【作者】 范晶华；

【导师】 闻建平；

【作者基本信息】 天津大学 ， 生物化工， 2007， 硕士

【摘要】 达托霉素(Daptomycin)是一种由13个氨基酸组成的新型脂肽类抗生素,其中十个氨基酸组成一个氨基酸环,另有3个氨基酸排列成线状,这三个氨基酸末端的L-色氨酸接着一个十碳癸酸。达托霉素由玫瑰孢链霉菌发酵生成,具有在体外抗绝大多数的临床革兰阳性菌的作用,主要用于耐药菌,如耐万古霉素的肠球菌(VRE),耐甲氧西林的金葡菌(MRSA)等。2003年,达托霉素被FDA批准在美国上市,主要用于皮肤和软组织感染治疗。目前国内达托霉素发酵的研究极少,产量较低、无法应用到商业生成中。因此,对达托霉素发酵进行深入的研究和开发具有重要意义。本文围绕如何提高达托霉素的产量和质量,对生产菌种的选育、培养基优化、发酵工艺等方面进行了系统研究,获得了如下结果:以玫瑰孢链霉菌LD-1为出发菌株,经硫酸二乙酯和激光诱变处理,并选用链霉素作为抗性筛选剂,最终选育出一株高产达托霉素的突变株LC-54,该菌在未优化条件下的达托霉素产量达到34.29mg/L,比出发菌株提高了194%。经验证,菌株LC-54的遗传性能稳定。研究了菌株LC-54的摇瓶分批发酵条件。确定了最佳种龄和培养条件,并利用SAS软件设计了较好的发酵培养基。考察了接种量、摇床转速、发酵培养基及初始pH对玫瑰孢链霉菌合成达托霉素的影响,对发酵培养基条件进行了优化。在最适发酵条件下,菌株LC-54摇瓶分批发酵生产达托霉素的产量可达到54.50mg/L,比初始条件(34.29mg/L)的产量提高了58.9%。研究了前体癸酸对玫瑰孢链霉菌合成达托霉素的影响,并确定了最佳添加量和添加时间。利用7.5 L发酵罐进行达托霉素的发酵放大研究,在优化条件下,流加前体,发酵罐中达托霉素产量可达到126.6mg/L,比摇瓶发酵提高了133%。以7.5L发酵罐分批发酵的试验数据为依据,对分批发酵动力学进行了研究,建立了Logistic非结构模型,该模型能较好地反映达托霉素的分批发酵过程。更多还原

【Abstract】 Daptomycin is a new lipopeptide antibiotic which contains a ring with teen amino acids and three amino acids with a 10-carbon decanoic acid attached to the terminal L-tryptophan. Daptomycin is produced by Streptomyces roseosporus, and its activity limited to gram-positive organisms, especially the antibiotic-resistant bacteria such as Staphvlococcus aureus (MRSA), vancomycin-resistant enterococci (VRE). Daptomycin was approved by FDA in 2003, and it was used in skin and soft tissue infection. However, the internal research of daptomycin production is rare, so the application of daptomycin is limited severely. Therefore, it is of great importance to enhance the research and development of daptomycin production process. In this work, the high productivity strain was selected for daptomycin production and the medium composition and fermentation conditions were optimized. The major research contents and results are as follows:Streptomyces roseosporus LD-1 was used as an original strain. After treated with diethylsulfate and laser irradiation in series, a mutant strain LD-54 was screened out by using the medium with selective pressure of streptomycin. Under the fermentation condition without being optimized, the production of daptomycin was 34.29 mg/L, which was 1.94 times higher than that of the parental strain LD-1. The strain is genetically stable.The batch fermentation of daptomycin production with the strain LC-54 in shake flask was studied. The optimal seed age and the fermentation medium for daptomycin production were designed by using SAS. Also, the effects of cultivation conditions such as inoculum, rotate speed, initial pH value, and culture medium on daptomycin production by Streptomyces roseosporus were evaluated. Under the optimum conditions, strain LC-54 could produce daptomycin 54.5mg/L by shaking-flask batch fermentation, which was 58.9% higher than that of initial conditions.The effect of precursor on daptomycin production was studied. A 7.5L bioreactor was used to study the daptomycin fermentation, the production of daptomycin reached 126.6 mg/L in the optimized operating condition, which was 133% higher than that in shaking-flask fermentation. A logistic model was adopted to simulate the experiment data from 7.5 L bioreactor batch fermentation. The model simulations for cell growth, daptomycin formation and total carbohydrate consumption were in good agreement with the experiment data.更多还原