衣藻叶绿体表达系统的改造和应用及木聚糖酶工程菌的发酵研究
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摘要
素有“绿色酵母”之称的莱茵哈德衣藻(Chlamydomonas reinhardtii)是一种单细胞小球状绿藻,具有一个大型的杯状叶绿体,其中含有上80个拷贝的叶绿体基因组,它是研究光合作用最简单的模式生物。莱茵哈德衣藻营养简单,代谢能力强,生长快(世代时间6-8小时)；在光照和CO2培养条件下能产生比常见陆生植物(如玉米)高得多的生物量,可以大规模产业化生产；其代谢途径比植物简单得多,不产生毒素和一些复杂化合物；同时也具有许多动、植物细胞共同特征,使蛋白质翻译后加工和正确折叠而产生有生物学活性的蛋白质,为超量表达外源蛋白提供了一个理想的空间。因此,开展衣藻叶绿体高效表达系统的研究和改造,不仅可以丰富衣藻叶绿体分子生物学研究内容,也具有极大的实际应用价值。本文以我国畜牧业中造成危害的口蹄疫病毒抗原基因为材料,构建了用于超量表达和增强免疫原性的叶绿体表达系统,并简化了衣藻叶绿体转化方法,为利用低等生物大规模生产动物口服疫苗奠定基础。主要研究结果如下：
1.构建了一系列的通用载体,为构建外源基因的衣藻叶绿体表达重组质粒简化了操作过程；
2.用电穿孔的方法将一系列衣藻叶绿体重组表达质粒导入到不同生理状态的感受细胞中,建立了一套简单高效的叶绿体转化策略,为衣藻的叶绿体转化提供了另一个选择方案。与粒子轰击技术相比,电穿孔法具有转化效率高、操作更为简便、得到转化子的周期更短而且真菌污染的可能性更小的优点。进一步分析说明：受体细胞细胞壁的降解与再生对电穿孔法介导的衣藻叶绿体转化有重要影响；
3.对转基因衣藻继代培养过程中转基因丢失情况的检测结果说明：叶绿体基因组上的转基因迟早会丢失,但在用5-氟-2-脱氧尿嘧啶处理的受体细胞得到的转基因衣藻中,转基因丢失的时间可以延长一倍。另外,提高培养基中的氮源也可以将转基因丢失的时间延长一个继代培养周期；
4.将编码霍乱毒素B亚基的CTB基因与口蹄疫病毒的主要表面抗原基因VP1融合成抗原基因CV1,构建了CV1基因的大肠杆菌重组表达质粒和衣藻叶绿体重组表达质粒。将大肠杆菌表达的CV1包涵体蛋白免疫兔得到了CV1蛋白的抗体,并用这个抗体检测到了衣藻叶绿体中表达的CV1抗原蛋白。转基因衣藻中CV1基因丢失情况的检测为衣藻替代转基因植物生产口蹄疫口服疫苗提供了工作基础。
木聚糖酶在食品、饲料、酿酒和造纸等现代工业领域发挥着重要作用,最近其在生物能源和燃料酒精方面的应用潜力更是吸引了越来越多的关注。本研究在3 L的小发酵罐中进行了一株高产木聚糖酶毕赤酵母工程菌GS115-xylCX8的初步发酵生产。受设备所提供的溶氧水平限制,只得到了140g/L细胞湿重的生物量,但经过大约150 h的甲醇诱导后,仍然获得了高达7340U/mL的产酶水平,是摇瓶诱导表达水平的4倍多,这说明本实验室构建的木聚糖酶工程菌GS115-xylCX8具有极其诱人的商业化生产潜力。
So-called "green yeast", Chlamydomonas reinhardtii is a unicellular spheroidal green alga with a single cup-shaped chloroplast, which has around 80 copies in its genome. The alga had become one of the simplest model organisms for its inexpensive cost of culture, robust metabolic capacity and rapid growth rate (approximately 6-8 h generation time). Large-scale production can be carried out because it yields much biomass more than conventional terrestrial plant (for example, corn) under illumination and CO2 supply. Furthermore, it has no toxin and complicated compound, and has too much simpler metabolic pathway than plant. In addition, the green alga is a perfect platform of recombinant protein overexpression because the cell shares some common physiological features, which can assembly protein into biological active complex by post-translational processing. For this, research and modification of efficient expression system in C.reinhardtii can not only enrich study on molecular biology of chloroplast, but also tremendously practical applications. In this research, the interest gene is VP1 antigen gene of foot-and-mouth disease virus, which resulted in substantial losses in Chinese stockbreeding. A chloroplast protein overexpression system with enhancing element of immunogenicity and a simpler transformation strategy of chloroplast was established in chlamydomonas, thus it become possible that inferior organism can be applicable to production of large-scale edible vaccine. The results as follows:
1. We constructed a series of compatible plasmids, which facilitate construction of chloroplast expression plasmid with interest gene in chlamydomonas;
2. A series of different expression plasmids had been introduced into recipient cell at different physiological status by electroporation. We achieve a simpler high-frequency transformation method, which provide an alternative for chloroplast transformation in C.reinhardtii. Compared to particle bombardment technology, the method has several advantages:higher transformation rate, simpler manipulation. more time-saving for transgenic line generation and lesser risk of fungal contamination. Further analysis demonstrated that degradation and regerenation of recipient cell wall have a crucial effect on chloroplast transformation by electroporation in C.reinhardtii.
3. The transgenic fragment was analyzed by PCR during subculture. The results showed that transgene integrated with chloroplast genome will be lost sooner or later. However, the process can be extended for doubling time when the recipient cell is treated by 5-fluorodeoxyuridine prior to transformation. On the other hand, the existence of transgene will be prolonged a period of subculture time in rich nitrogen sources medium.
4. The cholera toxin B gene and foot and mouth disease virus VP1 gene was fused with CV1 fragment, which is constructed into prokaryotic and chloroplast expression plasmids. The CV1 inclusion body expressed in E.coli was purified and immuned rabbit, which produced anti-CV1 serum. The antibody analyzed the recombinant CV1 antigen in chlamydomonas chloroplast by western blotting. The detection data of CV1 fragment in transgenic alga carried out element task for edible vaccine production using the green alga as target host, rather than higher plant.
Xylanases not only play an important role on a wide array of biotechnological and industrial applications in processes such as food, animal feed, liquor-making and paper pulp bleach, but also have great potentials in the bioconversion from lignocellulosic feedstocks to fuel-grade ethanol, which have attracted tremendous interests in the past few years. In this section, an engineering pichia yeast GS115-xylCX8 secreting high-activity xylanase was carried out scale zymolysis for combinant enzyme production in the fermentor (NBS 3 L). Although the wet cell weight of the yeast cells was only 140 g/L for dissolved oxygen limitation, the yield of the recombinant xylanase were 7340 U/mL after approximately 150 h methanol inducement. The yield of the fermentor was as more than 4-fold as that of the shake flask; it is promising to produce the recombinant xylanase commercially.
1.构建了一系列的通用载体,为构建外源基因的衣藻叶绿体表达重组质粒简化了操作过程；
2.用电穿孔的方法将一系列衣藻叶绿体重组表达质粒导入到不同生理状态的感受细胞中,建立了一套简单高效的叶绿体转化策略,为衣藻的叶绿体转化提供了另一个选择方案。与粒子轰击技术相比,电穿孔法具有转化效率高、操作更为简便、得到转化子的周期更短而且真菌污染的可能性更小的优点。进一步分析说明：受体细胞细胞壁的降解与再生对电穿孔法介导的衣藻叶绿体转化有重要影响；
3.对转基因衣藻继代培养过程中转基因丢失情况的检测结果说明：叶绿体基因组上的转基因迟早会丢失,但在用5-氟-2-脱氧尿嘧啶处理的受体细胞得到的转基因衣藻中,转基因丢失的时间可以延长一倍。另外,提高培养基中的氮源也可以将转基因丢失的时间延长一个继代培养周期；
4.将编码霍乱毒素B亚基的CTB基因与口蹄疫病毒的主要表面抗原基因VP1融合成抗原基因CV1,构建了CV1基因的大肠杆菌重组表达质粒和衣藻叶绿体重组表达质粒。将大肠杆菌表达的CV1包涵体蛋白免疫兔得到了CV1蛋白的抗体,并用这个抗体检测到了衣藻叶绿体中表达的CV1抗原蛋白。转基因衣藻中CV1基因丢失情况的检测为衣藻替代转基因植物生产口蹄疫口服疫苗提供了工作基础。
木聚糖酶在食品、饲料、酿酒和造纸等现代工业领域发挥着重要作用,最近其在生物能源和燃料酒精方面的应用潜力更是吸引了越来越多的关注。本研究在3 L的小发酵罐中进行了一株高产木聚糖酶毕赤酵母工程菌GS115-xylCX8的初步发酵生产。受设备所提供的溶氧水平限制,只得到了140g/L细胞湿重的生物量,但经过大约150 h的甲醇诱导后,仍然获得了高达7340U/mL的产酶水平,是摇瓶诱导表达水平的4倍多,这说明本实验室构建的木聚糖酶工程菌GS115-xylCX8具有极其诱人的商业化生产潜力。
So-called "green yeast", Chlamydomonas reinhardtii is a unicellular spheroidal green alga with a single cup-shaped chloroplast, which has around 80 copies in its genome. The alga had become one of the simplest model organisms for its inexpensive cost of culture, robust metabolic capacity and rapid growth rate (approximately 6-8 h generation time). Large-scale production can be carried out because it yields much biomass more than conventional terrestrial plant (for example, corn) under illumination and CO2 supply. Furthermore, it has no toxin and complicated compound, and has too much simpler metabolic pathway than plant. In addition, the green alga is a perfect platform of recombinant protein overexpression because the cell shares some common physiological features, which can assembly protein into biological active complex by post-translational processing. For this, research and modification of efficient expression system in C.reinhardtii can not only enrich study on molecular biology of chloroplast, but also tremendously practical applications. In this research, the interest gene is VP1 antigen gene of foot-and-mouth disease virus, which resulted in substantial losses in Chinese stockbreeding. A chloroplast protein overexpression system with enhancing element of immunogenicity and a simpler transformation strategy of chloroplast was established in chlamydomonas, thus it become possible that inferior organism can be applicable to production of large-scale edible vaccine. The results as follows:
1. We constructed a series of compatible plasmids, which facilitate construction of chloroplast expression plasmid with interest gene in chlamydomonas;
2. A series of different expression plasmids had been introduced into recipient cell at different physiological status by electroporation. We achieve a simpler high-frequency transformation method, which provide an alternative for chloroplast transformation in C.reinhardtii. Compared to particle bombardment technology, the method has several advantages:higher transformation rate, simpler manipulation. more time-saving for transgenic line generation and lesser risk of fungal contamination. Further analysis demonstrated that degradation and regerenation of recipient cell wall have a crucial effect on chloroplast transformation by electroporation in C.reinhardtii.
3. The transgenic fragment was analyzed by PCR during subculture. The results showed that transgene integrated with chloroplast genome will be lost sooner or later. However, the process can be extended for doubling time when the recipient cell is treated by 5-fluorodeoxyuridine prior to transformation. On the other hand, the existence of transgene will be prolonged a period of subculture time in rich nitrogen sources medium.
4. The cholera toxin B gene and foot and mouth disease virus VP1 gene was fused with CV1 fragment, which is constructed into prokaryotic and chloroplast expression plasmids. The CV1 inclusion body expressed in E.coli was purified and immuned rabbit, which produced anti-CV1 serum. The antibody analyzed the recombinant CV1 antigen in chlamydomonas chloroplast by western blotting. The detection data of CV1 fragment in transgenic alga carried out element task for edible vaccine production using the green alga as target host, rather than higher plant.
Xylanases not only play an important role on a wide array of biotechnological and industrial applications in processes such as food, animal feed, liquor-making and paper pulp bleach, but also have great potentials in the bioconversion from lignocellulosic feedstocks to fuel-grade ethanol, which have attracted tremendous interests in the past few years. In this section, an engineering pichia yeast GS115-xylCX8 secreting high-activity xylanase was carried out scale zymolysis for combinant enzyme production in the fermentor (NBS 3 L). Although the wet cell weight of the yeast cells was only 140 g/L for dissolved oxygen limitation, the yield of the recombinant xylanase were 7340 U/mL after approximately 150 h methanol inducement. The yield of the fermentor was as more than 4-fold as that of the shake flask; it is promising to produce the recombinant xylanase commercially.
引文
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