短密青霉生产霉酚酸发酵策略优化和代谢工程研究

发布时间：2018-01-15 04:16

本文关键词：短密青霉生产霉酚酸发酵策略优化和代谢工程研究　出处：《华东理工大学》2017年博士论文　论文类型：学位论文

【摘要】：临床医学发展至今,器官移植已经成为外科领域的研究重点,因此抗免疫排斥反应抗生素的研发应用水平需要进一步提高。霉酚酸(Mycophenolicacid,MPA)是短密青霉(Penicilliumbrevicompactum)次级代谢分泌产生的一种抗生素,它具有抗真菌、抗肿瘤和免疫抑制的作用。霉酚酸对次黄嘌呤单核苷酸脱氢酶具有可逆性抑制作用,并对淋巴细胞活性起到选择性抑制作用。它的2-乙基酯类衍生物——霉酚酸酯(Morpholinoethyl Ester of Mycophenolic Acid,MMF)是新一代免疫抑制剂,在器官移植和自身免疫性疾病的临床治疗方面展示了广泛的应用空间和前景。作为合成霉酚酸酯的前体化合物,提高霉酚酸工业化发酵生产水平具有重要的研究意义。本文对短密青霉ATCC16024在液体环境发酵生产霉酚酸初始培养基配方和补料策略进行优化,通过对菌种合成霉酚酸代谢途径的定向遗传改造,提高工业菌种的生产性能,并对丝状菌的发酵放大规律进行研究。(1)短密青霉发酵生产霉酚酸初始培养基配方优化运用Plackett-Burman设计法和响应面分析法,优化霉酚酸初始发酵培养基组分,最终确定优化后的发酵培养基配方:葡萄糖93.43 g/L,甘氨酸13.36 g/L,KH2PO4 3 g/L,MgSO4·7H2O 2g/L,L-蛋氨酸0.5g/L,微量元素。在7L发酵罐中发酵验证,霉酚酸产量达到1.72 g/L,实现了响应面优化预期效果,比初始培养基发酵产量提高了63.8%。(2)组合补料策略提高短密青霉发酵生产霉酚酸的产量对短密青霉ATCC16024生产霉酚酸发酵策略进行了优化。在7L发酵罐中,在发酵120 h 后即霉酚酸生产合成期,分别设计 CN-feeding strategy(F-CN)、pH-controlled strategy(F-pH)、Met-feeding strategy(F-Met)策略并对相关参数进行了考察分析。实验结果显示,三种策略控制下霉酚酸产量分别达到2.23 g/L、1.92g/L和2.03g/L,比分批发酵实验分别提高了29.65%、11.63%、18.02%。在上述基础上,建立了一种组合补料策略(F-CPM)。发酵120h后,通过三种方式控制发酵过程:1)补加质量比C/N=7:1的葡萄糖-甘氨酸;2)控制pH维持在6.5;3)补加蛋氨酸,使其浓度达到1.0 g/L。在本组合补料策略控制下,发酵240h时菌体最大干重(DCWmax)达到45.77g/L,312h时霉酚酸最大产量(Pmax)达到2.68 g/L,比分批发酵提高了55.81%。(3)透明颤菌血红蛋白基因在短密青霉菌中的克隆转化与表达通过根癌农杆菌LBA4404介导转化,编码透明颤菌血红蛋白的结构基因(Vitreoscilla Hemoglobingene,vgb)在短密青霉ATCC16024中克隆,从而表达透明颤菌血红蛋白(VHb),有效的改善菌体对氧的摄取能力,提高了霉酚酸的发酵产量。通过潮霉素(Hygromycin B)抗性筛选、基因鉴定和7L反应器中发酵验证得出,vgb基因在短密青霉中成功获得了克隆;VHb的表达提高了短密青霉的菌体密度,转化子霉酚酸产量达到2.18 g/L,比初始菌株提高了 27.5%。(4)短密青霉HMG-CoA裂解酶基因插入失活提高霉酚酸发酵产量运用巢式PCR和基因步移技术,首次获得了霉酚酸途径中的一个关键酶HMG-CoA裂解酶的全酶基因序列。同时设计对应简并引物获得了霉酚酸生物合成途径中其余4个关键酶的基因片段序列。利用根癌农杆菌LBA4404转化系统(ATMT),将HMG-CoA裂解酶基因进行了定向插入失活。潮霉素抗性筛选、基因鉴定等结果表明:HMG-CoA裂解酶基因定向敲除成功,霉酚酸产量达到2.94 g/L,较初始菌株提高了70.9%。ATMT转化系统在短密青霉发酵生产霉酚酸的遗传改造,为该菌通过基因工程方法提高霉酚酸发酵水平提供了强有力的工具。本课题阐述了提高短密青霉发酵生产霉酚酸产量的一系列方法。通过设计基于碳氮源配比分段式流加发酵策略,对于碳氮源的配比在菌体生长和霉酚酸生产阶段进行优化。深入探索霉酚酸合成代谢机理、发酵调控机理,跟踪菌丝形态、发酵液颜色、溶氧等参数,优化霉酚酸发酵策略。开展基于代谢工程技术的代谢网络关键酶研究,采用巢式PCR和基因步移等技术手段分析霉酚酸合成途径中基因簇和关键酶基因,通过对代谢途径中关键酶进行基因水平研究,进而对霉酚酸的发酵生产进行调控。
[Abstract]:The development of clinical medicine so far, organ transplantation has become the focus of research in the field of surgery, so the effect of anti rejection antibiotic development needs to be further improved. The application level of mycophenolic acid (Mycophenolicacid, MPA) is Penicillium brevicompactum (Penicilliumbrevicompactum) an antibiotic produced secondary metabolism, it has antifungal, antitumor and immune suppression effect of mycophenolic acid can inhibit the reversibility of inosine monophosphate dehydrogenase, and the lymphocyte activity inhibition to 2- ethyl ester - selectivity. Its derivatives of mycophenolate mofetil (Morpholinoethyl Ester of Mycophenolic Acid, MMF) is a new generation of immunosuppressive agents in clinical treatment of organ transplantation and autoimmune diseases show wide application space and Prospects. As a precursor in the synthesis of mycophenolate mofetil mycophenolic acid, improve the industrial fermentation raw The production level has important significance. This paper on Penicillium brevicompactum ATCC16024 in liquid fermentation of mycophenolic acid in initial culture medium and feeding strategy optimization, directed by genetic modification of strain synthesis of mycophenolic acid metabolism, improve the production performance of industrial strains, and the fermentation of filamentous bacteria amplification were studied. (1) Penicillium brevicompactum mycophenolic acid fermentation medium optimization method of initial analysis and response using the Plackett-Burman design method to optimize the formulation of mycophenolic acid in the initial fermentation medium components, and ultimately determine the optimized fermentation medium: glucose 93.43 g/L, glycine 13.36 g/L, KH2PO4 3 g/L, MgSO4 7H2O 2g/L trace elements, L- methionine 0.5g/L, verified in 7L. The fermentation in fermentor, mycophenolic acid production reached 1.72 g/L, achieve the expected effect of response surface optimization, the ratio of the initial fermentation medium yield increased by 63.8%. (2) Combined feeding strategy to improve Penicillium brevicompactum fermentation production of mycophenolic acid production were optimized for Penicillium brevicompactum ATCC16024 production of mycophenolic acid fermentation strategy. In 7L fermentor, 120 h after the fermentation of mycophenolic acid production synthesis phase, design CN-feeding strategy (F-CN), pH-controlled strategy (F-pH), Met-feeding strategy (F-Met) strategy and analysis of the relevant parameters. The experimental results show that the three strategies under the control of mycophenolic acid production reached 2.23 g/L, 1.92g/L and 2.03g/L, batch fermentation experiments were increased by 29.65%, 11.63%, 18.02%. on the basis of the above, the establishment of a combined feeding strategy (F-CPM). After 120h fermentation, the fermentation process control through three ways: 1) adding quality of glycine than C/N=7:1 glucose control; 2) pH maintained at 6.5; 3) adding methionine, the concentration reached 1 g/L. in the combined feeding strategy under control, Fermentation 240h cell maximum dry weight (DCWmax) reached 45.77g/L, 312h when the maximum yield of mycophenolic acid (Pmax) reached 2.68 g/L in batch fermentation increased by 55.81%. (3) of Vitreoscilla hemoglobin gene cloning in Penicillium brevicompactum in the transformation and expression of LBA4404 by Agrobacterium mediated transformation, gene structure encoding Vitreoscilla hemoglobin (Vitreoscilla Hemoglobingene, vgb) in Penicillium brevicompactum ATCC16024 cloning, and expression of Vitreoscilla hemoglobin (VHb), the ability of oxygen uptake was improved effectively, improve the fermentation yield of mycophenolic acid. Through hygromycin resistance screening (Hygromycin B), and 7L gene identification the reactor fermentation proved that VGB gene is successfully cloned in Penicillium brevicompactum; VHb expression enhanced Penicillium brevicompactum cell density, transformants of mycophenolic acid production reached 2.18 g/L, 27.5%. higher than the initial strain (4) short Dense Penicillium HMG-CoA lyase gene insertion inactivation of mycophenolic acid to improve the yield of fermentation by nested PCR and genome walking technique, for the first time the whole gene of mycophenolic acid in the pathway of a key enzyme of HMG-CoA lyase. At the same time corresponding to the design of degenerate primers obtained mycophenolic acid biosynthetic pathway gene fragment in the rest of the 4 key enzymes. Transformation system by Agrobacterium tumefaciens LBA4404 (ATMT), HMG-CoA lyase gene was inserted into inactivation. Hygromycin resistance screening, gene identification results showed that the HMG-CoA lyase gene directed knockout, mycophenolic acid production reached 2.94 g/L, increased by 70.9%.ATMT conversion system in Penicillium brevicompactum production by genetic modification of mycophenolic acid than the initial strain, provides a powerful tool for the bacteria to improve the fermentation level of mycophenolic acid by genetic engineering methods. This paper improved Penicillium brevicompactum A series of methods of production of mycophenolic acid production by fermentation. Based on the design of carbon and nitrogen source ratio of segmented flow and fermentation strategies, for the carbon and nitrogen source ratio is optimized in cell growth and mycophenolic acid production phase. Further exploration of mycophenolic acid synthesis mechanism, fermentation mechanism, tracking mycelia morphology, fermentation liquid color. The parameters such as dissolved oxygen, optimization of mycophenolic acid fermentation strategies. Research on key enzymes in the metabolic network of metabolic engineering based on the technology of using nested PCR and genome walking technique of gene cluster and key enzyme in the synthesis pathway of mycophenolic acid gene by gene level of key enzymes in the metabolic pathway, and regulation of fermentation the production of mycophenolic acid.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ927

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