柔红霉素C-14羟化酶基因的克隆、表达和应用研究
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摘要
柔红霉素和阿霉素是临床上重要的蒽环类抗生素，主要用于多种实体瘤和急性白血病的治疗，在临床上作为一线抗肿瘤药物使用。阿霉素是柔红霉素C-14位羟化的衍生产物，具有比柔红霉素更广的抗肿瘤谱和更小的毒副作用。目前工业上采用化学半合成法从柔红霉素转化生产阿霉素，不仅工艺复杂、转化率低，而且污染环境严重。本研究对微生物转化法替代现有工艺生产阿霉素进行了探索，从柔红霉素产生菌天蓝淡红链霉菌SIPI 1482中克隆了柔红霉素C-14羟化酶基因(doxA)，构建了doxA基因的链霉菌表达质粒，导入变铅青链霉菌TK24中获得了柔红霉素转化基因工程菌，并研究了doxA基因在工程菌中的表达，最后还对工程菌转化柔红霉素生成阿霉素的发酵工艺进行了初步研究。研究结果如下：
通过PCR方法，首次从天蓝淡红链霉菌SIPI 1482菌株基因组DNA中扩增出约1.4 kb大小的doxA基因片段，这在国内外还是首次报道；但是从波赛链霉菌SIPI 40141菌株中却没有扩增出任何片段。经过测序和序列比对分析，PCR扩增获得的doxA基因序列和链霉菌C5菌株来源doxA基因序列完全相同，而和公开发表的波赛链霉菌ATCC 29050来源doxA基因同源性为93.4％。
构建了一个新颖的链霉菌高拷贝表达载体质粒pYG504，以用于异源基因在链霉菌中的克隆和表达。该载体质粒采用了黑色素基因(编码酪氨酸酶)启动子(Pmel)和fd终止子，启动子下游的SphⅠ、BglⅡ、SacⅠ、XbaⅠ和HindⅢ单一酶切位点可以用于插入异源目的基因，并且在SphⅠ位点上游存在SD序列。
构建了五个doxA基因链霉菌表达质粒pYG57、pYG502、pYG503、pYG505和pYG506，使得doxA基因在上游的Pmel启动子、红霉素抗性基因启动子(PermE)或者两者的串联启动子，以及下游的fd终止子(fd terminator)控制之下。相应地将构建的表达质粒导入变铅青链霉菌TK24中获得五株基因工程菌株。
SDS-PAGE蛋白电泳实验证明，上述五个菌株都能够表达大小约45 kD的柔红霉素C-14羟化酶蛋白，并发现以在PermE启动子控制下的doxA基因表达量相对较高，而终止子对
＠中英文摘要
dd基因表达似乎没有影响。而对Tm4（pYG57）菌株中doxA基因表达方式的研究表明，
dd基因在 PermE启动子控制下可能是一种组成型控制表达，其表达量在接种后培养到
48－60 h左右最高并且维持相对稳定，并且表达的柔红霉素 Cl4羟化酶主要存在于菌丝体
细胞内，很少分泌到细胞外。
对所构建的doxA基因工程菌发酵转化柔红霉素的实验结果表明，它们都能将桑红霉素
转化为阿霉素和某一副产物组分，并且以 TK24（pYG505）菌株转化桑红霉素生成阿霉素
的能力相对较强。通过桑红霉素转化产物的紫外可见吸收光谱分析、LC／MS分析和‘H－NMR
＠分析，最终确定工程菌的柔红霉素转化产物中主要由阿霉素和13－M氢柔红霉素组成．其中
前者由工程菌细胞内的羟化酶转化细胞中的柔红霉素形成。后者由宿主菌本身C1 酮还原
酶转化桑红霉素形成。
对TK24（pYG57）工程菌株转化柔红霉素条件进行了初步优化，在优化树下工程菌
TK24巾YG57）对桑红霉素的转化率为 74．4％，是优化前的 3．l倍；而阿霉素和 13－一氢桑
红霉素之比从0．N提高到o．叩。
Daunorubicin and doxorubicin are clinically important anthracyclines and are primarily used as first line chemotherapeutic agents in treatment of a variety of neoplasias and adult myelogenous leukemia. Doxorubicin, as the C-14 hydroxylated derivative of daunorubicin, has a broader spectrum of anti-tumor activity, a lower toxicity and fewer side-effects compared with daunorubicin. Nowadays doxorubicin is produced through chemical semi-synthesis initiated from daunorubicin, which is complicated and has low conversion efficiency with bad environment pollution. In this research work, the technology of microbial conversion was studied in order to establish an environment friendly method for the production of doxorubicin. A doxA gene encoding Daunorubicin C-14 hydroxylase was cloned from a daunorubicin-producing strain Streptomyces coeruleorubidus SIPI 1482. Some plasmids for the expression of doxA gene were constructed and transformed into S. lividans TK24 to get engineered strains for daunorubicin conversion. Co
nditions for the expression of doxA gene and the conversion of daunorubicin into doxorubicin in these strains were investigated. All results are described as follows:
A DNA fragment containing doxA gene of approximate 1.4 kb was amplified for the fist time from SIPI 1482 strain by PCR method, but not from S. peucetius SIPI 40141. Sequence alignment indicated that the cloned doxA gene from SIPI 1482 strain was identical with that from S. sp. C5 strain, and had a 93.4 % homology with the published doxA gene from S. peucetius ATCC 29050.
A novel high-copied expression vector, plasmid pYG504 was constructed for the cloning and expression of heterologous gene in Streptomyces, in which some unique restriction enzyme sites for the insertion of heterlogous genes are flanked by a promoter of melanin gene (encoding tyrosinase gene) and a fd terminator and a Shine-Dalgarno sequence is also located in the upstream of SphI site.
Five plasmids for the expression of doxA gene, which were designated as pYG57, pYG502, pYG503, pYG505 and pYG506 were constructed so that the cloned doxA gene were under the control of either promoter of melanin gene or erythromycin resistant gene or their tandem promoters, and a fd terminator downstream. These plasmids were introduced into S. lividans TK24, respectively and five genetic engineering strains were constructed.
The SDS-PAGE electrophoresis indicated that all engineered strains containing different expression plasmid could apparently express a 45 kD band specific for daunorubicin C-14 hydroxylase. The expression level of doxA gene was much higher when the doxA gene was under the control of promoter PermE. However, it was found that fd terminator had few effect on the expression of doxA gene. The research work involving the expression manner of doxA gene in S. lividans TK24 with plasmid pYG57 implied that the promoter PermE may control its expression constitutively, the time for maximum expression emerged from 48 to 60 h after inoculation and cultivation and the expression level was kept relatively stable, furthermore, the recombinant hydroxylase existed mostly in mycelium cell but little in broth.
The bioconversion experiments of the engineered strains concluded they can convert daunorubicin into doxorubicin and an unknown product and the S. lividans TK24 bearing plasmid pYG505 strain has a relatively stronger ability to convert daunorubicin into doxorubicin among five strains. Identified by means of ultraviolet and visible spectra analysis, LC/MS analysis and 'H-NMR analysis, the converted products of daunorubicin by the engineered strain consisted of doxorubicin and 13-dihydrodaunorubicin. The former was produced by the intracellular recombinant hydroxylase and the latter by the host C-13 ketoreductase.
The bioconversion conditions of daunorubicin by S. lividans TK24 with plasmid pYG57 were optimized. According to these optimized conditions, the conversion efficiency of daunorubicin is as high as 74.4 %, which is 3.1 times of that of the control before optimization, a
通过PCR方法，首次从天蓝淡红链霉菌SIPI 1482菌株基因组DNA中扩增出约1.4 kb大小的doxA基因片段，这在国内外还是首次报道；但是从波赛链霉菌SIPI 40141菌株中却没有扩增出任何片段。经过测序和序列比对分析，PCR扩增获得的doxA基因序列和链霉菌C5菌株来源doxA基因序列完全相同，而和公开发表的波赛链霉菌ATCC 29050来源doxA基因同源性为93.4％。
构建了一个新颖的链霉菌高拷贝表达载体质粒pYG504，以用于异源基因在链霉菌中的克隆和表达。该载体质粒采用了黑色素基因(编码酪氨酸酶)启动子(Pmel)和fd终止子，启动子下游的SphⅠ、BglⅡ、SacⅠ、XbaⅠ和HindⅢ单一酶切位点可以用于插入异源目的基因，并且在SphⅠ位点上游存在SD序列。
构建了五个doxA基因链霉菌表达质粒pYG57、pYG502、pYG503、pYG505和pYG506，使得doxA基因在上游的Pmel启动子、红霉素抗性基因启动子(PermE)或者两者的串联启动子，以及下游的fd终止子(fd terminator)控制之下。相应地将构建的表达质粒导入变铅青链霉菌TK24中获得五株基因工程菌株。
SDS-PAGE蛋白电泳实验证明，上述五个菌株都能够表达大小约45 kD的柔红霉素C-14羟化酶蛋白，并发现以在PermE启动子控制下的doxA基因表达量相对较高，而终止子对
＠中英文摘要
dd基因表达似乎没有影响。而对Tm4（pYG57）菌株中doxA基因表达方式的研究表明，
dd基因在 PermE启动子控制下可能是一种组成型控制表达，其表达量在接种后培养到
48－60 h左右最高并且维持相对稳定，并且表达的柔红霉素 Cl4羟化酶主要存在于菌丝体
细胞内，很少分泌到细胞外。
对所构建的doxA基因工程菌发酵转化柔红霉素的实验结果表明，它们都能将桑红霉素
转化为阿霉素和某一副产物组分，并且以 TK24（pYG505）菌株转化桑红霉素生成阿霉素
的能力相对较强。通过桑红霉素转化产物的紫外可见吸收光谱分析、LC／MS分析和‘H－NMR
＠分析，最终确定工程菌的柔红霉素转化产物中主要由阿霉素和13－M氢柔红霉素组成．其中
前者由工程菌细胞内的羟化酶转化细胞中的柔红霉素形成。后者由宿主菌本身C1 酮还原
酶转化桑红霉素形成。
对TK24（pYG57）工程菌株转化柔红霉素条件进行了初步优化，在优化树下工程菌
TK24巾YG57）对桑红霉素的转化率为 74．4％，是优化前的 3．l倍；而阿霉素和 13－一氢桑
红霉素之比从0．N提高到o．叩。
Daunorubicin and doxorubicin are clinically important anthracyclines and are primarily used as first line chemotherapeutic agents in treatment of a variety of neoplasias and adult myelogenous leukemia. Doxorubicin, as the C-14 hydroxylated derivative of daunorubicin, has a broader spectrum of anti-tumor activity, a lower toxicity and fewer side-effects compared with daunorubicin. Nowadays doxorubicin is produced through chemical semi-synthesis initiated from daunorubicin, which is complicated and has low conversion efficiency with bad environment pollution. In this research work, the technology of microbial conversion was studied in order to establish an environment friendly method for the production of doxorubicin. A doxA gene encoding Daunorubicin C-14 hydroxylase was cloned from a daunorubicin-producing strain Streptomyces coeruleorubidus SIPI 1482. Some plasmids for the expression of doxA gene were constructed and transformed into S. lividans TK24 to get engineered strains for daunorubicin conversion. Co
nditions for the expression of doxA gene and the conversion of daunorubicin into doxorubicin in these strains were investigated. All results are described as follows:
A DNA fragment containing doxA gene of approximate 1.4 kb was amplified for the fist time from SIPI 1482 strain by PCR method, but not from S. peucetius SIPI 40141. Sequence alignment indicated that the cloned doxA gene from SIPI 1482 strain was identical with that from S. sp. C5 strain, and had a 93.4 % homology with the published doxA gene from S. peucetius ATCC 29050.
A novel high-copied expression vector, plasmid pYG504 was constructed for the cloning and expression of heterologous gene in Streptomyces, in which some unique restriction enzyme sites for the insertion of heterlogous genes are flanked by a promoter of melanin gene (encoding tyrosinase gene) and a fd terminator and a Shine-Dalgarno sequence is also located in the upstream of SphI site.
Five plasmids for the expression of doxA gene, which were designated as pYG57, pYG502, pYG503, pYG505 and pYG506 were constructed so that the cloned doxA gene were under the control of either promoter of melanin gene or erythromycin resistant gene or their tandem promoters, and a fd terminator downstream. These plasmids were introduced into S. lividans TK24, respectively and five genetic engineering strains were constructed.
The SDS-PAGE electrophoresis indicated that all engineered strains containing different expression plasmid could apparently express a 45 kD band specific for daunorubicin C-14 hydroxylase. The expression level of doxA gene was much higher when the doxA gene was under the control of promoter PermE. However, it was found that fd terminator had few effect on the expression of doxA gene. The research work involving the expression manner of doxA gene in S. lividans TK24 with plasmid pYG57 implied that the promoter PermE may control its expression constitutively, the time for maximum expression emerged from 48 to 60 h after inoculation and cultivation and the expression level was kept relatively stable, furthermore, the recombinant hydroxylase existed mostly in mycelium cell but little in broth.
The bioconversion experiments of the engineered strains concluded they can convert daunorubicin into doxorubicin and an unknown product and the S. lividans TK24 bearing plasmid pYG505 strain has a relatively stronger ability to convert daunorubicin into doxorubicin among five strains. Identified by means of ultraviolet and visible spectra analysis, LC/MS analysis and 'H-NMR analysis, the converted products of daunorubicin by the engineered strain consisted of doxorubicin and 13-dihydrodaunorubicin. The former was produced by the intracellular recombinant hydroxylase and the latter by the host C-13 ketoreductase.
The bioconversion conditions of daunorubicin by S. lividans TK24 with plasmid pYG57 were optimized. According to these optimized conditions, the conversion efficiency of daunorubicin is as high as 74.4 %, which is 3.1 times of that of the control before optimization, a
引文
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