纤维堆囊菌基因转化体系建立及埃博霉素生物发酵和纯化工艺研究
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摘要
来源于纤维堆囊菌(Sorangium cellulosum)二级代谢产物的埃博霉素是一种新型抗癌剂,机制与紫杉醇相似,能够稳定微管蛋白,抑制细胞的分裂和增殖。但由于它具有更好的水溶性、简单的化学结构,并且对紫杉醇的耐受细胞表现出很大活性以及不会象紫杉醇那样产生内毒素等的优点,激发了科研人员和制药公司的极大热情,成为化学界、生物界和临床医学研究的热点。在化学全合成、生物发酵、生物的合成改造、活性与构象关系的推导、基因簇异源表达等方面取得了卓越的成就。当前已经有5种埃博霉素类活性物进入II期和III期临床评估,作为新的第三代化疗药物走向市场指日可待。
但作为埃博霉素的来源菌—纤维堆囊菌,因为传代时间长、菌株特异性强、耐药范围广、聚集生长和可使用的分子标记少等不利因素,使得对纤维堆囊菌基因操作极为困难,对埃博霉素合成的基因调控、操纵子的工作方式,转录的控速部分等问题的认知近于空白。除此之外,到目前为止人们还没有在纤维堆囊菌乃至粘细菌中找到一个能自主复制,独立于宿主细胞的质粒。以上种种原因成为限制对纤维堆囊菌所产生的丰富的、具有生物活性二级代谢产物进行研究和利用的瓶颈。
本文以埃博霉素的阳性菌株——S. cellulosum So ce90为对象,研究了该菌株的发酵条件和纯化的工艺流程,并通过利用广谱可移动质粒pRK415构建含有EpoPro片段(埃博霉素合成酶基因中epoA起始密码子(GTG)上游(-890～-1bp)区域)和荧光蛋白基因的pRP-GFP载体,建立了针对该菌株的基因转化体系。主要结论如下:
①以高效液相色谱为检测手段,埃博霉素的产量为指标,比较9种不同培养基,筛选出适合S. cellulosum So ce90的培养基和发酵条件。适宜培养基(II号发酵液)的主要成分是:2g/L脱脂大豆粉,10g/L马铃薯淀粉,8g/L脱脂奶粉,2g/L酵母浸膏,2g/L葡萄糖,8 mg/L Na-FeIII-EDTA,1 g/L MgSO4 ? 7H2O,1g/L CaCl2 ? 2H2O,11.5g/L HEPES,2%(v/v)树脂XAD-16。用KOH调节pH值为7.4。最适种子培养液采用在30oC,180rpm培养4天得到的G52发酵液。以5%的接种量添加到II号发酵液中,发酵8天达到最高产率。
②埃博霉素分离和纯化工艺是首先采用100%异丙醇将埃博霉素从树脂中洗脱出来,通过乙酸乙脂和石油醚(1:1)的混合有机液进行萃取后,SOURCETM 30反相层析,最后利用C18反相半制备柱,高效液相制备得到埃博霉素A和B。经质谱,紫外光谱和红外光谱等验证结构,与文献报道一致。
③利用结合转移的方式将载体pRP-GFP从E.coli S17-1中导入到S. cellulosum So ce90中,在四环素(50μg/ml)和卡那霉素(10μg/ml)的选择压力下筛选转化子。对转化子的Southern杂交证明该质粒在宿主菌中并没有被整合到宿主菌的基因组上。这是首次发现能在粘细菌中独立存在且自主复制的质粒。因此,此质粒将为分离及分析埃博霉素代谢相关基因提供有力而便利的工具,从这个意义上说,这个质粒的获得是对S. cellulosum So ce90的遗传及分子生物学研究上取得的突破。
④结合转移的效率主要和融合时间相关,而融合前的热击并不影响转化率。最佳的融合时间是60-65h,转移效率达到9×10-6。但是,当融合时间高于70小时或低于40小时,获得转化子极少。
⑤转化子稳定性实验表明,当宿主细胞维持在有四环素的环境中时,pRP-GFP能长期存在,并且稳定表达;而在没有抗生素的压力下,子代中质粒将出现严重的丢失,繁殖到50代的时候,细胞中质粒的含有率仅仅达到73%。
⑥采用荧光显微镜能观察到转化子呈现绿色荧光。这是荧光蛋白首次被导入到纤维堆囊菌中,并且使之得到很好表达,为纤维堆囊菌的分子操纵提供了一个快捷而便利的分子标记。
⑦通过比较载体pRK415-GFP(不含EpoPro片段)和pRP-GFP在宿主菌中GFP的表达与否,证明了EpoPro具有启动活性。这是首次得到埃博霉素合成酶基因上具有启动活性的片段,为进一步分析埃博霉素的操纵子及其基因的功能调节等研究打下基础。
Epothilones, the secondary metabolites produced from the myxobacterium Sorangium cellulosum, are the potential anticancer drugs that stabilize microtubules and inhabit mitosis of cells in a manner similar to paclitaxel (Taxol). Epothilones have superior features relative to paclitaxel, including more water solubility, simpler molecular structure, non-endotoxicity and effectivity against tumors resistant to paclitaxel. These advantages make epothilones likely successors to paclitaxel and become a hotspot of clinic research. Great progresses have been made in total chemical synthesis, biological fermentation, biosynthesis and derivation, relation between activity and conformation and heterologous expression of the gene clusters. Currently five Epothilone derivatives are been initiated to phase II or III clinical trials to finally evaluate the anticancer potential. Being surrogates to replace taxol in treatment of multidrug resistant tumors, it will not be long before epothilones become marketable.
However, S. cellulosum, the producer of epothilones, is difficult to engineer, due to the long doubling time, the specificity of bacterial strain, the broad drugs resistance, the aggregating growth, the low efficiency of introducing DNA into the bacterium and the limited number of molecular tools and markers that have been developed. There is no inkling as to the regulation and control of the epothilones biosynthesis gene clusters and the model of operon. Moreover, no endogenous plasmid had been found in S. cellulosum, and few attempts to transfer an autonomously replicating exogenous plasmid into this species have been reported. All these restricted the research and application of the abundant secondary metabolites with bioactivities produced by the bacterial.
In our research, S. cellulosum So ce90, a positive strain for epothilone, was used. The research involved its fermentation conditions, purification technics of epothilone in the fermented solution, construction of plasmid pRP-GFP derived from pRK415 with a green fluorescent protein gene and an EpoPro fragment spanning from -890 to -1 relative to the start codon (GTG) of epoA (a part of epothilone synthase gene clusters), and the establishment of gene transformation system for the bacterial. The main conclusions are as follows:
①To select the optimum medium and the conditions of culture, S. cellulosum So ce90 was cultured in nine medium, and the production of epothilone in the fermented solutions was measured by HPLC. The optimum medium included 2g/L soy grits, 10g/L potato starch, 8g/L skim milk powder, 2g/L yeast extract, 2g/L glucose, 8 mg/L Na-FeIII-EDTA, 1 g/L MgSO4 ? 7H2O, 1g/L CaCl2 ? 2H2O, 11.5g/L HEPES,2%(v/v) the resin XAD-16. The pH of the medium was adjusted to 7.4 with KOH before autoclaving. The optimum seed cultures, which were obtained from the fermented solution cultured in 180rpm, 30oC for 4 days, were added to the mediums by 5%. The maximum of epothilone could be gained after 8 days fermentation.
②The separation and purification technics of epothilone are as follows. Firstly, the active materials were eluted with 100% isopropanol. And then, the active materials were extracted with ethyl acetate and petroleum ether (1:1). Lastly isolation of pure epothilone A and epothilone B were achieved by chromatography on a SOURCETM 30RPC colunm and C18 reversed-phase column. Structure of epothilone A and epothilone B were further confirmed by MS, UV and IR.
③Plasmid pRP-GFP was transform from E.coli S17-1 to S. cellulosum So ce90 by conjugation, and the transformants were selected by tetracycline (50μg/ml) and kanamycin sulfate (10μg/ml). The results of the Southern blots analyses confirmed that the plasmid could be replicated autonomously without being integrated into the chromosome of S. cellulosum So ce90. To date, it is the first plasmid which has been described in myxobacteria. Therefore, the work will provide a powerful tool for isolating and analyzing the genes involved in epothilone metabolism. In this sense, this work may be a breakthrough in the genetics and molecular biology of S. cellulosum So ce90.
④The conditions that might induce an increase in the transfer frequency were investigated. The frequency did not change by heating the S. cellulosum cells after transconjugation. But the mating time was more important for transfer frequency, of which the highest frequency was observed in the range of 60-65 h. The frequency of transfer to S. cellulosum So ce90 (calculated per viable Sorangium cell) was in the range 9×10-6. However, when the mating time was less than 40 h or more than 70 h, few transformants were found.
⑤The stability of the S. cellulosum So ce90 transformants containing the plasmid pRP-GFP was tested. The fluorescence found in the subculture indicated that the plasmid was stably maintained in the bacteria with selective pressure. However, only 73% of the cells retained fluorescence at the 50th generation in the case of the antibiotic-free incubation.
⑥In this study, the GFP reporter system in S. cellulosum was also first established. The stable expression of GFP in S. cellulosum was a rapid and convenient marker for the conjugation screening of the transformants of the bacterium.
⑦The comparison of the expression of gfp within pRP-GFP with that within pRK415-GFP (without EpoPro fragment) in S. cellulosum confirmed that the EpoPro fragment had the function of a promoter that could drive the expression of the gene in the bacterium. This would be a foundation for the analyses of the operon of epothilone synthase gene clusters and the research about the regulation and control of the gene.
但作为埃博霉素的来源菌—纤维堆囊菌,因为传代时间长、菌株特异性强、耐药范围广、聚集生长和可使用的分子标记少等不利因素,使得对纤维堆囊菌基因操作极为困难,对埃博霉素合成的基因调控、操纵子的工作方式,转录的控速部分等问题的认知近于空白。除此之外,到目前为止人们还没有在纤维堆囊菌乃至粘细菌中找到一个能自主复制,独立于宿主细胞的质粒。以上种种原因成为限制对纤维堆囊菌所产生的丰富的、具有生物活性二级代谢产物进行研究和利用的瓶颈。
本文以埃博霉素的阳性菌株——S. cellulosum So ce90为对象,研究了该菌株的发酵条件和纯化的工艺流程,并通过利用广谱可移动质粒pRK415构建含有EpoPro片段(埃博霉素合成酶基因中epoA起始密码子(GTG)上游(-890～-1bp)区域)和荧光蛋白基因的pRP-GFP载体,建立了针对该菌株的基因转化体系。主要结论如下:
①以高效液相色谱为检测手段,埃博霉素的产量为指标,比较9种不同培养基,筛选出适合S. cellulosum So ce90的培养基和发酵条件。适宜培养基(II号发酵液)的主要成分是:2g/L脱脂大豆粉,10g/L马铃薯淀粉,8g/L脱脂奶粉,2g/L酵母浸膏,2g/L葡萄糖,8 mg/L Na-FeIII-EDTA,1 g/L MgSO4 ? 7H2O,1g/L CaCl2 ? 2H2O,11.5g/L HEPES,2%(v/v)树脂XAD-16。用KOH调节pH值为7.4。最适种子培养液采用在30oC,180rpm培养4天得到的G52发酵液。以5%的接种量添加到II号发酵液中,发酵8天达到最高产率。
②埃博霉素分离和纯化工艺是首先采用100%异丙醇将埃博霉素从树脂中洗脱出来,通过乙酸乙脂和石油醚(1:1)的混合有机液进行萃取后,SOURCETM 30反相层析,最后利用C18反相半制备柱,高效液相制备得到埃博霉素A和B。经质谱,紫外光谱和红外光谱等验证结构,与文献报道一致。
③利用结合转移的方式将载体pRP-GFP从E.coli S17-1中导入到S. cellulosum So ce90中,在四环素(50μg/ml)和卡那霉素(10μg/ml)的选择压力下筛选转化子。对转化子的Southern杂交证明该质粒在宿主菌中并没有被整合到宿主菌的基因组上。这是首次发现能在粘细菌中独立存在且自主复制的质粒。因此,此质粒将为分离及分析埃博霉素代谢相关基因提供有力而便利的工具,从这个意义上说,这个质粒的获得是对S. cellulosum So ce90的遗传及分子生物学研究上取得的突破。
④结合转移的效率主要和融合时间相关,而融合前的热击并不影响转化率。最佳的融合时间是60-65h,转移效率达到9×10-6。但是,当融合时间高于70小时或低于40小时,获得转化子极少。
⑤转化子稳定性实验表明,当宿主细胞维持在有四环素的环境中时,pRP-GFP能长期存在,并且稳定表达;而在没有抗生素的压力下,子代中质粒将出现严重的丢失,繁殖到50代的时候,细胞中质粒的含有率仅仅达到73%。
⑥采用荧光显微镜能观察到转化子呈现绿色荧光。这是荧光蛋白首次被导入到纤维堆囊菌中,并且使之得到很好表达,为纤维堆囊菌的分子操纵提供了一个快捷而便利的分子标记。
⑦通过比较载体pRK415-GFP(不含EpoPro片段)和pRP-GFP在宿主菌中GFP的表达与否,证明了EpoPro具有启动活性。这是首次得到埃博霉素合成酶基因上具有启动活性的片段,为进一步分析埃博霉素的操纵子及其基因的功能调节等研究打下基础。
Epothilones, the secondary metabolites produced from the myxobacterium Sorangium cellulosum, are the potential anticancer drugs that stabilize microtubules and inhabit mitosis of cells in a manner similar to paclitaxel (Taxol). Epothilones have superior features relative to paclitaxel, including more water solubility, simpler molecular structure, non-endotoxicity and effectivity against tumors resistant to paclitaxel. These advantages make epothilones likely successors to paclitaxel and become a hotspot of clinic research. Great progresses have been made in total chemical synthesis, biological fermentation, biosynthesis and derivation, relation between activity and conformation and heterologous expression of the gene clusters. Currently five Epothilone derivatives are been initiated to phase II or III clinical trials to finally evaluate the anticancer potential. Being surrogates to replace taxol in treatment of multidrug resistant tumors, it will not be long before epothilones become marketable.
However, S. cellulosum, the producer of epothilones, is difficult to engineer, due to the long doubling time, the specificity of bacterial strain, the broad drugs resistance, the aggregating growth, the low efficiency of introducing DNA into the bacterium and the limited number of molecular tools and markers that have been developed. There is no inkling as to the regulation and control of the epothilones biosynthesis gene clusters and the model of operon. Moreover, no endogenous plasmid had been found in S. cellulosum, and few attempts to transfer an autonomously replicating exogenous plasmid into this species have been reported. All these restricted the research and application of the abundant secondary metabolites with bioactivities produced by the bacterial.
In our research, S. cellulosum So ce90, a positive strain for epothilone, was used. The research involved its fermentation conditions, purification technics of epothilone in the fermented solution, construction of plasmid pRP-GFP derived from pRK415 with a green fluorescent protein gene and an EpoPro fragment spanning from -890 to -1 relative to the start codon (GTG) of epoA (a part of epothilone synthase gene clusters), and the establishment of gene transformation system for the bacterial. The main conclusions are as follows:
①To select the optimum medium and the conditions of culture, S. cellulosum So ce90 was cultured in nine medium, and the production of epothilone in the fermented solutions was measured by HPLC. The optimum medium included 2g/L soy grits, 10g/L potato starch, 8g/L skim milk powder, 2g/L yeast extract, 2g/L glucose, 8 mg/L Na-FeIII-EDTA, 1 g/L MgSO4 ? 7H2O, 1g/L CaCl2 ? 2H2O, 11.5g/L HEPES,2%(v/v) the resin XAD-16. The pH of the medium was adjusted to 7.4 with KOH before autoclaving. The optimum seed cultures, which were obtained from the fermented solution cultured in 180rpm, 30oC for 4 days, were added to the mediums by 5%. The maximum of epothilone could be gained after 8 days fermentation.
②The separation and purification technics of epothilone are as follows. Firstly, the active materials were eluted with 100% isopropanol. And then, the active materials were extracted with ethyl acetate and petroleum ether (1:1). Lastly isolation of pure epothilone A and epothilone B were achieved by chromatography on a SOURCETM 30RPC colunm and C18 reversed-phase column. Structure of epothilone A and epothilone B were further confirmed by MS, UV and IR.
③Plasmid pRP-GFP was transform from E.coli S17-1 to S. cellulosum So ce90 by conjugation, and the transformants were selected by tetracycline (50μg/ml) and kanamycin sulfate (10μg/ml). The results of the Southern blots analyses confirmed that the plasmid could be replicated autonomously without being integrated into the chromosome of S. cellulosum So ce90. To date, it is the first plasmid which has been described in myxobacteria. Therefore, the work will provide a powerful tool for isolating and analyzing the genes involved in epothilone metabolism. In this sense, this work may be a breakthrough in the genetics and molecular biology of S. cellulosum So ce90.
④The conditions that might induce an increase in the transfer frequency were investigated. The frequency did not change by heating the S. cellulosum cells after transconjugation. But the mating time was more important for transfer frequency, of which the highest frequency was observed in the range of 60-65 h. The frequency of transfer to S. cellulosum So ce90 (calculated per viable Sorangium cell) was in the range 9×10-6. However, when the mating time was less than 40 h or more than 70 h, few transformants were found.
⑤The stability of the S. cellulosum So ce90 transformants containing the plasmid pRP-GFP was tested. The fluorescence found in the subculture indicated that the plasmid was stably maintained in the bacteria with selective pressure. However, only 73% of the cells retained fluorescence at the 50th generation in the case of the antibiotic-free incubation.
⑥In this study, the GFP reporter system in S. cellulosum was also first established. The stable expression of GFP in S. cellulosum was a rapid and convenient marker for the conjugation screening of the transformants of the bacterium.
⑦The comparison of the expression of gfp within pRP-GFP with that within pRK415-GFP (without EpoPro fragment) in S. cellulosum confirmed that the EpoPro fragment had the function of a promoter that could drive the expression of the gene in the bacterium. This would be a foundation for the analyses of the operon of epothilone synthase gene clusters and the research about the regulation and control of the gene.
引文
[1] http://www.who.int/mediacentre/factsheets/fs297/zh/
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