纤维堆囊菌GSUV3-205发酵生产埃博霉素过程中关键因子的探索及优化
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摘要
粘细菌是一类具有复杂多细胞社会学行为的革兰氏阴性细菌,能够合成多种具有抗肿瘤、抗真菌、抗细菌、免疫调节等生物活性的天然产物。研究人员已经从粘细菌次级代谢物中鉴定出100多种基本化学结构和500多种结构类似物。微生物来源的小分子物质中约有3.5%来源于粘细菌,粘细菌代谢产物的化学结构和生物活性的多样性甚至可以与著名的链霉菌类群和芽孢杆菌类群相比拟。
纤维堆囊菌(Sorangium cellulosum)属于粘球菌目、堆囊菌亚目、多囊菌科、堆囊菌属,可以通过降解纤维素进行生长。据我们了解,纤维堆囊菌拥有迄今为止发现的最大的原核生物基因组,如纤维堆囊菌So ce56的基因组大小约为13Mb。目前研究结果显示,在庞大的基因组中有大量基因被”奢侈”的用来进行次级代谢,它合成的次级代谢产物几乎占所有已发现粘细菌种类的一半,并且几乎100%的菌株都具有合成生物学活性物质的能力。因此,纤维堆囊菌不但具有重要的基础研究价值,而且还具有诱人的应用开发前景。
埃博霉素(epothilones)是粘细菌纤维堆囊菌(Sorangium cellulosum)产生的一类聚酮类次级代谢产物。与目前临床上广泛应用的抗肿瘤化疗药物紫杉醇相比,它具有相似的稳定微管活性,而且对紫杉醇耐药的肿瘤细胞具有活性。目前至少有五个埃博霉素类化合物(ixabepilone, patupilone, BMS-310705, KOS-862和ZK-EPO)已经进行了Ⅰ-Ⅲ期临床实验,而且目前已经有一种产品(ixabepilone)得到了美国FDA的权威认证。但是,目前埃博霉素的发酵生产能力成为该药研发的一个主要瓶颈。
菌株GSUV3-205是本实验室通过Genome Shuffling技术得到的一株具有较高产量的埃博霉素产生菌,具有一定的的工业应用前景。但是无论是GSUV3-205还是在国际上其他实验室的发酵生产菌株,埃博霉素的实际生产量相对于发酵培养体系、底物利用率以及生物量都存在着转化效率低的问题,它们还是有很大的提升空间的。而且,通过长时间的传代培养,重组菌株GSUV3-205的埃博霉素产生能力发生了很大的退化。因此,如何能够在发酵体系中充分挖掘菌株的埃博霉素的合成能力成为研究者最为感兴趣的问题之一,所以对于纤维堆囊菌合成埃博霉素过程中某些关键因子的研究,对纤维堆囊菌资源的开发利用和埃博霉素走向产业化都具有巨大的意义。
本论文以本实验室的一株埃博霉素产生菌纤维堆囊菌GSUV3-205为研究材料,以提高菌株的埃博霉素合成能力为主要目标,主要从菌株的培养条件、营养条件、生长状态等方面分别进行了一系列系统的改进和优化,并取得了良好的结果。
对于每一种能够合成有应用价值的代谢产物的微生物来说,发酵过程改良都是从实验室理论研究走向工业产业化道路的必要阶段,因此本文的研究工作首先分两个阶段分别对埃博霉素产生菌纤维堆囊菌GSUV3-205的发酵培养条件和营养条件进行了优化。
在第一阶段,我们结合GSUV3-205的生理特性,主要对接种量、发酵时间、种子培养及接种的状态、树脂添加时间、发酵培养转速等培养条件因素进行了系列考察。根据不同接种量下的埃博霉素产生曲线,确定了合适的接种量(5×109个细胞/50m1)和发酵培养时间(9天)；通过对不同接种量时不同种子培养接种状态的埃博霉素的产量进行分析,确定了最优的种子培养接种状态(种子200rpm培养5天)；通过单因子分析树脂添加时间和发酵培养转速这两个条件,得出了较有利于高产埃博霉素的条件,即灭菌时添加树脂和200rpm发酵培养。
第二个阶段,我们利用统计学优化方法中的响应面设计法以epothilone B的产量为响应值对GSUV3-205生产埃博霉素的发酵培养基成分进行了优化。这个阶段主要是通过两个大的步骤来实现的。首先,我们在前期使用的EPM培养基的基础上进行了营养成分的拓展和初步优化,即：利用单因子实验考察了21种EPM培养基中不包含的营养成分对epothilone B合成的影响,获得了7种较有利于合成埃博霉素的营养物质；然后利用Plackett-Burman和Box-Behnken响应面设计把通过单因子实验拓展的7种营养成分和EPM培养基中原有的所有成分进行了优化,通过重要营养成分的数学建模和响应面分析,获得了一个初步优化的培养基。其成分是：糊精0.3%、蔗糖0.05%、葡萄糖0.08%、大豆粉0.17%、脱脂奶粉0.1%、七水硫酸镁0.1%、无水氯化钙0.1%、EDTA-Fe3+溶液2m1/L、微量元素液0.5ml/L、pH7.2、吸附树脂2%(v/v)。可以看出,与EPM培养基相比(土豆淀粉0.2%、葡萄糖0.2%、大豆粉0.2%、脱脂奶粉0.1%、七水硫酸镁0.1%、无水氯化钙0.1%、EDTA-Fe3+溶液1ml/L、微量元素液1ml/L、VB12 0.5mg/l、pH7.2、吸附树脂2%(v/v)),优化培养基中复杂碳源由土豆淀粉换成糊精且增加了添加量,同时增加了一个新的简单碳源即二糖物质蔗糖,并对绝大部分成分的含量进行了调整。利用这个培养基epothilone B的产量可以达到46.5mg/1,较原始产量11.3 mg/1提高了3.1倍。此后,我们对初步优化的培养基又进行了进一步的系统优化：基于这个新的培养体系,利用FFD设计考察了这个体系各个组成成分对epothilone B产量的影响。发现三个因子糊精(X2)、脱脂奶粉(X6)和硫酸镁(X8)对epothilone B的产量具有显著性影响。利用CCD这种五水平的响应面设计法对这三个因素进行了进一步研究,建立了相应的二次回归模型：Y=51.69+17.03×X2+1.17×X6+0.70×X8-4.55×X2×X2+7.01×X2×X6-2.48×X6×X6-0.69×X2×X8-2.01×X6×X8+0.52×X8×X8利用SAS 8.0软件的RSREG过程对这个模型的统计学意义进行分析,说明了建立的数学模型和实验结果有好的相关性。通过模型可以看出糊精对于epothilone B的产量具有显著影响,糊精和脱脂奶粉对epothilone B产量的影响具有较强的交互作用。最后获得的优化培养基成分为：糊精0.63%、蔗糖0.05%、葡萄糖0.08%、大豆粉0.17%、脱脂奶粉0.26%、七水硫酸镁0.32%、无水氯化钙0.1%、EDTA-Fe3+溶液2ml/L、微量元素液0.5m1/L、pH7.0、吸附树脂2%(v/v)。So0157-2利用此培养体系发酵培养,可产生82.0mg/1的epothilone B,较初步优化培养基(46.5mg/1)又提高了76.3%。这是首次在能够产生埃博霉素的本源菌纤维堆囊菌中进行的系统发酵过程优化,并把GSUV3-205的埃博霉素产量水平提高到了较高的水平,为下一步开展发酵罐中试和最终的工业化生产奠定了坚实的基础。
本文随后的工作结合相关的文献报道,将有可能与纤维堆囊菌发酵合成埃博霉素过程密切相关的小分子物质,梯度添加到优化后的发酵培养基中,探讨了它们对GSUV3-205埃博霉素合成能力的影响。
研究表明,铁离子、甜菜碱类物质以及埃博霉素合成的前体物质乙酸盐、丙酸盐、甲基丙二酸、琥珀酸、半胱氨酸等物质的过量添加,都会对GSUV3-205合成埃博霉素的过程有抑制作用。但是在适当的添加时间和添加量时,这些小分子物质的存在会促进GSUV3-205埃博霉素的合成。比如,在发酵第三天添加0.05%的98%的盐酸甜菜碱时,埃博霉素产量有16%的提高；在添加10 mM的乙酸钠、甲基丙二酸、半胱氨酸混合溶液时,epothilone A和B的产量分别提高了68%和270%。由此看见,这些小分子物质的添加是与So0157-2的埃博霉素合成能力密切相关的。其中,我们首次将与epothilone B合成的前体物质甲基丙二酸单酰-CoA具有相同碳链骨架的甲基丙二酸添加到发酵培养中,并取得了良好的效果：在添加5 mM的甲基丙二酸时,epothilone B的产量比对照提高了86%。这些结果为进一步利用连续或半连续发酵技术进行前体物质添加提供了基础,同时也为GSUV3-205发酵生产埃博霉素的产物选择提供了思路。
GSUV3-205在液体培养基中聚团生长的特性成为该菌株大规模发酵及产业化道路上的重要障碍,同时这对于GSUV3-205的某些性质研究及遗传操作方面来说也是一个巨大的难题。我们希望通过生物学手段对GSUV3-205进行性状改造,在保持埃博霉素产生能力不变的情况下使其能够在液体培养基中分散生长。这样一方面可以提高单位培养基中的生物量从而提高埃博霉素的单位产量,加快其工业化应用的进程,另一方面,还能够给我们进一步探索GSUV3-205的性质及遗传操作带来极大的方便。
因此我们从生物驯化和遗传改造这两个角度进行了获取GSUV3-205液体分散生长突变株的尝试。通过特殊设计的驯化流程对GSUV3-205进行了长时间的驯化培养。经过8个循环的驯化培养,我们最终得到了一株在M26液体培养基中生长状态有些分散的菌株GSUV3-205XH。尽管通过一系列的验证及分析发现,GSUV3-205XH生长非常缓慢而且几乎没有埃博霉素产生能力,但是今后我们可以结合genome shuffling的思路方法利用这个菌株与埃博霉素高产菌株进行原生质体融合从而对菌株进行育种改良,则有可能会得到具有多重优良性状(分散生长、代时短、合成次级代谢产物能力突出)的突变菌株。
随后,我们通过分析可能与GSUV3-205聚团生长密切相关的基因,选择目前还没有在纤维堆囊菌中进行过相关研究的pilA基因和epsD基因,通过克隆基因的同源片段构建了pCCPA和pCCED两种插入失活重组质粒。通过使用添加抗生素的接合转移体系,获得了70株抗性接合菌株。此后我们对其进行液体生长状态进行验证,发现这些抗性菌株经过长期培养并不能够在液体培养基中分散生长。在通过质粒营救进而对质粒的插入位点进行验证时,我们发现接合质粒并没有整合到我们预期的基因位点,因此在一些现有的实验数据的基础上,我们提出了纤维堆囊菌可以根据外界环境压力吸收外源DNA并随机整合到其染色体基因组上的推论。但是目前我们尚缺乏更确凿的证据来阐明这一理论的具体机制。
Myxobacteria are Gram-negative, unicellular gliding bacteria, which exhibit complicated multicellular social behavior. The microbes are capable of producing large numbers of bioactive compounds that show anticancer, antibacterial, fungicidal or immune-modulating activities. Thus far, about 100 basic structures and 500 structural analogs have been discovered in myxobacteria and have been fully characterized chemically, which account for about 3.5% of the presently known secondary metabolites of microbial origin. The diversity of the chemical structures and action mechanisms of the metabolites from myxobacteria can even be compared with those from Streptomyces and Bacillus.
Sorangium cellulosum is a species of myxobacteria belonging to the Myxococcales order, Sorangineae suborder, Polyangiaceae class, Sorangium genus and it can grow by degradation of cellulose. As we know, Sorangium has the largest prokaryotic genome hitherto, for instance, Sorangium cellulosum So ce56 harbors the genome with a size of approximately 13 Mb. As reported, over half of these genes are involved in the production of secondary metabolites. In attation, almost half of the bioactive secondary metabolites of myxobacteria were identified from Sorangium, and nearly all of the Sorangium strains were found to produce these bioactive compounds. Therefore, myxobacteira are not only important in fundamental studies, but also alluring by industrial application.
Epothilones, which are naturally produced by the myxobacterium Sorangium cellulosum, are anticancer agents that mimic the anticancer mechanisms of paclitaxel (i.e. microtubule stabilization), it also has activity to the tumor cells which are resistant to paclitaxel. Up to now, there are at least five epothilones or chemically modified derivatives of epothilone undergoing evaluation in clinical trials and one has already been authorized for clinical use by the U.S. Food and Drug Administration. However, the research and development of epothilone drugs is seriously limited by difficulties in their production.
The Sorangium GSUV3-205 strain was obtained by mutation and high-throughput screening based on the technology of genome shuffling. Our previous bioactivity screening analysis confirmed that GSUV3-205 is a promising strain for the production of bioactive secondary metabolites. However, the epothilone production became low by GSUV3-205 because of strain degradation and the epothilone production of other epothilone producer strains was very low in contrast to the biomass and substrate in the medium. Thus, it has become the most interesting questions for researchers that how to explore the potential of epothilone production by natural producers Sorangium in fermentation system.
In this thesis, we optimized and improved the culture conditions, nutrition and growth characteristic to improve the production of epothilones by S. cellulosum GSUV3-205 that had been confirmed by the previous study in our laboratory to be an epothilone producer, the results were excellent.
Process improvement is an important method to improve the production of metabolites of industrial microbiology for every metabolites producer. Therefor, this article firstly optimized the nutrition and culture conditions in the process of biosynthesis of epothilone by S. cellulosum GSUV3-205 though two phases.
PhaseⅠ, based on the physiological characteristics of GSUV3-205, we investigated some culture condition factors such as inoculum size, fermentation time, seed condition when inoculating, adding time of resin, shaker speed in the process of epothilone production. We determined the appropriate inoculum size and fermentation time (108/ml,9d) by formulating synthetic curve of epothilone at different inoculum size. Afterwards, we determined the appropriate seed condition (200rpm,5d) by analyzing the titer of epothilone on the different seed conditions at different inoculum size. Finally, we established the suitable adding time of resin (before sterilization) and shaker speed (200rpm) by single-factor design.
PhaseⅡ, We statistical optimized the medium components in order to maximize epothilone B production by S. cellulosum GSUV3-205 using multiple steps of the response surface methodology. The optimization experiments include two steps. Firstly, based on GFM medium used in initial experiments, we extended the nutrition components of fermentation medium by single-factor design, Plackett-Burman design and Box-Behnken RSM design, and obtained a primary optimized medium which components includes dextrin 3.0g; sucrose 0.5g; glucose 0.8g; soy powder 1.7g; Slim milk powder 1.0g; MgSO4.7H2O 1.0g; CaCl2 1.0g; EDTA-Fe3+ 2ml and trace element solution 0.5ml; distilled water 1000ml; Amberlite XAD-16 resin (Rohm and Haas) 2%(v/v); pH 7.2. In this medium, compared the GFM medium, complex carbon resource is 0.3% dextrin instead of 0.2% potato power and a new component sucrose was added, and the content of most other components were adjusted. The yield of epothilone B by GSUV3-205 in this medium reached 46.5mg/L, which was about 4.1 folds than the yield in the GFM medium. Then, we optimized in detail the components of the primary optimized medium. Based on this new incubation system, we elucidated the medium components that significantly affect epothilone B production with a fractional factorial design (FFD), and found three factor (Dextrin, Slim milk powder, MgSO4) were important for epothilone B production. These significant ingredients were optimized by central composite design (CCD). A statistical model: (Y=51.69+17.03×X2+1.17×X6+0.70×Xg-4.55×X2×X2+7.01×X2×X6-2.48×X6×X6-0.69×X2×X8-2.01×X6×X8+0.52×X8×X8) was established for the description of the relationships between the medium components and epothilone B production using statistical analysis system. The statistical signification of the model indicated that the experimental model was in good agreement with the experimental results. The optimal medium for producing epothilone B was obtained by RSM and canonical analysis of mathematical model using SAS. The components of the optimal medium includes dextrin 6.3g; sucrose 0.5g; glucose 0.8g; soy powder 1.7g; Slim milk powder 2.6g; MgSO4.7H2O 3.2g; CaCl2 1.0g; EDTA-Fe3+ 2ml and trace element solution 0.5ml; distilled water 1000ml; Amberlite XAD-16 resin (Rohm and Haas) 2%(v/v); pH 7.2. Epothilone B production in the optimal medium reached 82.0mg/L about 76.3% more than that in the primary optimized medium by GSUV3-205. For the first time, we optimized the process of biosynthesis of epothilone in the natural producer Sorangium. Meantime, we greatly improved the yield of epothilone and provided a way to the industrial production of epothilone.
Based on the literatures reported, we investigated some small molecules to examine the influences on biosynthesis of epothilone. The results showed that epothilone biosynthesis was inhibit when the small molecules, such as iron ions, betaine, acetate, propionate, methylmalonic acid, succinic acid and cysteine, was excess in the medium. However, the existence of some small molecules could stimulate the biosynthesis of epothilone by GSUV3-205 while they were added with appropriate amount at appropriate time. For instance, the epothilone production increased 16% when 0.05% of betaine hydrochloride was added to the medium in the third day of fermentation, and the yield of epothilone A and B increased 68% and 270% respectively while10 mM solution including acetate, methylmalonic acid and cysteine was added to the medium. In addition, we, for the first time, added methylmalonic acid which had a same carbon chain skeleton to the precursor of epothilone B methylpropionyl-CoA to the fermentation medium and achieved good results that the yield of epothilone B increased 86% in contrast to the control while 5 mM methylmalonic acid was added to the medium. These results provided a basis for further use of continuous fermentation technology with adding precursors, but also provide a guideline for selecting the metabolites from GSUV3-205 product.
S. cellulosum GSUV3-205 is agglomerate in the liquid culture; this character is a formidable inconvenience for the industrial production and is a difficult problem for genetic research on the strain. Accordingly, we hope to use biological methods to make GSUV3-205 dispersed in liquid culture while maintaining the ability of epothilone synthesis. This can increase the biomass to improve epothilone production, thus speeding up the process of its industrial application, on the other hand, it can bring great convenience when we further explore GSUV3-205 though molecular biology methods.
We tried to get a mutant that was dispersed in liquid culture through biological domestication and genetic manipulation on GSUV3-205. First, we designed a special domestication process of GSUV3-205, after 8 cycles of domestication; we finally got one mutant GSUV3-205XH which was dispersed in M26 liquid medium. Although GSUV3-205XH grow very slow and lost the ability to synthesize epothilone, we can use this strain to proceed protoplast fusion with the strain which has good ability to synthesize epothilone to get a mutant which both has the ability to synthesize epothilone and grow dispersed in liquid culture on basis of theory of genome shuffling in future.
Afterwards, we chose epsD and pilA gene, which might be closely related to the aggregation of GSUV3-205 and had not reported in Sorangium yet, to built two insertion inactivation plasmid pCCPA and pCCED by cloning the homologous fragments. We obtained 70 resistant strains by conjugation, then verified the status of these strains in liquid culture and found that all of these resistant strains had no ability to grow dispersed. In addition, we tested the insertion site of plasmids by the method plasmid rescue and the results showed that the plasmid did not insert to the right site of the genome of GSUV3-205. Thus, we proposed a corollary that Sorangium could absorb and random integrate exogenous DNA into the genome of chromosome at ambient pressure which caused the large genome of Sorangium. However, we were lack of more conclusive evidence to clarify the specific mechanism of this theory.
纤维堆囊菌(Sorangium cellulosum)属于粘球菌目、堆囊菌亚目、多囊菌科、堆囊菌属,可以通过降解纤维素进行生长。据我们了解,纤维堆囊菌拥有迄今为止发现的最大的原核生物基因组,如纤维堆囊菌So ce56的基因组大小约为13Mb。目前研究结果显示,在庞大的基因组中有大量基因被”奢侈”的用来进行次级代谢,它合成的次级代谢产物几乎占所有已发现粘细菌种类的一半,并且几乎100%的菌株都具有合成生物学活性物质的能力。因此,纤维堆囊菌不但具有重要的基础研究价值,而且还具有诱人的应用开发前景。
埃博霉素(epothilones)是粘细菌纤维堆囊菌(Sorangium cellulosum)产生的一类聚酮类次级代谢产物。与目前临床上广泛应用的抗肿瘤化疗药物紫杉醇相比,它具有相似的稳定微管活性,而且对紫杉醇耐药的肿瘤细胞具有活性。目前至少有五个埃博霉素类化合物(ixabepilone, patupilone, BMS-310705, KOS-862和ZK-EPO)已经进行了Ⅰ-Ⅲ期临床实验,而且目前已经有一种产品(ixabepilone)得到了美国FDA的权威认证。但是,目前埃博霉素的发酵生产能力成为该药研发的一个主要瓶颈。
菌株GSUV3-205是本实验室通过Genome Shuffling技术得到的一株具有较高产量的埃博霉素产生菌,具有一定的的工业应用前景。但是无论是GSUV3-205还是在国际上其他实验室的发酵生产菌株,埃博霉素的实际生产量相对于发酵培养体系、底物利用率以及生物量都存在着转化效率低的问题,它们还是有很大的提升空间的。而且,通过长时间的传代培养,重组菌株GSUV3-205的埃博霉素产生能力发生了很大的退化。因此,如何能够在发酵体系中充分挖掘菌株的埃博霉素的合成能力成为研究者最为感兴趣的问题之一,所以对于纤维堆囊菌合成埃博霉素过程中某些关键因子的研究,对纤维堆囊菌资源的开发利用和埃博霉素走向产业化都具有巨大的意义。
本论文以本实验室的一株埃博霉素产生菌纤维堆囊菌GSUV3-205为研究材料,以提高菌株的埃博霉素合成能力为主要目标,主要从菌株的培养条件、营养条件、生长状态等方面分别进行了一系列系统的改进和优化,并取得了良好的结果。
对于每一种能够合成有应用价值的代谢产物的微生物来说,发酵过程改良都是从实验室理论研究走向工业产业化道路的必要阶段,因此本文的研究工作首先分两个阶段分别对埃博霉素产生菌纤维堆囊菌GSUV3-205的发酵培养条件和营养条件进行了优化。
在第一阶段,我们结合GSUV3-205的生理特性,主要对接种量、发酵时间、种子培养及接种的状态、树脂添加时间、发酵培养转速等培养条件因素进行了系列考察。根据不同接种量下的埃博霉素产生曲线,确定了合适的接种量(5×109个细胞/50m1)和发酵培养时间(9天)；通过对不同接种量时不同种子培养接种状态的埃博霉素的产量进行分析,确定了最优的种子培养接种状态(种子200rpm培养5天)；通过单因子分析树脂添加时间和发酵培养转速这两个条件,得出了较有利于高产埃博霉素的条件,即灭菌时添加树脂和200rpm发酵培养。
第二个阶段,我们利用统计学优化方法中的响应面设计法以epothilone B的产量为响应值对GSUV3-205生产埃博霉素的发酵培养基成分进行了优化。这个阶段主要是通过两个大的步骤来实现的。首先,我们在前期使用的EPM培养基的基础上进行了营养成分的拓展和初步优化,即：利用单因子实验考察了21种EPM培养基中不包含的营养成分对epothilone B合成的影响,获得了7种较有利于合成埃博霉素的营养物质；然后利用Plackett-Burman和Box-Behnken响应面设计把通过单因子实验拓展的7种营养成分和EPM培养基中原有的所有成分进行了优化,通过重要营养成分的数学建模和响应面分析,获得了一个初步优化的培养基。其成分是：糊精0.3%、蔗糖0.05%、葡萄糖0.08%、大豆粉0.17%、脱脂奶粉0.1%、七水硫酸镁0.1%、无水氯化钙0.1%、EDTA-Fe3+溶液2m1/L、微量元素液0.5ml/L、pH7.2、吸附树脂2%(v/v)。可以看出,与EPM培养基相比(土豆淀粉0.2%、葡萄糖0.2%、大豆粉0.2%、脱脂奶粉0.1%、七水硫酸镁0.1%、无水氯化钙0.1%、EDTA-Fe3+溶液1ml/L、微量元素液1ml/L、VB12 0.5mg/l、pH7.2、吸附树脂2%(v/v)),优化培养基中复杂碳源由土豆淀粉换成糊精且增加了添加量,同时增加了一个新的简单碳源即二糖物质蔗糖,并对绝大部分成分的含量进行了调整。利用这个培养基epothilone B的产量可以达到46.5mg/1,较原始产量11.3 mg/1提高了3.1倍。此后,我们对初步优化的培养基又进行了进一步的系统优化：基于这个新的培养体系,利用FFD设计考察了这个体系各个组成成分对epothilone B产量的影响。发现三个因子糊精(X2)、脱脂奶粉(X6)和硫酸镁(X8)对epothilone B的产量具有显著性影响。利用CCD这种五水平的响应面设计法对这三个因素进行了进一步研究,建立了相应的二次回归模型：Y=51.69+17.03×X2+1.17×X6+0.70×X8-4.55×X2×X2+7.01×X2×X6-2.48×X6×X6-0.69×X2×X8-2.01×X6×X8+0.52×X8×X8利用SAS 8.0软件的RSREG过程对这个模型的统计学意义进行分析,说明了建立的数学模型和实验结果有好的相关性。通过模型可以看出糊精对于epothilone B的产量具有显著影响,糊精和脱脂奶粉对epothilone B产量的影响具有较强的交互作用。最后获得的优化培养基成分为：糊精0.63%、蔗糖0.05%、葡萄糖0.08%、大豆粉0.17%、脱脂奶粉0.26%、七水硫酸镁0.32%、无水氯化钙0.1%、EDTA-Fe3+溶液2ml/L、微量元素液0.5m1/L、pH7.0、吸附树脂2%(v/v)。So0157-2利用此培养体系发酵培养,可产生82.0mg/1的epothilone B,较初步优化培养基(46.5mg/1)又提高了76.3%。这是首次在能够产生埃博霉素的本源菌纤维堆囊菌中进行的系统发酵过程优化,并把GSUV3-205的埃博霉素产量水平提高到了较高的水平,为下一步开展发酵罐中试和最终的工业化生产奠定了坚实的基础。
本文随后的工作结合相关的文献报道,将有可能与纤维堆囊菌发酵合成埃博霉素过程密切相关的小分子物质,梯度添加到优化后的发酵培养基中,探讨了它们对GSUV3-205埃博霉素合成能力的影响。
研究表明,铁离子、甜菜碱类物质以及埃博霉素合成的前体物质乙酸盐、丙酸盐、甲基丙二酸、琥珀酸、半胱氨酸等物质的过量添加,都会对GSUV3-205合成埃博霉素的过程有抑制作用。但是在适当的添加时间和添加量时,这些小分子物质的存在会促进GSUV3-205埃博霉素的合成。比如,在发酵第三天添加0.05%的98%的盐酸甜菜碱时,埃博霉素产量有16%的提高；在添加10 mM的乙酸钠、甲基丙二酸、半胱氨酸混合溶液时,epothilone A和B的产量分别提高了68%和270%。由此看见,这些小分子物质的添加是与So0157-2的埃博霉素合成能力密切相关的。其中,我们首次将与epothilone B合成的前体物质甲基丙二酸单酰-CoA具有相同碳链骨架的甲基丙二酸添加到发酵培养中,并取得了良好的效果：在添加5 mM的甲基丙二酸时,epothilone B的产量比对照提高了86%。这些结果为进一步利用连续或半连续发酵技术进行前体物质添加提供了基础,同时也为GSUV3-205发酵生产埃博霉素的产物选择提供了思路。
GSUV3-205在液体培养基中聚团生长的特性成为该菌株大规模发酵及产业化道路上的重要障碍,同时这对于GSUV3-205的某些性质研究及遗传操作方面来说也是一个巨大的难题。我们希望通过生物学手段对GSUV3-205进行性状改造,在保持埃博霉素产生能力不变的情况下使其能够在液体培养基中分散生长。这样一方面可以提高单位培养基中的生物量从而提高埃博霉素的单位产量,加快其工业化应用的进程,另一方面,还能够给我们进一步探索GSUV3-205的性质及遗传操作带来极大的方便。
因此我们从生物驯化和遗传改造这两个角度进行了获取GSUV3-205液体分散生长突变株的尝试。通过特殊设计的驯化流程对GSUV3-205进行了长时间的驯化培养。经过8个循环的驯化培养,我们最终得到了一株在M26液体培养基中生长状态有些分散的菌株GSUV3-205XH。尽管通过一系列的验证及分析发现,GSUV3-205XH生长非常缓慢而且几乎没有埃博霉素产生能力,但是今后我们可以结合genome shuffling的思路方法利用这个菌株与埃博霉素高产菌株进行原生质体融合从而对菌株进行育种改良,则有可能会得到具有多重优良性状(分散生长、代时短、合成次级代谢产物能力突出)的突变菌株。
随后,我们通过分析可能与GSUV3-205聚团生长密切相关的基因,选择目前还没有在纤维堆囊菌中进行过相关研究的pilA基因和epsD基因,通过克隆基因的同源片段构建了pCCPA和pCCED两种插入失活重组质粒。通过使用添加抗生素的接合转移体系,获得了70株抗性接合菌株。此后我们对其进行液体生长状态进行验证,发现这些抗性菌株经过长期培养并不能够在液体培养基中分散生长。在通过质粒营救进而对质粒的插入位点进行验证时,我们发现接合质粒并没有整合到我们预期的基因位点,因此在一些现有的实验数据的基础上,我们提出了纤维堆囊菌可以根据外界环境压力吸收外源DNA并随机整合到其染色体基因组上的推论。但是目前我们尚缺乏更确凿的证据来阐明这一理论的具体机制。
Myxobacteria are Gram-negative, unicellular gliding bacteria, which exhibit complicated multicellular social behavior. The microbes are capable of producing large numbers of bioactive compounds that show anticancer, antibacterial, fungicidal or immune-modulating activities. Thus far, about 100 basic structures and 500 structural analogs have been discovered in myxobacteria and have been fully characterized chemically, which account for about 3.5% of the presently known secondary metabolites of microbial origin. The diversity of the chemical structures and action mechanisms of the metabolites from myxobacteria can even be compared with those from Streptomyces and Bacillus.
Sorangium cellulosum is a species of myxobacteria belonging to the Myxococcales order, Sorangineae suborder, Polyangiaceae class, Sorangium genus and it can grow by degradation of cellulose. As we know, Sorangium has the largest prokaryotic genome hitherto, for instance, Sorangium cellulosum So ce56 harbors the genome with a size of approximately 13 Mb. As reported, over half of these genes are involved in the production of secondary metabolites. In attation, almost half of the bioactive secondary metabolites of myxobacteria were identified from Sorangium, and nearly all of the Sorangium strains were found to produce these bioactive compounds. Therefore, myxobacteira are not only important in fundamental studies, but also alluring by industrial application.
Epothilones, which are naturally produced by the myxobacterium Sorangium cellulosum, are anticancer agents that mimic the anticancer mechanisms of paclitaxel (i.e. microtubule stabilization), it also has activity to the tumor cells which are resistant to paclitaxel. Up to now, there are at least five epothilones or chemically modified derivatives of epothilone undergoing evaluation in clinical trials and one has already been authorized for clinical use by the U.S. Food and Drug Administration. However, the research and development of epothilone drugs is seriously limited by difficulties in their production.
The Sorangium GSUV3-205 strain was obtained by mutation and high-throughput screening based on the technology of genome shuffling. Our previous bioactivity screening analysis confirmed that GSUV3-205 is a promising strain for the production of bioactive secondary metabolites. However, the epothilone production became low by GSUV3-205 because of strain degradation and the epothilone production of other epothilone producer strains was very low in contrast to the biomass and substrate in the medium. Thus, it has become the most interesting questions for researchers that how to explore the potential of epothilone production by natural producers Sorangium in fermentation system.
In this thesis, we optimized and improved the culture conditions, nutrition and growth characteristic to improve the production of epothilones by S. cellulosum GSUV3-205 that had been confirmed by the previous study in our laboratory to be an epothilone producer, the results were excellent.
Process improvement is an important method to improve the production of metabolites of industrial microbiology for every metabolites producer. Therefor, this article firstly optimized the nutrition and culture conditions in the process of biosynthesis of epothilone by S. cellulosum GSUV3-205 though two phases.
PhaseⅠ, based on the physiological characteristics of GSUV3-205, we investigated some culture condition factors such as inoculum size, fermentation time, seed condition when inoculating, adding time of resin, shaker speed in the process of epothilone production. We determined the appropriate inoculum size and fermentation time (108/ml,9d) by formulating synthetic curve of epothilone at different inoculum size. Afterwards, we determined the appropriate seed condition (200rpm,5d) by analyzing the titer of epothilone on the different seed conditions at different inoculum size. Finally, we established the suitable adding time of resin (before sterilization) and shaker speed (200rpm) by single-factor design.
PhaseⅡ, We statistical optimized the medium components in order to maximize epothilone B production by S. cellulosum GSUV3-205 using multiple steps of the response surface methodology. The optimization experiments include two steps. Firstly, based on GFM medium used in initial experiments, we extended the nutrition components of fermentation medium by single-factor design, Plackett-Burman design and Box-Behnken RSM design, and obtained a primary optimized medium which components includes dextrin 3.0g; sucrose 0.5g; glucose 0.8g; soy powder 1.7g; Slim milk powder 1.0g; MgSO4.7H2O 1.0g; CaCl2 1.0g; EDTA-Fe3+ 2ml and trace element solution 0.5ml; distilled water 1000ml; Amberlite XAD-16 resin (Rohm and Haas) 2%(v/v); pH 7.2. In this medium, compared the GFM medium, complex carbon resource is 0.3% dextrin instead of 0.2% potato power and a new component sucrose was added, and the content of most other components were adjusted. The yield of epothilone B by GSUV3-205 in this medium reached 46.5mg/L, which was about 4.1 folds than the yield in the GFM medium. Then, we optimized in detail the components of the primary optimized medium. Based on this new incubation system, we elucidated the medium components that significantly affect epothilone B production with a fractional factorial design (FFD), and found three factor (Dextrin, Slim milk powder, MgSO4) were important for epothilone B production. These significant ingredients were optimized by central composite design (CCD). A statistical model: (Y=51.69+17.03×X2+1.17×X6+0.70×Xg-4.55×X2×X2+7.01×X2×X6-2.48×X6×X6-0.69×X2×X8-2.01×X6×X8+0.52×X8×X8) was established for the description of the relationships between the medium components and epothilone B production using statistical analysis system. The statistical signification of the model indicated that the experimental model was in good agreement with the experimental results. The optimal medium for producing epothilone B was obtained by RSM and canonical analysis of mathematical model using SAS. The components of the optimal medium includes dextrin 6.3g; sucrose 0.5g; glucose 0.8g; soy powder 1.7g; Slim milk powder 2.6g; MgSO4.7H2O 3.2g; CaCl2 1.0g; EDTA-Fe3+ 2ml and trace element solution 0.5ml; distilled water 1000ml; Amberlite XAD-16 resin (Rohm and Haas) 2%(v/v); pH 7.2. Epothilone B production in the optimal medium reached 82.0mg/L about 76.3% more than that in the primary optimized medium by GSUV3-205. For the first time, we optimized the process of biosynthesis of epothilone in the natural producer Sorangium. Meantime, we greatly improved the yield of epothilone and provided a way to the industrial production of epothilone.
Based on the literatures reported, we investigated some small molecules to examine the influences on biosynthesis of epothilone. The results showed that epothilone biosynthesis was inhibit when the small molecules, such as iron ions, betaine, acetate, propionate, methylmalonic acid, succinic acid and cysteine, was excess in the medium. However, the existence of some small molecules could stimulate the biosynthesis of epothilone by GSUV3-205 while they were added with appropriate amount at appropriate time. For instance, the epothilone production increased 16% when 0.05% of betaine hydrochloride was added to the medium in the third day of fermentation, and the yield of epothilone A and B increased 68% and 270% respectively while10 mM solution including acetate, methylmalonic acid and cysteine was added to the medium. In addition, we, for the first time, added methylmalonic acid which had a same carbon chain skeleton to the precursor of epothilone B methylpropionyl-CoA to the fermentation medium and achieved good results that the yield of epothilone B increased 86% in contrast to the control while 5 mM methylmalonic acid was added to the medium. These results provided a basis for further use of continuous fermentation technology with adding precursors, but also provide a guideline for selecting the metabolites from GSUV3-205 product.
S. cellulosum GSUV3-205 is agglomerate in the liquid culture; this character is a formidable inconvenience for the industrial production and is a difficult problem for genetic research on the strain. Accordingly, we hope to use biological methods to make GSUV3-205 dispersed in liquid culture while maintaining the ability of epothilone synthesis. This can increase the biomass to improve epothilone production, thus speeding up the process of its industrial application, on the other hand, it can bring great convenience when we further explore GSUV3-205 though molecular biology methods.
We tried to get a mutant that was dispersed in liquid culture through biological domestication and genetic manipulation on GSUV3-205. First, we designed a special domestication process of GSUV3-205, after 8 cycles of domestication; we finally got one mutant GSUV3-205XH which was dispersed in M26 liquid medium. Although GSUV3-205XH grow very slow and lost the ability to synthesize epothilone, we can use this strain to proceed protoplast fusion with the strain which has good ability to synthesize epothilone to get a mutant which both has the ability to synthesize epothilone and grow dispersed in liquid culture on basis of theory of genome shuffling in future.
Afterwards, we chose epsD and pilA gene, which might be closely related to the aggregation of GSUV3-205 and had not reported in Sorangium yet, to built two insertion inactivation plasmid pCCPA and pCCED by cloning the homologous fragments. We obtained 70 resistant strains by conjugation, then verified the status of these strains in liquid culture and found that all of these resistant strains had no ability to grow dispersed. In addition, we tested the insertion site of plasmids by the method plasmid rescue and the results showed that the plasmid did not insert to the right site of the genome of GSUV3-205. Thus, we proposed a corollary that Sorangium could absorb and random integrate exogenous DNA into the genome of chromosome at ambient pressure which caused the large genome of Sorangium. However, we were lack of more conclusive evidence to clarify the specific mechanism of this theory.
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