【作者】 曾丽香；

【导师】 曾庆平；

【作者基本信息】 广州中医药大学 ， 中医临床基础， 2010， 硕士

【摘要】 青蒿素的化学本质是倍半萜内酯过氧化物,它是由我国科技工作者于20世纪70年代从传统中药青蒿中分离纯化的抗疟单体。作为一种次生代谢产物,青蒿素在青蒿中的含量极低。同时,野生青蒿资源非常有限,人工种植青蒿要占用大量耕地,化学合成青蒿素的反应复杂、成本高、毒性大,无法实现工业化生产,于是国内外兴起了一股利用基因工程微生物生产青蒿素的热潮。可是,到目前为止,还没有一种基因工程微生物能实现青蒿素的全合成,而只能合成青蒿素的前体。在成功构建表达青蒿紫穗槐二烯合酶基因(ADS)的酵母工程菌并获得青蒿素前体紫穗槐二烯的基础上,本研究进一步将青蒿素合成所需的细胞色素P450单加氧酶基因(CYP71AV1)和青蒿醛双键还原酶基因(DBR2)与ADS一起导入酵母菌中,通过PCR、双酶切和重测序确认了携带上述青蒿素合成基因的重组质粒在酵母工程菌中的存在,并利用表型鉴定和RT-PCR证实了重组青蒿素合成基因的功能表达。酸化水相GC-MS分析表明,酵母工程菌的发酵产物比野生型酵母明显增加,其中三基因转化酵母(ADS+CYP71AV1+DBR2)的产物多于双基因转化酵母(ADS+CYP71AV1),而双基因转化酵母(ADS+CYP71AV1)的产物又多于单基因转化酵母(ADS),表明不同青蒿合成酶基因在酵母工程菌中的表达导致了不同青蒿素前体的合成。有机相GC-MS分析测得紫穗槐二烯的生成,其含量为1.7μg/mL。为了将酵母合成的青蒿素前体通过酶促反应转变成青蒿素,本研究将上述不同酵母工程菌的正己烷抽提物与冷处理青蒿无细胞酶混合液保温进行生物转化,并通过HPLC测定其青蒿素含量。结果表明,转基因酵母生物转化产物中的青蒿素含量显著提高,最高达到3.44mg/mL,这是迄今为止青蒿素含量提高幅度最大的青蒿素高产代谢工程研究之一。本研究的创新之处在于：(1)成功培育能同时表达3个青蒿素合成酶基因的新型酵母工程菌；(2)将酵母工程菌生产的青蒿素前体通过生物转化技术转变成青蒿素；(3)建立了利用微生物(酵母)和植物(青蒿)相互合作生产青蒿素的“二步法”。本研究为充分发挥工业发酵规模性与农业种植经济性的双重优势大幅度提高青蒿素产量提供了理论依据,同时为初步解决目前青蒿素原料供给紧张局面满足全球抗疟药市场需求提出了一个可行的解决办法。更多还原

【Abstract】 The chemical nature of artemisinin is the sesquiterpene lactone peroxide, which is an antimalarial monomer isolated and purified from the traditional Chinese medicinal herb Artemisia annua by Chinese scientists in 1970’s. As a kind of secondary metabolites, artemisinin scarcely accumulates in A. annua. Additionally, the natural resource of A. annua is extremely rare; plantation of A. annua occupys a large area of cultivated fields; and chemical biosynthesis of artemisinin cannot be readily industrialized due to involving a complicated, costly and toxic process. In consequence, production of artemisinin in engineered microorganisms has been attempted worldwide although no artemisinin per se rather than artemisinin precursors has been synthesized in any engineered microorganisms up to the present. With success in construction of the engineered yeast that expresses amorphadiene synthase gene (ADS) of A. annua and production of the artemisinin precursor amorphadiene, we further introduce two other necessary genes for artemisinin biosynthesis, cytochrome P450 monooxygenase gene (CYP71AV1) and artemisinic aldehyde double-bond reductase gene (DBR2), into the yeast with ADS. The presence of the recombinant plasmid containing A. annua genes in the engineered yeast was verified by PCR, double digestion and re-sequencing, while the functional expression of recombinant artemisinin biosynthetic genes was validated by phenotyping and RT-PCR. GC-MS analysis of the acidic water phase indicated that fermtative products in three-type of engineered yeast strains were more than those in the wild-type yeast, in which products in the three-gene-transferred yeast were more than those in the two-gene-transferred yeast, whereas products in the two-gene-transferred yeast were more than those in the one-gene-transferred yeast, demonstrating that the expression of different artemisinin biosynthetic genes in engineered yeast cells led to the biosynthesis of distinct artemisinin precursors. GC-MS analysis of the organic phase confirmed the presence of amorphadiene with 1.7μg/mL. To enzymatically convert the yeast-produced artemisinin precursors into artemisinin, biotransformation by incubation of the hexane extract from each engineered yeast with the cell-free enzyme mixture of cold-acclimed A. annua was carried out and artemisinin content was monitored by HPLC. As results, artemisinin content in biotransformation products of transgenic yeast strains was 3.28mg/mL, accounting for approximately 10 folds higher than the control, which may represent one of the most enhanced artemisinin production projects of metabolic engineering aiming at artemisinin overproduction. The creative outcomes of the present study are that:(1) a novel engineered yeast that simultaneously express three artemisinin biosynthetic genes has been available; (2) artemisinin precursors produced by engineered yeast strains have been converted to artemisinin by the biotransformation procedure; (3) a "two-step method" for production of artemisinin by the interplay between the microorganism (yeast) and the plant(A. annua) has been established. The prospective applications of these achievements are providing thereotic supports for harnessing dual merits of the scaled industrial fermentation and the economic agricultural plantation to realize enhanced artemisinin production and to resolving the predicment in the insufficient artemisinin supply.更多还原