【作者】 王军；

【导师】 闻建平；

【作者基本信息】 天津大学 ， 生物化工， 2017， 硕士

【摘要】 FK506（他克莫司）是一种多种链霉菌产生的23元大环内酯类免疫抑制剂。其能够广泛用于预防移植器官的排斥和自身免疫性疾病的治疗,从1994年获得FDA的批准在固体移植器官的应用到今天已经取得了明显的效果,逐步取代了环孢素的使用,并成为了世界上的畅销药之一。外源添加豆油的方法,能极大地促进大环内酯类的产量^[29-31],尤其是能够有效提高FK506的产量^[23-25]。本文中外加豆油到筑波链霉菌的发酵培养基中,产量提高了约88.8%,但是从分子内部角度揭示豆油对FK506高产的代谢机制研究未见文献报导,我们首次对这种高产机制进行深入研究。为了研究这种高产机制,采用比较蛋白组学和代谢组学两种组学结合的方法,综合分析胞内酶、代谢物和代谢途径这三个相关的方面。二维凝胶电泳对筑波链霉菌的总蛋白进行分离,总共有500多个蛋白点。这些蛋白点中,总共有77个蛋白点差异性表达,其中74个蛋白点在豆油添加的条件下有较高的水平,3个蛋白点有较低的水平,通过MALDI-TOF/TOF-MS鉴定这77个差异蛋白点,气相色谱-质谱仪测定了66种胞内代谢物,偏最小二乘法分析与FK506合成最相关的18种关键代谢物。蛋白质组和代谢组学的分析表明,外源添加的豆油作为补充碳源,不仅可以加强FK506前体代谢,而且能够强化与途径相关的转录调控,翻译和应激反应,表明了豆油为FK506合成提供了一个更好的胞内代谢环境。根据以上两种组学的综合分析,20种影响FK506合成的相关代谢物和前体添加到豆油培养基中进一步提高FK506的产量。其中,赖氨酸,柠檬酸,莽草酸和丙二酸能够极大地促进FK506产量和生物量。当添加0.01g/L丙二酸到豆油培养基时效果最好,生物量是12.4±0.4g/L,FK506产量达到165.05±3.6 mg/L。更多还原

【Abstract】 FK506(tacrolimus)is a 23-membered polyketide macrolide produced by various Streptomyces species.It widely used to prevent rejection and treat the autoimmune diseases.FK506 has gradually replaced the utilization of cyclosporine since the FDA approval in 1994,and now it becomes one of the best-selling medicines for treating solid-organ transplantation in the world.Feeding soybean oil has great effect on the production of macrolides,especially for FK506.In this study,feeding soybean oil into the fermentation culture of S.tsukubaensis improved FK506 production by 88.8 %.However,it has not been reported from molecular mechanism in other literature on FK506 overproduction by soybean oil,this is the first time for us to in-depth analysis.In order to study the mechanism of overproduction,we combined comparative proteomics with metabolomics,comprehensive analysis three aspects of intracellular enzymes,metabolites and metabolic pathways.To decipher the overproduction mechanism,comparative proteomic and metabolomic analysis was carried out.More than 500 protein spots were isolated in this study.A total of 77 protein spots with differential expression.And 74 spots were present at higher levels in soybean oil-fed samples,while only three protein spots showed higher abundances under the control condition.MALDI-TOF/TOF-MS was used to identified and function annotations of these protein spots.And 66 intracellular metabolites were measured by GC-MS.18 key metabolites was related FK506 biosynthesis in PLS analysis.The analysis of proteome and metabolome indicated that feeding soybean oil as a supplementary carbon source could not only strengthen the FK506 precursor metabolism and energy metabolism,but also tune the pathways related to transcriptional regulation,translation and stress response,suggesting a better intracellular metabolic environment for the synthesis of FK506.Based on these analyses,twenty key metabolites and precursors of FK506 were supplemented into the soybean oil medium.Among them,lysine,citric acid,shikimic acid and malonic acid performed excellently for promoting the FK506 production and biomass.Especially,the addition of malonic acid achieved the highest FK506 production 165.05 ± 3.6 mg/L and the biomass reached 12.4 ± 0.4g/L.更多还原