柔红霉素高产菌种选育及其生物转化的研究

【作者】 刘连碧；

【导师】 朱宝泉； 朱春宝；

【作者基本信息】 上海医药工业研究院 ， 微生物药学， 2000， 博士

【摘要】 柔红霉素（daunomycin）是由波赛链霉菌（S.peucetius）或天蓝淡红链霉菌（S.coeruleorubidus）等放线菌次级代谢产生的一种蒽环类抗肿瘤抗生素。它具有抗革兰氏阴性菌、革兰氏阳性菌、肿瘤及病毒的作用，并有免疫抑制活性。阿霉素（adriamycin）是柔红霉素的C-14位羟化衍生物，临床上使用的阿霉素是以柔红霉素为前体经化学合成获得。与柔红霉素相比，阿霉素的抗肿瘤谱比柔红霉素广，毒副作用更低，具有更高的应用价值。柔红霉素和阿霉素已广泛应用于肿瘤化疗，已成为某些肿瘤化疗的首选药物，有巨大的经济价值和社会效益。本研究工作的主要目的是提高柔红霉素产生菌Streptomycescoeruleorubidus SIPI-1482的产抗能力，以及开发用于生物转化法生产阿霉素的生物反应系统。 在柔红霉素高产菌菌种选育过程中，我们采用了“核糖体工程技术”。具体工作是应用链霉素和卡那霉素抗性辅助筛选方法，以天蓝淡红链霉菌SIPI-1482野生型菌株株为出发菌，首先确定其对链霉素和卡那霉素的MIC。然后用含链霉素（6μg/ml）或卡那霉素（15μg/ml）的高氏一号合成培养基筛选SIPI-1482菌株抗性自发突变株，TLC和HPLC检测突变株产柔红霉素的水平。实验结果发现，在SIPI 1482菌株的链霉素抗性自发突变株中，正向突变的频率为34％，同时获得了产抗能力是SIPI 1482菌株1.63倍左右的突变株。卡那霉素用于SIPI1482菌株选育中获得的抗性突变株数量少，突变株产抗能力提高幅度小，正突变率低，与链霉素相比用于高产菌种选育的效果相对较差。比较了链霉素抗性自发突变株与卡那霉素自发突变株之间的差异，并对这些差异作了理论上探讨。依据前人的研究结果合成PCR引物来分析突变株Str⁺51的核糖体结构基因，从分子水平研究该突变株柔红霉素生产能力的提高与其核糖体结构之间的关系。 在提高SIPI-1482产抗水平的菌种选育中，采用的另一个方法是离子注入诱变育种。SIPI1482为出发菌株，以N⁺作为辐射致变剂，离子注入条件为：辐射室的真空度10^-3Pa，离子能量60kev，辐射处理的剂量率10¹⁴N⁺/秒，总辐射剂量1×10¹⁴N⁺/cm²。处理后的孢子直接在G1培养基平皿上培养，摇瓶接种发酵，TLC及HPLC测定突变株的发酵水平。结果发现，经离子注入处理后，菌株的形态变异丰富，同时获得了柔红霉素生产能力提高15倍的突变株SIPI1482M2。比较了亲株SIPI-1482以及突变株的菌落形态特性，并对另一突变株SIPI-1482M1进行产阿霉素排除研究。试验发现高产突变株在平皿上培养时，菌株产生的红色蒽环类代谢产物大量分泌到培养基中。通过抽提亲株与突变株的总DNA进行 上海医药工业研究院博士论文 酶切试验，高产交变株SIPI．1482M2酶切图谱存在明显的差异。 在本论文的第二部分主要从事柔红霉素的生物转化研究。提高宿主菌StrCptomyn。 Jlljwll TK抡对柔红霉素的耐受性的试验时，采用紫外和NTG两种诱变剂对TKy菌株 进行诱变，以柔红霉素临界致死浓度筛选法和柔红霉素均匀浓度梯度筛选法对交变株进行 筛选与验证。试验中发现，柔红霉素对 TK24的 MIC为 spg／ml，UV对 TK24的最佳致变 剂量为905。培养基的种类，培养时问，抱子萌发与否等因素均对试验结果有一定的影啊。 经过多次重复诱变与筛选，最终获得了耐20pg／ml柔红霉素的稳定突变株。 以Strohl等发表的doxA基因序列设计PCR引物，从SIPll482以及S．CS菌株克隆dox’4 基因没有获得成功，并对此进行详细分析。采用pHZ 060，pIJ702，pHZ1351等质粒为载体， 构建了四个带 dnr厂 doxA基因的表达质粒 pYG50，pYG55，pYG56，pYG57。转化 TK64后获 得重组工程菌，生物转化试验表明：带以 pHZ 060为载体的重组质粒虽然能够获得转化于， 但是将柔红霉素添加到 pYG50／DHS Q和 pYG50／TK64的培养液中，均不能获得目的产物一 一阿霉素。而带PYG55，PYG56，PYG57质粒的TK64重组菌株转化液的抽提物中均有对应 于阿霉素标准样品的斑点，但不同质粒的生物转化效率不同。对pYG57／TK64的生物转化 产物进行HPLC、TLC以及HPLC—UV等项目分析，发现转化产物与阿霉素标准品完全相 同，同时测得的柔红霉素转化为阿霉素的转化效率为48．4％。将pYG57侣—N18工 程菌与SIPll482，共发酵试验，未能获得阿霉素。而质粒pYG57虽然能够转化SIPll482 菌株，但该质粒在SIPll482菌株中不能稳定存在。抽提pYG57沼 中的质粒，酶切 电泳表明，质粒pYG57被破坏。更多还原

【Abstract】 Daunomycin is one of the antitumor antibiotics, which belongs to anthracyclines and is produced by Streptomyces peucetius or Streptomyces coeruleorubidus. Its bioactivities include antibacterial, antitumor and immunosuppression. Adriamycin (also named doxorubicin) is a daunomycin derivative obtained by hydroxylation at c-i 4 and is produced from daunomycin by semi-synthesis methods in industry. Adriamycin has wider antitumor spectrum and fewer side effects as compared with daunomycin. They are the first line antitumor antibiotics in the chemotherapy of various cancers at present. The main purpose of this study is to improve the productivity of daunomycin producer, Streptomyces coeruleorubidus SIPL 1482, as well as to develop a biological system so as to produce adriamycin by bio-conversion. In the study of strain breeding by a novel technology called ribosome engineering, a screening method based on the resistance of strain to streptomycin or kanamycin was applied to improve the productivity of daunomycin by? Streptomyces coeruleorubidus var. Zhengding SIPI 1482. Streptomycin-resistant (str~) colonies occurred on G1 medium containing 6p.g/ml streptomycin showed a high frequency of positive mutation (34%). The highest one named Str5 1 demonstrated about 1.63 folds increase in daunomycin productivity as compared with parent strain. Initiative time for daunomycin biosynthesis by mutant Str~5 1 is about 24 hours earlier than that of the parent strain. When kanamycin was used to screen resistant mutants, the mutation frequency is much less than that with streptomycin. A distinctive character between these mutants is that kanamyein resistant mutant secretes something to inactivate kanamycin into medium but not for streptomycin resistant mutants. A pair of PCR primers was used to clone ribosomal gene from mutant Str5 1, however none of the desired results was obtained. The relationship between the improvement of daunomycin productivityand the level of streptomycin resistance was also discussed in this section. A mutagenie method of ion-beam implantation was adopted in the breeding of SIPI 1482. The spores of parent strain were treated in the conditions as follows: vacuum degree (1O3Pa), the energy of ion (6Okev), dosage rate (1O拁 N~/s ) and total dosage (1 X lO N/cm2). A mutant named S1P11482M2 was selected and it was found that its productivity is 15-folds higher than that of parent strain based on flask fermentation and 1-LPLC analysis. TLC and HPLC analysis was used to identify whether or not mutant SIPI 1 482M 1 produces any adriamycin. The colony characters between parent and mutant were also compared and it was found that mutant SIPI 1 482M2 could secrete great deal of red anthracycline metabolites into medium. The total DNAs of both parent and mutant SIPI 1 482M2 were isolated and digested completely by BamI-ll, and the electrophoresis characteristic of the SJPI 1482 M2 is obviously different from that of parent. In order to increase the resistance of Streptomyces lividans TK24 to daunomyc in, two kinds of mutagen (UV and NTG) were used in the strain breeding. Positive mutants were selected by a method of critical drug lethal concentration on the G 1 medium. Finally a stable mutant which exhibits the level of resistance up to 20p.gIml daunomyciri was obtained through several generations of mutation and selection. Other conditions such as type of medium, cultural time, spore pre-germination and so on were thoroughly investigated. It was found that artificial mutagenesis更多还原