青蒿醛脱氢酶基因克隆及青蒿琥酯抗菌增敏作用研究
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摘要
目的：
青蒿素以高效抗疟作用最为著名,但它也具有抗肿瘤、抗炎症(自身免疫病)等多种功效。由于世界市场供应的青蒿素主要来自我国人工种植的青蒿,其青蒿素含量偏低,生产成本较高,加之种植规模逐年萎缩,因而难以完全满足全球疟疾感染者对青蒿素类抗疟药的庞大需求,于是利用基因工程微生物大规模生产青蒿素成为当前最有希望的解决方案。本研究还对青蒿琥酯的抗菌作用进行了分子药理学评价,为利用抗生素增敏剂克服“超级细菌”日益严重的抗生素耐药性提供新思路。
方法：
本研究从青蒿中克隆了一个新的青蒿素前体合成基因——青蒿醛脱氢酶基因-1(ALDH1),并经双酶切、扩增和重测序予以确认。通过构建pESC-ALDH1表达载体(含尿嘧啶合成基因)并导入酿酒酵母YPH501菌株(尿嘧啶缺陷型)后,获得能在SD-U(尿嘧啶缺陷培养液)中生长的转化子。将酵母工程菌培养48-72h后,加入半乳糖处理12h诱导ALDH1基因表达,由青蒿试管苗制备无细胞提取液,对酵母发酵产物进行生物转化。
在青蒿琥酯抗菌增敏作用研究体外实验中我们检测经过缺氧适应的地衣芽孢杆菌产生的NO值和生长状态(OD值)以及单纯添加抗生素和抗生素和青蒿琥酯联用时NO值和过氧化氢酶活性。在体内实验中以大肠杆菌和地衣芽孢杆菌分别作为革兰氏阴性菌(G-)和阳性菌(G+)的代表,通过粪便菌落培养和计数或血清NO含量测定,在活菌饲喂导致肠道细菌感染的小鼠中评价了青蒿琥酯对抗生素的体内增敏效果。
结果：
通过气象色谱-质谱分析可见,转化样品中的青蒿素含量高于对照样品,初步证实青蒿ALDH1基因已在酵母细胞中实现功能表达。
青蒿琥酯抗菌作用体外实验结果可见青蒿琥酯与一氧化氮合酶(NOS)抑制剂L-NMMA一样,可阻断缺氧及缺氧+冷处理诱导的细菌一氧化氮(NO)合成,也能抑制细菌过氧化氢酶(CAT)活性。体内实验可见,青蒿琥酯不仅能提高头孢菌素对G-细菌和利福平对G+细菌的杀伤效果,而且还可辅助氨苄青霉素抑制抗药性细菌的繁殖。
结论：
以上结果为进一步创建青蒿素全合成微生物平台奠定了基础,为克服“超级细菌”的抗生素抗性提供了新思路。
Objective
While artemisinin is most famous as a highly effective anti-malarial drug, it can also possess other therapeutic efficacies including anti-tumor and anti-inflammation (autoimmune diseases). Artemisinin that has been supplied in the global anti-malarial market is mainly manufactured from field cultivated Artemisia annua in China. Due to the limitation of lower artemisinin content, higher production cost, and less scale of plantation annually, much need for artemisinin-derived drugs can not be fully met for all malarial patients worldwide. Large-scale production of artemisinin in genetically engineered microorganisms, therefore, has become the most promising solution. This study has also evaluated the anti-bacterial role of artesunate from the viewpoint of molecular pharmacology.
Methods
The present study has reported the cloning of a new artemisinin precursor gene, artemisinic aldehyde dehydrogenase1(ALDH1), which was clarified by double digestions, amplification, and re-sequencing. Upon constructing the expression vector pESC-ALDH1(containing a uracil synthetic gene), ALDH1was introduced into YPH501of Saccharomyces cerevisiae (a deficient strain of uracil biosynthesis) and a transformed colony was available in SD-U (a uracil-deficient medium). After the engineered yeast was cultured for48-72h, galactose was added and incubated for12h to induce the overexpression of ALDH1gene. A cell-free extract was prepared from seedlings of in vitro cultured A. annua for biotransformation of the yeast fermentation product.
Like the nitric oxide synthase (NOS) inhibitor L-NMMA, artesunate can block the induction of nitric oxide (NO) synthesis by hypoxia and hypoxia +cold treatments. thereby elevating intracellular hydrogen peroxide titers and facilitating bacterial apoptosis. Choosing Escherichia coli and Bacilus licheniformis as representatives of Gram negative bacteria (G-) and positive bacteria (G+), and through stool culture and colony counting or serum NO determination, the in vivo sensitization effect of antibiotics by artesunate was evaluated in mice with gastrointestinal infection due to live bacterial feeding.
Results
By GC-MS quantification, it was noted that artemisinin content is higher in transformed samples than control samples, thereby preliminarily confirming ALDH1gene from A. annua being functionally expressed in yeast cells.
While bacteria protect themselves from antibiotic killing through releasing NO, artesunate enhances the sensitivity of bacteria to antibiotics by inhibiting bacterial NOS activity, and artesunate also inhibits bacterial catalase (CAT) activity. Consequently, artesunate accelerates the killing effect of both cefotaxime against G and rifampicin against G+.Additionally, it also assists ampicillin to repress the propagation of drug tolerant bacteria.
Conclusion
These results should shed light on the further establishment of the microbial platform for full-synthesis of artemisinin, and have paved a new path towards abrogation of superbug's antibiotic resistance.
青蒿素以高效抗疟作用最为著名,但它也具有抗肿瘤、抗炎症(自身免疫病)等多种功效。由于世界市场供应的青蒿素主要来自我国人工种植的青蒿,其青蒿素含量偏低,生产成本较高,加之种植规模逐年萎缩,因而难以完全满足全球疟疾感染者对青蒿素类抗疟药的庞大需求,于是利用基因工程微生物大规模生产青蒿素成为当前最有希望的解决方案。本研究还对青蒿琥酯的抗菌作用进行了分子药理学评价,为利用抗生素增敏剂克服“超级细菌”日益严重的抗生素耐药性提供新思路。
方法：
本研究从青蒿中克隆了一个新的青蒿素前体合成基因——青蒿醛脱氢酶基因-1(ALDH1),并经双酶切、扩增和重测序予以确认。通过构建pESC-ALDH1表达载体(含尿嘧啶合成基因)并导入酿酒酵母YPH501菌株(尿嘧啶缺陷型)后,获得能在SD-U(尿嘧啶缺陷培养液)中生长的转化子。将酵母工程菌培养48-72h后,加入半乳糖处理12h诱导ALDH1基因表达,由青蒿试管苗制备无细胞提取液,对酵母发酵产物进行生物转化。
在青蒿琥酯抗菌增敏作用研究体外实验中我们检测经过缺氧适应的地衣芽孢杆菌产生的NO值和生长状态(OD值)以及单纯添加抗生素和抗生素和青蒿琥酯联用时NO值和过氧化氢酶活性。在体内实验中以大肠杆菌和地衣芽孢杆菌分别作为革兰氏阴性菌(G-)和阳性菌(G+)的代表,通过粪便菌落培养和计数或血清NO含量测定,在活菌饲喂导致肠道细菌感染的小鼠中评价了青蒿琥酯对抗生素的体内增敏效果。
结果：
通过气象色谱-质谱分析可见,转化样品中的青蒿素含量高于对照样品,初步证实青蒿ALDH1基因已在酵母细胞中实现功能表达。
青蒿琥酯抗菌作用体外实验结果可见青蒿琥酯与一氧化氮合酶(NOS)抑制剂L-NMMA一样,可阻断缺氧及缺氧+冷处理诱导的细菌一氧化氮(NO)合成,也能抑制细菌过氧化氢酶(CAT)活性。体内实验可见,青蒿琥酯不仅能提高头孢菌素对G-细菌和利福平对G+细菌的杀伤效果,而且还可辅助氨苄青霉素抑制抗药性细菌的繁殖。
结论：
以上结果为进一步创建青蒿素全合成微生物平台奠定了基础,为克服“超级细菌”的抗生素抗性提供了新思路。
Objective
While artemisinin is most famous as a highly effective anti-malarial drug, it can also possess other therapeutic efficacies including anti-tumor and anti-inflammation (autoimmune diseases). Artemisinin that has been supplied in the global anti-malarial market is mainly manufactured from field cultivated Artemisia annua in China. Due to the limitation of lower artemisinin content, higher production cost, and less scale of plantation annually, much need for artemisinin-derived drugs can not be fully met for all malarial patients worldwide. Large-scale production of artemisinin in genetically engineered microorganisms, therefore, has become the most promising solution. This study has also evaluated the anti-bacterial role of artesunate from the viewpoint of molecular pharmacology.
Methods
The present study has reported the cloning of a new artemisinin precursor gene, artemisinic aldehyde dehydrogenase1(ALDH1), which was clarified by double digestions, amplification, and re-sequencing. Upon constructing the expression vector pESC-ALDH1(containing a uracil synthetic gene), ALDH1was introduced into YPH501of Saccharomyces cerevisiae (a deficient strain of uracil biosynthesis) and a transformed colony was available in SD-U (a uracil-deficient medium). After the engineered yeast was cultured for48-72h, galactose was added and incubated for12h to induce the overexpression of ALDH1gene. A cell-free extract was prepared from seedlings of in vitro cultured A. annua for biotransformation of the yeast fermentation product.
Like the nitric oxide synthase (NOS) inhibitor L-NMMA, artesunate can block the induction of nitric oxide (NO) synthesis by hypoxia and hypoxia +cold treatments. thereby elevating intracellular hydrogen peroxide titers and facilitating bacterial apoptosis. Choosing Escherichia coli and Bacilus licheniformis as representatives of Gram negative bacteria (G-) and positive bacteria (G+), and through stool culture and colony counting or serum NO determination, the in vivo sensitization effect of antibiotics by artesunate was evaluated in mice with gastrointestinal infection due to live bacterial feeding.
Results
By GC-MS quantification, it was noted that artemisinin content is higher in transformed samples than control samples, thereby preliminarily confirming ALDH1gene from A. annua being functionally expressed in yeast cells.
While bacteria protect themselves from antibiotic killing through releasing NO, artesunate enhances the sensitivity of bacteria to antibiotics by inhibiting bacterial NOS activity, and artesunate also inhibits bacterial catalase (CAT) activity. Consequently, artesunate accelerates the killing effect of both cefotaxime against G and rifampicin against G+.Additionally, it also assists ampicillin to repress the propagation of drug tolerant bacteria.
Conclusion
These results should shed light on the further establishment of the microbial platform for full-synthesis of artemisinin, and have paved a new path towards abrogation of superbug's antibiotic resistance.
引文
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