【作者】 陈希杨；

【导师】 岑沛霖； 徐志南；

【作者基本信息】 浙江大学 ， 生物化工， 2011， 博士

【摘要】 雷帕霉素(Rapamycin)又名西罗莫司(Sirolimus),是由吸水链霉菌(S. hygroscopicus)产生的新型三烯含氮大环内酯抗生素。雷帕霉素除作为免疫抑制剂外,目前还广泛应用于器官移植、皮肤病、心脏病等领域。本文以实验室保存的低产雷帕霉素产生菌为出发菌株,通过传统诱变育种技术,结合高通量筛选手段,辅以培养基优化,大幅度提高了雷帕霉素产生菌的生产能力,并在发酵罐进行了中试及放大。同时,采用原生质体育种及基因组重排等新技术对雷帕霉素产生菌进行选育,并初步探索了雷帕霉素的异源合成。试验首先初步优化了雷帕霉素产生菌的培养基和培养条件,确定了最优的发酵培养基为：4%葡萄糖,1%甘油,1%黄豆饼粉,0.15%L-Lys,0.5%K2HPO4, 0.5%KH2PO4,0.5%氯化钠,0.2%硫酸镁,0.15%硫酸亚铁。较优的摇瓶发酵条件是：摇瓶装液量为20ml/250ml,培养温度为280C,接种后培养5天收获菌体。在以上最优条件下,原始菌株发酵液中雷帕霉素产量最高可达120mg/L。为提高雷帕霉素产量,开展了雷帕霉素产生菌的诱变育种研究。首先考察了紫外线、DES、NTG等不同诱变剂的剂量效应。然后通过单一诱变剂连续诱变、不同诱变剂交替诱变、不同诱变剂连续诱变等多种诱变方式,筛选到产量为279mg/L的诱变菌株D7-804,比出发菌株提高了156%。进一步发展了基于96孔板培养的雷帕霉素突变株的高通量筛选新技术,通过对约100,000个单菌落的快速高效筛选,筛选到高产突变菌株N5632,其最高产量达到445mg/L在以上高产菌选育和摇瓶发酵优化的基础上,开展了在120L发酵罐上的雷帕霉素发酵的放大研究。结果表明,初始搅拌转速设定为200rpm,在进入产物合成期后转速升至400rpm,控制整个发酵过程中溶氧不低于10%,雷帕霉素产量最高达到412mg／L,基本达到了摇瓶发酵的平均水平。在此基础上,开展了采用多种补料方式提高发酵水平的研究。结果表明,40h为最佳补料时间,采用多组分组合补料方式能够最大幅度提高发酵水平,其中组合补料甘油与磷酸盐的方式,不仅能够保持发酵液的pH值的相对稳定,而且能有效提高雷帕霉素产量,发酵水平最高可达800mg／L以上。最后,将这一新型补料发酵工艺在20吨发酵罐上进行工业放大,雷帕霉素发酵水平也达到了700mg／L,产量较本课题开始时的最初发酵水平提高了4倍以上。为了最大程度地提高雷帕霉素高产菌选育的效率,开展了基因组重排技术改造吸水链霉菌的遗传性能,提高雷帕菌素发酵水平的研究。首先确定了雷帕霉素产生菌原生质体制备、再生和融合的较优条件；然后通过吸水链霉菌原生质体诱变、融合以及与红霉素生产菌原生质体的种间融合等技术,得到具有不同遗传特性的产雷帕菌素产量较高的突变株。最后,对7株雷帕霉素高产突变株进行一轮基因组重排育种,结果筛选到一株产量较高菌株(GS-1437),雷帕霉素产量达到445mg/L,比最初出发菌株产量提高了59.5%,充分显示了基因组重排技术在改造链霉菌遗传特性方面的巨大潜力,与传统诱变育种方法比较,具有快速高效等显著优点。。开展了雷帕霉素生物合成基因镞在异源宿主菌中表达及生物合成雷帕霉素的研究。在已构建的三种雷帕霉素异源合成系统中,白色链霉菌S. albus 28-1-1发酵产物样品表现出与雷帕霉素标准品类似的抗菌生物活性。通过初步的结构鉴定和分析,该发酵产物样品中的未知生物活性物质是一种生物活性类似雷帕霉素但结构上有较大区别的新化合物。进一步从转录水平上对异源宿主菌中雷帕霉素生物合成基因镞中的各个基因进行了转录分析。通过提取发酵72h的菌体RNA,采用RT-PCR技术分析各基因在转录水平上的差异,S. albus 28-1-1中绝大部分雷帕霉素生物合成基因未能被正常转录,仅有下游的rapG,rapF和rapD显示与参照系统相同的转录信号。这一研究结果对在异源宿主菌中生物合成雷帕霉素及其他新颖抗生素具有重要的参考意义。更多还原

【Abstract】 Rapamycin (Sirolimus) is a triene macrolide antibiotic produced by Streptomyces hygroscopicus. Besides its wide application as an effective immunosuppressive agent, other important bioactivities have made rapamycin a potential drug lead for novel pharmaceutical development. However, the low titer of rapamycin in the original producer strain limits further industrialization efforts and restricts its use for other applications.We optimized the fermentation medium of the original high-yield mutant S. hygroscopicus with regard to the formation of rapamycin, and the optimal fermentation medium was formulated. The optimal medium:4% glucose,1% glycerol, 1% soybean meal,0.15% L-Lys,0.5% K2HPO4,0.5% KH2PO4,0.5% NaCl,0.2% MgSO4,0.15% FeSO4. Under these optimal fermentation conditions, shake bottle loading of 20ml/250ml, culture temperature of 28℃, and havest cell mass after cultivated for 5 days, the rapamycin production was improved to 120mg/L.Predicated on knowledge of the metabolic pathways related to rapamycin biosynthesis in S. hygroscopicus, we have designed a rational screening approach to generate a rapamycin high producer strain. The breeding of the rapamycin producer was carried out by isolating high-producing strains from colonies after exposure to the mutagens of UV, NTG or/and DES. The resultant strain produced rapamycin at 279mg/L in the shake flask scale. Then a novel high-throughput cultivation method was developed to rapidly screen large numbers of rapamycin-producing mutants of Streptomyces hygroscopicus by duplicate culturing of isolates on the surfaces of agar-solidified 96 wells in microtiter plates. By integrating 96-well solid cultivation and the bioassay, we screened more than 100,000 isolates and one mutant produced 445mg rapamycin/L was screened out, which was double the yield of parent strain used in the submerged fermentation process.The fermentation processes were further scaled up in 120 L and 20,000 L fermentors, respectively at the pilot plant. Selected fermentation factors including agitation speed, pH, and on-line supplementation were systematically evaluated. A fed-batch strategy was established to maximize rapamycin production. With these efforts, an optimized fermentation process in the larger scale fermentor was developed. In the final fermentation titer, the rapamycin productivity was 812 mg/L in the 120 L fermentor and 783 mg/L in the 20,000 L fermentor, respectively.Several protoplasts-related techniques including protoplasts mutation, intraspecies, and interspecies protoplasts fusion were carried out to improve the rapamycin productivity in S. hygroscopicus. The interspecies fusion of protoplasts of rapamycin producer S. hygroscopicus D7-804 and erythreumycin producer S. erythreus ZJU325 could have brought about one high-yield (345 mg/L) rapamycin producer with 23.6% higher than that of the parental strain. After genome shuffling work, two high-yielding strains were isolated and the highest productivity of rapamycin attained 445mg/L. The systematic research of protoplast-related techniques has established an applicable way to generate high-yield strains from original microorganisms which can only produce low amount of expected natural products, without information of target gene clusters and gene sequences. Comparing with traditional screening method, genome shuffling is much rapid and higher efficiency to obtain higher productivity strain.The biosynthetic gene cluster of rapamycin(RAP) has been respectively integrated into three Streptomyces mode hosts S. albus, S. ceolicolor M512, and S. lividans K4-114 by BAC-based technology to generate the RAP heterologous production systems. The bioassay results suggested that olny S. albus 28-1-1 from the constructed three heterologous systems has clearly showed the antibiotic activity similar to RAP. But the parallel HPLC and LC-MS analyses did not detect any trace of the production. Subsequent RT-PCR analyses has indicated that the majority of RAP biosynthetic genes were not transcripted, only three downstream genes rapG, rapF and rapD showed transcription signal in S. albus. Thus, insufficient or improper regulation of RAP genes could be a critical reason that limited the normal gene transcription. This exploring study will be a base for efficient biosynthesis of rapamycin and its similarities in the future.更多还原