribA时，菌株marinacarboline A产量提高约一倍。体外生化实验证实NfMcbB能够催化L-Trp和methylglyoxal生成1-acetyl-3-carboxy-β-carboline和1-acetyl-β-carboline，表明NfMcbB为一个新发现的Pictet-Spengler酶。同步利用构建的基因组挖掘平台，定向激活了另外一株放线菌Actinoalloteichus sp. AHMU CJ021中免疫抑制剂浅蓝霉素A的生物合成，提高其效价约14.6倍，达618.61±16.29 mg/L。初步的药理学实验证实浅蓝霉素A能调节人肾小管上皮细胞(HK2)炎症反应，后续可作为肾损伤和炎症的相关研究药物。本项目的开展不但建立起一个系统的海洋放线菌产物发掘技术平台，也为团队后续开发具有自主知识产权的海洋药物提供宝贵的先导化合物。