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4. Discussion The global antifungal market was estimated at $9.4 billion in 2010 and is expected to grow at a rate of 1.9% during 2010-2017. The major class of antifungal compound includes azoles, polyenes, pyrimidines, allyamines and echinodians. The antifungal therapies intend eradicating fungi through several action mechanisms, mainly involving destruction of cell wall and inhibition of cell division. There is a significant unmet need of novel anti- fungal compounds, because the present portfolio of treatments interact unfavorably with other medication, have resistance problem, low spectrum of activity, are fungistatic as opposed to fungicidal and are often toxic. Hence research has been focused on development of potent and safe antifungal compounds, either synthetic or from microbial sources [24]. In the present study, we exploited the marine resources for isolation of actinomycetes strains producing antifungal compounds. In the course of study, the marine invertebrate associated active acti- nomycetes strain PM0525875 was isolated and identified as Actinoalloteichus cyanogriseus. The antifungal principle from this strain was characterized as Caerulomycin A that showed potent in-vitro activity against pathogenic and drug resistant Candida strains. Caerulomycin A was first isolated from S. caeruleus [25] [26] as an antibiotic. Later it was reported to have anti-cancer activity [27]. In-vitro and in-vivo activity of Caerulomycin A against Entamoeba histolytica has been reported [28]. Natural derivatives of Caerulomycin A has also been reported [29]. Caerulomycin B and C, a new 2,2′-dipyridyl derivatives were isolated from Streptomyces caeruleus supplemented with 1 mM L-tryptophan. This group also reported the isolation of Caerulomycin D [19]. Additional natural derivatives of Caerulomycin F-K were isolated from marine derived actinomycetes (Actinoalloteichus cyanogriseus) that showed anticancer and antibacterial activity [29]. Lin et al. identified the biosynthetic gene cluster for Caerulomycin A from ma- rine actinomycetes Actinoalloteichus cyanogriseus. It mainly consists of unusual hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) [30]. The biosynthesis of Caerulomycin A based on enzy- matic modifications leading to structural changes, have been studied [31]. Chemical synthesis of Caerulomycin A-C has also been reported [32] [33]. The immunosuppressive activity of Caerulomycin A and its natural ana- logues has been reported [34]. Although Caerulomycin A has not been entered in clinic so far, may be due to its limitations, it could be the ideal molecule for generating semi synthetic druggable derivatives having potential application as an anticancer, antifungal or immunosuppressive drug. The strain and the optimized fermentation process described here could be useful for large scale production of this molecule for further drug development studies. 5. Conclusion In our in-house screening program in the quest of novel and superior antifungal compounds, an actinomycetes strain PM0525875 was isolated from a marine invertebrate. The active principle was characterized to be Caeru- lomycin A. Minimum inhibitory concentration (MIC) of the compound was found in the range of 0.39 - 1.56 μg/ml against pathogenic fungal test strains. The phylogenetic analysis of producer strain using 16S rRNA se- quence showed closest match with Actinoalloateichus cyanogriseus. Overall, Caerulomycin A was isolated from marine invertebrate-associated Actinoalloteichus sp. using optimized medium and fermentation conditions. |
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bxzc1233楼2014-10-22 19:33:49
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4 讨论 2010年全球抗真菌市场估计值为94亿美元，且在2010至2017年间有望以1.9%的增长率持续增长。 抗菌剂的主要成分含唑类、多烯类、嘧啶类、以及烯胺类 。抗真菌疗法试图通过多种作用机制—主要是破坏细胞壁及抑制细胞的分解—来根除真菌。新型抗真菌化合物的需求量远未得到满足，其原因是现有的组合疗法内部机制间存在相互抵制，且作用范围小，其与抗菌剂的作用相悖，甚至会使抗菌剂失效。 因而有研究致力于开发高效、安全的抗真菌化合物，包括合成及从细菌中提取[24]。在本研究中，我们开发利用了细菌源进行放线菌株的隔离，来制取抗真菌化合物。我们在试验中将海洋无脊椎动物的相关抗菌性菌株进行了隔离，并确定其为海洋来源放线菌。该菌株的抗菌性原理类似于浅蓝霉素 A，其对致病性和耐药性念珠菌菌株表现出很强的体外活性。 浅蓝霉素 A是一种首次由S. caeruleus [25] [26]分离的抗生素。之后有报道称其有抗癌作用[27]。浅蓝霉素 A的体内抗痢疾变形虫作用已经报道[28]。还有报道指出浅蓝霉素 A的人工合成方法[29]。 浅蓝霉素B与C是由青蓝链霉菌补充以1mM左旋色氨酸分离而得的一种新型联吡啶衍生物。该课题组还报道了浅蓝霉素D 的分离提取[19]。其他自然衍生物浅蓝霉素F-K均分离自海洋来源放线菌(称为Actinoalloteichus cyanogriseus)，具有抗癌性及抗菌性[29]。 Lin等鉴定了分离自海洋放线菌Actinoalloteichus cyanogriseus的浅蓝霉素A的生物基因组。结果发现该浅蓝霉素A主要含不常见的杂聚酮合酶（PKS）及无核糖体型肽合成酶（NRPS）[30]. 浅蓝霉素A 的生物合成以对酶的修饰为基础实现其结构的改变，已有相关研究[31].浅蓝霉素A-C的化学合成法已有报道[32][33]. 浅蓝霉素A免疫抑制性及其自然界类似物已有报道[34]。虽然浅蓝霉素A至今还未用于临床——可能由于其限制性——但其仍可作为制取半合成药物衍生物的理想分子，将成为一种有应用潜力的抗癌、杀菌或免疫抑制性的药物。 5 结论 我们为了获得新型、优化的抗真菌化合物设定了室内筛选程序，利用该程序从一种海洋无脊椎动物中分离出一段放线菌主链PM0525875。其活性成分为浅蓝霉素A。该化合物经对抗致病真菌的测试，得出其最低抑菌浓度为0.39-1.56μg/ml。利用16S rRNA对产生菌进行系统分析，结果表明其与Actinoalloteichus cyanogriseus（海洋来源放线菌）有相关匹配性。综上所述，本研究利用最优方法及最佳发酵条件，从海洋无脊椎联合放线菌浅蓝霉素A中分离得到了浅蓝霉素A。 |
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