【作者】 徐波；

【导师】 岑沛霖； 金志华；

【作者基本信息】 浙江大学 ， 生物化工， 2008， 博士

【摘要】 普那霉素（pristinamycin）是由始旋链霉菌（Streptomycespristinaespiralis）产生的一种链阳性菌素类（Streptogramin）抗生素,包含普那霉素Ⅰ（PⅠ）和普那霉素Ⅰ（PⅡ）两类化合物,对大多数革兰氏阳性菌,如MRSA、MRSE等均具有较强的杀菌活性,且抗生素后效应较长,被视为治疗顽固性革兰氏阳性菌感染的特选药物。目前,普那霉素已经在国外实现商品化生产,我国还未进行工业生产。因此,作为药效优良的抗生素,对普那霉素进行深入的研究和开发具有重要意义。本论文以提高普那霉素的发酵水平为目标,对始旋链霉菌的基因组重排、高产菌株培养基成分和培养条件优化以及高产菌产物合成分子机理等进行了深入细致的研究,获得了如下主要研究结果:1.以S.pristinaespiralis 0957作为原始菌株,利用紫外诱变获得四株普那霉素产量和耐受性都有所提高的菌株作为基因组重排的出发菌株。对始旋链霉菌原生质体制备和再生过程进行了研究,确定了合适的制备方案为:添加0.6%的甘氨酸到种子培养基中培养菌丝,用2%的溶菌酶在32℃下酶解1.5h。始旋链霉菌的原生质体制备率和原生质体再生率分别达到了95.8%和18.1%。2.对四株出发菌株（M-23、M-79、M-113和M-156）连续以原生质融合的方式进行基因组重排,然后以普那霉素自身耐受性的提高作为筛选依据,获得了一株高产突变菌株G4-17,它的原生质体对普那霉素耐受性高达100μg/mL,摇瓶培养时普那霉素产量达到0.89g/L,比产量最高的亲本菌株提高了89.4%,比原始出发菌株提高了145.9%。通过连续传代实验,确定高产菌株G4-17的遗传稳定性良好。3.研究了S.pristinaespiralis G4-17菌株的摇瓶发酵条件,通过正交设计对发酵培养基进行了优化;考察了摇瓶培养条件对菌体生长和产物合成的影响。优化后的发酵培养基组成为:可溶性淀粉3.0%、蔗糖1.0%、葡萄糖0.5%、麦芽糖0.8%、黄豆饼粉1.2%、蛋白胨0.4%、鱼粉1.2%、酵母粉0.6%、（NH₄）2SO₄ 0.2%、MgSO₄ 0.35%、KH₂PO₄ 0.02%、CaCO₃0.4%、NaNO₃ 0.075%;优化的摇瓶培养条件为:初始pH值为6.5,在25mL/250mL三角瓶中以6%接种量接入经过36h培养的种子培养液,在25℃、220rpm条件下进行培养60h,S.pristinaespiralis G4-17的普那霉素产量达到了1.21g/L,比优化前的产量（0.89g/L）提高了40.0%。在5-L发酵罐上,优化后的培养条件更有利于高产菌种G4-17合成,G4-17在培养60h时普那霉素产量达到最大值1.237g/L。4.利用ApaⅠ/TaqⅠ双酶切的AFLP技术对3支筛选出的普那霉素高产菌株以及它们的原始菌株CGMCC 0957进行了遗传多态性研究。结果显示,高产菌株间具有类似的AFLP指纹图谱,相比而言,高产菌株与原始菌株间的AFLP指纹图谱差异稍大。因此,在这些高产菌株特别是产量水平类似的菌株中存在类似的变异机理导致了普那霉素的产量提高。另外,AFLP分析还表明基因组重排技术较传统诱变育种更容易使微生物菌株产生基因组水平的变异,因此,利用基因组重排技术进行微生物育种工作较传统诱变育种技术更为有效。5.利用RT-PCR技术检测了普那霉素生物合成相关基因及其抗性基因在高产菌株G4-17和原始菌株CGMCC 0957表达的丰度变化,分析了这些基因表达水平与合成普那霉素的关系。结果显示PⅠ组分生物合成相关基因snbA在高产菌株的整个发酵进程中始终保持高丰度表达,而原始菌株只在发酵前期（24～48h）高丰度表达,随后逐渐降低。因此snbA基因的持续高表达与高产菌株的PⅠ组分高产密切相关。PⅡ组分生物合成基因snaB基因在在这两个菌株的发酵过程中表达丰度变化与snbA基因的表达变化类似,表明snaB基因的持续高表达与高产菌株的PⅡ组分高产密切相关。其它两个PⅡ组分生物合成基因snaA、snaC在高产菌株和原始菌株的发酵过程中无明显差异。研究还发现,对普那霉素产生抗性的ptr基因在高产菌株的整个发酵进程中表达丰度较高,而在原始菌株中仅在发酵中后期（48～96h）保持较高丰度表达。因此,ptr基因在高产菌株开始合成普那霉素之前就已经高表达,从而建立起了自我保护性抗性,这种抗性的提前建立与普那霉素高产密切相关。另外,普那霉素生物合成的调控基因spbR在高产菌株和原始菌株发酵过程中的基因表达丰度没有显著变化,表明这个调控基因的表达与普那霉素产量变化无显著相关性。6.利用蛋白质组学的一些基本研究手段对始旋链霉菌细胞裂解方法,裂解液的选择进行了实验比较,结果发现,液氮研磨法和硫脲裂解液更适合始旋链霉菌细胞内总蛋白的提取;对不同发酵时间高产菌株和原始菌株、抗生素产生前期与抗生素产生高峰期始旋链霉菌细胞内总蛋白含量和种类的差异进行了比较,结果发现,高产菌株G4-17总蛋白质含量高于原始菌株CGMCC 0957,同一菌株在发酵生产前期的蛋白质含量比后期高。更多还原

【Abstract】 Pristinamycin which produced by streptomyces pristinaespiralis is a streptogramin of natural origin comprising two components, prsitinamycin I and prsitinamycin II. Pristinamycin not only has strong antibacterial activity against gram-positive bacteria, including methicillin-resistant strains of Staphylococcus aureus and Staphylococcus epidermidis, but also exhibits a prolonged post-antibiotics effect. So it is considered as the specially selected medicament against the stubborn gram-positive infection. Pristinamycin has been commercially produced in overseas and have not been accomplished in China currently. Therefore, it is very valuable to investigate and develop pristinamycin further as a new generation of antibiotic.The aim of this work is to increase the productivity of pristinamycin fermentation. After thorough and particularity study in strain improvement by genome shuffling, medium composition and culture condition optimization, pristinamycin productivity is increased greatly. Moreover, molecular mechanisms of yield enhancement of pristinamycin and proteomics in Streptomyces pristinaespiralis were anlaysed in the paper.The main research contents and results are as follows:1. Streptomyces pristinaespiralis CGMCC 0957, used as an original strain, was stepwise treated with ultraviolet light （UV）. Four mutant strains with higher yield and tolerance of pristinamycin were selectively starting strain of genome shuffling. Then the formation and regeneration of protoplasts were studied. When streptomyces pristinaespiralis were cultured with 0.6 % glycine in advance and treated with 2% lysozyme for 1.5h at 32℃, the protoplast formation rate was 95.8%. The protoplast regeneration was 18.1%.2. Four mutant strain, M-23、M-79、M-113、M-156, were subjected for recursive protoplast fusion and selection for improved resistance to the product antibiotic in a genome shuffling format. A 100-μg/mL pristinamycin resistant recombinant, G 4-17, was obtained after four rounds of protoplast fusion, and its production of pristinamycin reached 0.89 g/L, which was increased by 89.4% and 145.9% in comparison with that of the highest parent strain M-156 and the original strain CGMCC 0957, respectively. The subculture experiments indicated that the hereditary character of high producing S. pristinaespiralis G 4-17 was stable.3. The pristinamycins fermentation in shake flasks by Streptomyces pristinaespiralis G4-17 was studied. The production medium was optimized by orthogonal design experimental methods. Also, the effect of culture conditions in shake flasks on the pristinamycins production was evaluated. The optimized medium and culture conditions were as follows: soluble starch 3.0%, sucrose 1.0%, glucose 0.5%, maltose 0.8%, soybean flour 1.2%, peptone 0.4%, fish flour 1.2%, yeast extract 0.6%, （NH₄）₂SO₄ 0.2%, MgSO₄·7H₂O 0.35%, KH₂PO₄ 0.02%, NaNO₃ 0.075%, CaCO₃ 0.4%; inoculum age of 36 h; inoculum level of 6%; initial pH value of 6.5; temperature of 25℃; shaking speed of 220 rpm. Under the above optimized conditions, S. pristinaespiralis G4-17 could produce 1.21g/L pristinamycins after 60 h cultivation in the shake flask, which was 40.0% higher than that before optimization.4. Amplified fragment length polymorphism （AFLP） with the double-enzyme ApaI/TaqI was used to analyze genomic variability between high pristinamycin-producing recombinants of S. pristinaespiralis obtained by genome shuffling and their ancestral strain CGMCC 0957. The AFLP fingerprints showed together that there were some polymorphism between these high yield recombinants and their ancestor, and that there was similar polymorphism among these recombinants. Nevertheless, the unique polymorphic bands, which be absent in the ancestor, could be distinguished from all the recombinants. In addication, AFLP analysis showed that variation of organism at the genomic level happens more extensively and easily by genome shuffling than by induced mutagenesis. 5. In order to analyze gene expression changes related to the enhanced antibiotic yield, genes involved in the biosynthesis of PI or PII, and resistance to pristinamycin were investigated by reverse transcription PCR （RT-PCR） between the high-yield recombinants and their ancestral strain CGMCC 0957. The results showed the persistent expression of snbA and snaB involved in the biosynthesis of PI and PII component respectively in the recombinant had a close correlation to the increase of the antibiotic production. It might imply that snbA was a key gene for the biosynthesis of PI component, while snaB was another key gene for the PII biosynthesis. Other two genes snaA and snaC displayed no obvious changes at this time course of fermentation between the recombinant and the ancestor. The ptr gene involved in pristinamycin resistance started ahead high-level expression before the onset of pristinamycin production for the recombinant, also leading to the increase of the antibiotics yield. The spbR gene regulating the antibiotic production did not showed obvious expression change between the recombinant and the ancestor.6. To extract and identify the total protein in the cell of Streptomyces pristinaespiralis, the application and effects of some conventional proteomics methods are compared. The results showed that liquid nitrogen grounding method and high lysis buffer were much suitable for Streptomyces pristinaespirali. And the content of total protein in the cell of G4-17 was more than that of CGMCC 0957 in the whole fermentation process. Compared with fermentation anaphase, the content of total protein in the same strain was higher in earlier fermentation.更多还原