阿霉素的微生物转化及dauU基因的阻断突变研究
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摘要
阿霉素是柔红霉素C-14位羟化的衍生物，具有比柔红霉素更广的抗肿瘤谱和更小的毒副作用，在临床上作为一线抗肿瘤药物使用，主要用于多种实体瘤和急性白血病的治疗，仅阿霉素在世界范围内的市场已超过2亿美元。目前阿霉素的工业生产主要采用化学半合成法，从微生物发酵产生的柔红霉素出发须经过七步反应获得阿霉素，收率较低且污染环境。因此，研究酶法和微生物转化及生物合成直接生产阿霉素的新工艺将是技术进步和环境友好的必由之路。本研究对阿霉素合成的两种方法进行了探索，一是酶或微生物转化，即通过优化表达系统及引入DNR／DXR抗性基因来改善工程菌的阿霉素的产量；二是通过柔红霉素产生菌旁路代谢途径的阻断来研究对阿霉素合成的影响。
研究结果如下：
将来自天蓝淡红链霉菌SIPI-1482的编码柔红霉素C-14羟化酶的doxA基因克隆至一高效表达载体pET32a中，构建了表达载体pYG914，Western blotting实验证明该融合蛋白在大肠杆菌中表达正确，SDS-PAGE电泳检测表达蛋白主要为包涵体。对包涵体复性和酶分离纯化条件进行了研究，最终获得了单一条带的目的蛋白。通过引入辅因子再生系统尝试体外酶法转化阿霉素，实验结果表明，由于所涉及的影响因素太多，大肠杆菌系统不适合阿霉素的转化。
通过PCR方法从SIPI-1482中扩增了含核糖体结合位点大小为1.3Kb的编码C-14柔红霉素羟化酶的doxA基因，编码SnpR蛋白的snpR基因及受SnpR调控的snpA启动子序列。与GenBank报道的来自链霉菌(S．sp．)C5的相关DNA序列比对表明，doxA基因和snpA启动子序列的同源性均为100％，而snpR的同源性为99.4％，氨基酸序列同源性为96.5％，有8个氨基酸的差异。
构建了三个链霉菌表达质粒pYG908、pYG915和pYG927，使得doxA基因分别在上游受SnpR激活的snpA启动子、红霉素抗性基因突变启动子(ermEp~*)及诱导型启动子tipA控制之下，导入变铅青链霉菌(S．lividans)TK24获得了三株含doxA基因的工程菌。SDS-PAGE蛋白电泳证明，它们都能够表达大小约45KD的柔红霉素C-14羟化酶蛋白。工程菌发酵转化实验表明，它们都能将柔红霉素转化为阿霉素，其中，含pYG927／ET的工程菌的转化能力相对较强。对该工程菌发酵转化条件进行了初步研究，当底物(柔红霉素)浓度为2μg／ml时，工程菌的阿霉素产量为0.25μg／ml，转化率为12％。
为提高工程菌转化中底物的浓度，尝试将来自链霉菌S．peucetius ATCC 29050(另一株柔红霉素产生菌)的柔红霉素抗性基因之一drrC克隆至载体pYG934(含dnrV与doxA的串连片段)并导入变铅青链霉菌TK24，转化试验表明，含drrC的工程菌可在10μg／ml的底物浓度下进行转化，阿霉素的产量为0.3μg／ml，比2μg／ml底物浓度时的产量提高了
Doxorubicin, a C-14 hydroxylated derivative of daunorubicin, is primarily used as first line chemotherapeutic agent in treatment of a variety of neoplasias and adult myelogenous leukemia. Compared with daunorubicin, doxorubicin has a broader spectrum of anti-tumor activity, lower toxicity and fewer side-effects. The world market of doxorubicin exceeded two hundreds of millions of dollars. Currently, doxorubicin is produced by semi-synthesis which is initiated from daunorubicin. The relative low productivity and high pollution make it possible to study on enzymatic biotransformation and fermentation production of doxorubicin, which is the essential way to friendly environment and technology advancement. In this research, two methods were studied for the production of doxorubicin. One was optimization on microbial conversion system by means of choosing different promoters and/or introducing DNR/DXR resistance gene into the host to increase the yield of doxorubicin. The other was disruption of dauU gene responsible for a shunt pathway in the daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482 so as to explore the possibility of direct production of doxorubicin by fermentation.
Results are described as follows:
doxA gene coding for daunorubicin C-14 hydroxylase was amplified by PCR from Streptomyces coeruleorubidus SIPI-1482 genomic DNA and was cloned into a E.coli expression vector pET32a. The expressed protein was confirmed by western blotting and it showed that most of the fusion protein was inclusion body. Thus, the process of renaturation including washing, dissolving, refolding and purification of inclusion body were studied and a single band of purified protein was obtained. In vitro biotransformation of doxorubicin was studied in E.coli by adding co-factor regeneration system. The result showed E.coli is not a suitable host due to uncertain impact factor in the system.
doxA gene containing a strong ribosome binding site GGAGG was PCR amplified from SIPI-1482 strain. A gene encoding SnpR and snpA promoter sequence were also amplified from SIPI-1482 strain. Sequence alignment indicated that the cloned doxA gene and the snpA promoter from SIPI-1482 were identity with that from S. sp. C5, while snpR from SIPI-1482 had a 99.4% DNA homology and 96.5% amino acid homology with the published sequence from S. sp. C5, respectively.
Three novel expression plasmids pYG908, pYG915, pYG927 were constructed for the cloning and expression of doxA in Streptomyces lividans so that the transcription of doxA was
研究结果如下：
将来自天蓝淡红链霉菌SIPI-1482的编码柔红霉素C-14羟化酶的doxA基因克隆至一高效表达载体pET32a中，构建了表达载体pYG914，Western blotting实验证明该融合蛋白在大肠杆菌中表达正确，SDS-PAGE电泳检测表达蛋白主要为包涵体。对包涵体复性和酶分离纯化条件进行了研究，最终获得了单一条带的目的蛋白。通过引入辅因子再生系统尝试体外酶法转化阿霉素，实验结果表明，由于所涉及的影响因素太多，大肠杆菌系统不适合阿霉素的转化。
通过PCR方法从SIPI-1482中扩增了含核糖体结合位点大小为1.3Kb的编码C-14柔红霉素羟化酶的doxA基因，编码SnpR蛋白的snpR基因及受SnpR调控的snpA启动子序列。与GenBank报道的来自链霉菌(S．sp．)C5的相关DNA序列比对表明，doxA基因和snpA启动子序列的同源性均为100％，而snpR的同源性为99.4％，氨基酸序列同源性为96.5％，有8个氨基酸的差异。
构建了三个链霉菌表达质粒pYG908、pYG915和pYG927，使得doxA基因分别在上游受SnpR激活的snpA启动子、红霉素抗性基因突变启动子(ermEp~*)及诱导型启动子tipA控制之下，导入变铅青链霉菌(S．lividans)TK24获得了三株含doxA基因的工程菌。SDS-PAGE蛋白电泳证明，它们都能够表达大小约45KD的柔红霉素C-14羟化酶蛋白。工程菌发酵转化实验表明，它们都能将柔红霉素转化为阿霉素，其中，含pYG927／ET的工程菌的转化能力相对较强。对该工程菌发酵转化条件进行了初步研究，当底物(柔红霉素)浓度为2μg／ml时，工程菌的阿霉素产量为0.25μg／ml，转化率为12％。
为提高工程菌转化中底物的浓度，尝试将来自链霉菌S．peucetius ATCC 29050(另一株柔红霉素产生菌)的柔红霉素抗性基因之一drrC克隆至载体pYG934(含dnrV与doxA的串连片段)并导入变铅青链霉菌TK24，转化试验表明，含drrC的工程菌可在10μg／ml的底物浓度下进行转化，阿霉素的产量为0.3μg／ml，比2μg／ml底物浓度时的产量提高了
Doxorubicin, a C-14 hydroxylated derivative of daunorubicin, is primarily used as first line chemotherapeutic agent in treatment of a variety of neoplasias and adult myelogenous leukemia. Compared with daunorubicin, doxorubicin has a broader spectrum of anti-tumor activity, lower toxicity and fewer side-effects. The world market of doxorubicin exceeded two hundreds of millions of dollars. Currently, doxorubicin is produced by semi-synthesis which is initiated from daunorubicin. The relative low productivity and high pollution make it possible to study on enzymatic biotransformation and fermentation production of doxorubicin, which is the essential way to friendly environment and technology advancement. In this research, two methods were studied for the production of doxorubicin. One was optimization on microbial conversion system by means of choosing different promoters and/or introducing DNR/DXR resistance gene into the host to increase the yield of doxorubicin. The other was disruption of dauU gene responsible for a shunt pathway in the daunorubicin-producing strain Streptomyces coeruleorubidus SIPI-1482 so as to explore the possibility of direct production of doxorubicin by fermentation.
Results are described as follows:
doxA gene coding for daunorubicin C-14 hydroxylase was amplified by PCR from Streptomyces coeruleorubidus SIPI-1482 genomic DNA and was cloned into a E.coli expression vector pET32a. The expressed protein was confirmed by western blotting and it showed that most of the fusion protein was inclusion body. Thus, the process of renaturation including washing, dissolving, refolding and purification of inclusion body were studied and a single band of purified protein was obtained. In vitro biotransformation of doxorubicin was studied in E.coli by adding co-factor regeneration system. The result showed E.coli is not a suitable host due to uncertain impact factor in the system.
doxA gene containing a strong ribosome binding site GGAGG was PCR amplified from SIPI-1482 strain. A gene encoding SnpR and snpA promoter sequence were also amplified from SIPI-1482 strain. Sequence alignment indicated that the cloned doxA gene and the snpA promoter from SIPI-1482 were identity with that from S. sp. C5, while snpR from SIPI-1482 had a 99.4% DNA homology and 96.5% amino acid homology with the published sequence from S. sp. C5, respectively.
Three novel expression plasmids pYG908, pYG915, pYG927 were constructed for the cloning and expression of doxA in Streptomyces lividans so that the transcription of doxA was
引文
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4. Furuya K, Huthinson CR. The DrrC protein of Streptomyces peueetius, a UvrA-like protein, is a DNA-binding protein whose gene is induced by daunorubicin. FEMS Lett, 1998, 168: p. 243-249.
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