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温特曲霉中抗肿瘤四降二萜培养条件优化及海洋来源真菌简青霉次级代谢产物研究
徐蕊
Subtype博士
Thesis Advisor王斌贵
2016-05
Degree Grantor中国科学院大学
Place of Conferral北京
Degree Discipline海洋生物学
Keyword温特曲霉 四降二萜 条件优化 简青霉 次级代谢
Other Abstract
    微生物来源的天然产物结构多样、活性显著,在其基础上所开发的新药在人类保卫健康、战胜疾病的过程中发挥了十分重要的作用。海洋是生命的起源。海洋真菌由于生活在高压、高盐、低温、低光照、寡营养的海洋环境条件下,逐步进化出与陆生微生物不同的遗传和代谢机制,从而能够代谢产生多种结构新颖、活性显著的次级代谢产物,包括抗肿瘤、抗菌、抗病毒等化合物以及抗生素和生物毒素等,已经引起了人们越来越多的关注。
    本课题组从热带马尾藻中分离得到一株内生真菌温特曲霉(Aspergillus wentii EN-48),从该菌株的次级代谢产物中分离得到了一系列具有显著抗肿瘤活性的四降二萜类化合物。为了提高四降二萜类化合物的产量,本论文对菌株A. wentii EN-48产四降二萜类化合物的发酵条件进行了优化。对EN-48进行了多相系统鉴定,绘制了3个目标化合物wentilactoneA 、wentilactone B以及asperolide A的标准曲线,通过单因素试验研究了培养基、装液量、发酵时间、发酵液初始pH、盐度、温度和光照条件对EN-48中四降二萜类抗肿瘤化合物产量的影响,通过响应面实验,利用BBD设计原理,对影响EN-48中四降二萜类化合物产量的三个关键条件(发酵液初始pH、盐度和发酵时间)进行了三因素三水平的发酵条件优化,确定了最优发酵条件并对所用实验模型进行了验证,3个目标化合物wentilactone A 和wentilactone B以及asperolide A的产量分别达到13.4、6.5和4.5mg/L,分别为原产量12倍、13.5倍和14倍。通过分析温特曲霉的基因组信息,我们找到了温特曲霉中四降二萜类化合物的生物合成基因簇以及生物合成激活子TdtR,并推导了该类化合物的生物合成途径,进一步的研究还有待进行。
    综合利用硅胶柱层析、凝胶柱层析、pTLC、重结晶等多种分离手段对温特曲霉EN-48 wickerham培养基摇床动态发酵产物进行了分离,利用核磁共振(NMR)、质谱(MS)、单晶衍射(X-ray)、旋光等多种分析手段鉴定了11个单体化合物,并对得到的单体化合物进行了卤虫致死和DPPH自由基清除活性测试,化合物AM1~AM5表现出不同程度的DPPH自由基清除活性,但未表现出明显的卤虫致死活性。
    为了从海洋环境中继续寻找新的潜力菌株,对从采集自海南、南麂岛和青岛等地的海藻、红树林及其根际土壤样品进行了菌株分离。从上述样品中共分离得到不同种属的海洋真菌57株,并使用3种培养基对所得到的菌株进行了小规模静置发酵筛选,以筛选菌株粗提物的HPLC、TLC和NMR分析情况作为评价标准筛选出多株产量较大、次级代谢产物丰富的潜力菌株,并对红树林根际土壤来源的简青霉MA-332进行了次级代谢产物及生物活性研究。综合利用硅胶柱层析、凝胶柱层析、RP柱层析、pTLC、重结晶等多种分离技术,以及核磁共振(NMR)、质谱(MS)、单晶衍射(X-ray)、旋光等多种分析手段从简青霉MA-332大米培养基静置发酵产物中分离鉴定了9个单体化合物,包括3个异香豆素类新化合物(PS1*~PS3*),1个新的吉玛琓型倍半萜化合物(PS4*),和1个新天然产物(PS5)。对分离得到的单体化合物进行了抗菌和DPPH自由基清除活性测试,异香豆素类新化合物(PS1*~PS3*)对多株致病细菌和真菌表现出广谱的抑制活性,其中以PS1*抗菌活性最为显著,对Escherichia coli,Pseudomonas aeruginosa,Vibrio parahaemolyticus,Vibrio harveyi 和 Colletotrichum gloeosprioides的MIC值均为4μg/mL。
    本论文通过单因素法和响应面法对海藻内生真菌温特曲霉EN-48 产四降二萜类抗肿瘤化合物的发酵条件进行了优化,提高了目标化合物产量,为进一步的活性研究提供了充足的单体化合物,并为温特曲霉发酵产物优化及四降二萜类化合物产量优化提供了参考。通过对温特曲霉EN-48 以及红树林根际土壤来源真菌简青霉MA-332 的次级代谢产物研究,分离鉴定了20 个化合物的结构,包括4 个新结构以及1 个新天然产物,并对分离得到的部分单体化合物进行了活性测试,发现部分化合物具有较好的抑菌和DPPH 自由基清除活性,丰富了海洋天然产物化学研究内容,为海洋活性物质的有效利用提供了依据。
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   Due to the various structures and potent pharmaceutical activities,microbial natural products play an important role in human fighting diseases and protecting health. The ocean is the mother of life. Living in the high pressure, high salinity, low temperature, less light and oligotrophic marine environment, marine fungi have evolved new genetic and metabolic mechanism and able to produce novel secondary metabolites with potent anti-tumor, anti-microbial, antiviral and antibiotic bioactivities, which has attracted more and more attention.
   Recently, our group obtained several new tetranorlabdane diterpenoids withpotent anti-cancer activities from a marine alga-derived endophytic fungus A. wentii EN-48. However, the yields of tetranorlabdane diterpenoids in A. wentii were relatively low under static cultures, which limited further investigations. In order to enhance the production of the anti-tumor agents tetranorlabdane diterpenoids, the fermentation conditions of A. wentii EN-48 were optimized. In this dissertation, the fungus was identified through systematic identification, and standard curves of wentilactone A, wentilactone B and asperolide A were established. Different media, medium volume, salinity, initial pH, culture time, temperature, and light conditionswere tested by singal-factor experiment. And according to the results of single-factor experiments, effects of three factors (salinity, initial pH, and culture time) were chosen for further optimization employing response surface methodology (RSM) based on Box-Behnken design. Under the optimized culture condition, the yields of wentilactone A, wentilactone B and asperolide A were 13.4, 6.5 and 4.5 mg/L, respectively, and 12-, 13.5- and 14- fold higher than before.
    From the cultures of A. wentii EN-48, 14 compounds were isolated and identified by repeated column chromatography on silica gel, RP-18, Sephadex LH-20, preparative thin layer chromatography and preparative high liquid chromatography as well as by recrystallization. Compounds AM1~AM5 showed different DPPH radical-scavenging activitives, but no brine shrimp lethality activity.
   In this dissertation, 57 marine-derived fungi were obtained and screened under different medium for further investigation. The fermentation extracts were evaluated on the basis of TLC, HPLC and NMR analysis. As a result, Penicillium simplicissimum MA-332, a marine fungus derived from the rhizosphere of the mangrove Bruguiera sexangula var. Rhynchopetala, were selected for further chemical investigation. From the cultures of Penicillium simplicissimum MA-332, 9 compounds were isolated and identified, including three new dihydroisocoumarin derivatives (PS1*~PS3*), one new sesquiterpene (PS4*) and a new natural products diphenyl ether (PS5). The isolated compounds were screened for antimicrobial, and DPPH radical-scavenging activity, and PS1*~PS3* exhibited broad-spectrum activities against human and aquatic pathogenic bacteria and plant pathogenic fungi. PS1 exhibited highest activities against E. coli, P. aeruginosa, V. parahaemolyticus, V.harveyi, and C. gloeosprioides, each with MIC value of 4 μg/mL.
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/116978
Collection实验海洋生物学重点实验室
Affiliation中国科学院海洋研究所
Recommended Citation
GB/T 7714
徐蕊. 温特曲霉中抗肿瘤四降二萜培养条件优化及海洋来源真菌简青霉次级代谢产物研究[D]. 北京. 中国科学院大学,2016.
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