喷他霉素新生产菌的发现及产量提高

Discovery and production improvement of new pentamycin-producing bacteria

摘要:

【背景】喷他霉素(pentamycin)是一种多烯大环内酯类抗生素，对白色念珠菌(Candida albicans)、阴道毛滴虫(Trichomonas vaginalis)和其他几种病原菌有显著的抑制活性，在瑞士已被注册用于治疗阴道念珠菌病、滴虫病和混合感染。【目的】发掘新的喷他霉素生物合成基因簇及其生产菌株，并通过基因工程改造方法提高其产量，为进一步提高喷他霉素产量、推动其工业化生产应用提供新思路及理论基础。【方法】通过BLASTp分析从细菌基因组中搜索喷他霉素已知生物合成酶的同源蛋白，进一步筛选出可能合成喷他霉素及其类似物filipin的新基因簇，使用多位点序列分析(multi-locus sequence analysis, MLSA)方法比较其所在宿主菌株的基因组相似性。利用组成型启动子kasOp*过表达LuxR家族转录调控基因ptnF以提高喷他霉素的产量。【结果】从细菌基因组中发掘获得了27个新的含有喷他霉素/filipin生物合成基因簇的菌株。并通过培养基筛选、发酵及代谢产物分析等证实了其中的米修链霉菌(Streptomyces misionensis)能够合成喷他霉素、filipin III等一系列化合物。进一步通过基因工程方法将喷他霉素的产量较野生菌株提高了4.34倍，达到101.7 mg/L，并且喷他霉素及filipin类似物总量达到146.1 mg/L，较野生菌株提高了3.28倍。【结论】本研究系统分析了细菌基因组中喷他霉素类天然产物生物合成基因簇及其所在菌株的多样性，通过实验证实其中的米修链霉菌能够合成喷他霉素及filipin类天然产物。此外，通过基因工程改造实现了喷他霉素产量的提高。本研究为构建更优的喷他霉素工业化生产菌株提供了新的元件、途径、菌株及理论基础。

关键词:次级代谢产物;喷他霉素;生物合成基因簇;基因工程改造

Abstract:

[Background] Pentamycin is a polyene macrolide with significant inhibitory activities against Candida albicans, Trichomonas vaginalis, and several other vaginal pathogens and has been registered in Switzerland for treating vaginal candidiasis, trichomoniasis, and mixed infections. [Objective] To explore new biosynthetic gene clusters and production strains and improve the yield of pentamycin by genetic engineering, providing new ideas and a theoretical basis for further increasing the yield and promoting the industrial production and application of pentamycin. [Methods] BLASTp was used to search for the homologous proteins of known enzymes involved in the biosynthesis of pentamycin from bacterial genomes, and new gene clusters for synthesizing pentamycin and its analogues were identified. Then, multi-locus sequence analysis (MLSA) was performed to compare the genomic similarity among the strains possessing the biosynthetic gene clusters. The constitutive promoter kasOp* was used to overexpress the positive transcriptional regulatory gene ptnF for increasing pentamycin production. [Results] A total of 27 new strains containing the biosynthetic gene clusters of pentamycin/filipin were discovered from the bacterial genome. The results from medium screening, fermentation, and metabolite analysis indicated that Streptomyces misionensis can synthesize multiple compounds such as pentamycin and filipin III. The genetic engineering increased the yield of pentamycin by 4.34 folds (reaching 101.7 mg/L) compared with that of the original strain. The total production of pentamycin and filipin analogues by the engineered strain reached 146.1 mg/L, which was 3.28 times higher than that of the original strain. [Conclusion] This study systematically analyzed the biosynthetic gene clusters of pentamycin in bacterial genomes and the diversity of the strains carrying these gene clusters. The experimental data confirmed that S.misionensis can produce natural products including pentamycin and filipin. In addition, the production of pentamycin was improved by genetic engineering. This study provides new elements, pathways, strains, and theoretical foundations for constructing elite strains for the industrial production of pentamycin.

Key words:secondary metabolites;pentamycin;biosynthetic gene cluster;genetic engineering