龟裂霉素新产生菌株的鉴定及其产量优化

Identification and yield improvement of a new rimocidin-producing strain

摘要:

【背景】龟裂霉素(rimocidin)是一种具有广谱抗真菌活性的四烯大环内酯类化合物，对多种真菌性植物病害具有较强的防治作用，因此具有开发成农用抗生素药物的潜力。目前已经陆续发现几株链霉菌可以生产龟裂霉素，如龟裂链霉菌(Streptomyces rimosus) M527，但是龟裂霉素的低产量仍限制其在农用抗生素方面的开发。【目的】系统揭示链霉菌基因组中龟裂霉素生物合成基因簇(rim基因簇)的分布及特征，发现新的龟裂霉素生产菌株，并基于基因工程等策略提高其产量。【方法】采用BLASTp在链霉菌基因组中搜索龟裂霉素已知生物合成酶的同源蛋白，进一步筛选出预测可合成龟裂霉素的基因簇及所在菌株。使用多位点序列分析方法(multi-locus sequence analysis, MLSA)评价含有龟裂霉素生物合成基因簇的菌株的进化关系。基于代谢产物分析鉴定新的龟裂霉素生产菌株，并通过筛选发酵培养基和过表达调控蛋白RimoR2来提高龟裂霉素的产量。【结果】共发掘获得36个新的含有龟裂霉素生物合成基因簇的链霉菌菌株。并通过培养基筛选、发酵及代谢产物分析等证实其中的白色链霉菌(Streptomyces albofaciens) JCM 4342能够合成龟裂霉素及其类似物CE-108。经过培养基优化的龟裂霉素产量为172.5 mg/L。此外，过表达调控蛋白RimoR2后龟裂霉素的产量较出发菌株提高了2.3倍，达到397.1 mg/L。【结论】本研究系统分析了链霉菌中龟裂霉素生物合成基因簇及其所在菌株的分布特点，并通过实验证实其中的S. albofaciens JCM 4342能够合成龟裂霉素及其类似物CE-108。此外，通过基因工程方法实现了龟裂霉素产量的提高。本研究为构建更优的龟裂霉素高产菌株提供了新的元件、菌株和理论基础，将有助于推动龟裂霉素的进一步应用开发研究。

Abstract:

[Background] Rimocidin is a tetraene macrolide with broad-spectrum antifungal activities, there being developed into a potential agricultural antibiotic. Until now, several Streptomyces producers (such as S.rimosus M527) have been discovered, while the low yield of rimocidin limits the application of these strains in the development of the agricultural antibiotic. [Objective] To systematically reveal the distribution and characteristic of the biosynthetic gene clusters (BGCs) for rimocidin (rim BGCs) in Streptomyces genomes, identify a new strain which can produce rimocidin, and improve its yield via genetic engineering and other strategies. [Methods] We employed BLASTp to search for the homologous proteins of the known synthetases of rimocidin in Streptomyces genomes to discover the strains with predicted rim BGCs and capable of producing rimocidin. The multi-locus sequence analysis (MLSA) was conducted to analyze the phylogenetic relationships among these strains and publicly available Streptomyces strains. A new rimocidin-producing strain was identified based on the metabolite analysis and the titer of rimocidin was increased by medium optimization and RimoR2 overexpression. [Results] A total of 36 Streptomyces strains carrying rim BGCs were predicted, in which S.albofaciens JCM 4342 was capable of producing rimocidin and its congener CE-108. The medium optimization led to a rimocidin titer of 172.5 mg/L. Moreover, the RimoR2 overexpression increased the rimocidin titer by 2.3 folds, which reached 397.1 mg/L. [Conclusion] This study systematically revealed the distribution of rim BGCs in Streptomyces. We confirmed that S.albofaciens JCM 4342 can produce rimocidin and its congener CE-108. Moreover, the titer of rimocidin was improved by genetic engineering. Our findings provide new catalytic elements, strains, and a theoretical basis for the design and construction of strains with high yields of rimocidin and the application and development of rimocidin.