本文關(guān)鍵詞：短密青霉生產(chǎn)霉酚酸發(fā)酵策略優(yōu)化和代謝工程研究　出處：《華東理工大學》2017年博士論文　論文類型：學位論文

  更多相關(guān)文章： 霉酚酸 短密青霉 發(fā)酵 農(nóng)桿菌介導 vgb

【摘要】：臨床醫(yī)學發(fā)展至今,器官移植已經(jīng)成為外科領(lǐng)域的研究重點,因此抗免疫排斥反應(yīng)抗生素的研發(fā)應(yīng)用水平需要進一步提高。霉酚酸(Mycophenolicacid,MPA)是短密青霉(Penicilliumbrevicompactum)次級代謝分泌產(chǎn)生的一種抗生素,它具有抗真菌、抗腫瘤和免疫抑制的作用。霉酚酸對次黃嘌呤單核苷酸脫氫酶具有可逆性抑制作用,并對淋巴細胞活性起到選擇性抑制作用。它的2-乙基酯類衍生物——霉酚酸酯(Morpholinoethyl Ester of Mycophenolic Acid,MMF)是新一代免疫抑制劑,在器官移植和自身免疫性疾病的臨床治療方面展示了廣泛的應(yīng)用空間和前景。作為合成霉酚酸酯的前體化合物,提高霉酚酸工業(yè)化發(fā)酵生產(chǎn)水平具有重要的研究意義。本文對短密青霉ATCC16024在液體環(huán)境發(fā)酵生產(chǎn)霉酚酸初始培養(yǎng)基配方和補料策略進行優(yōu)化,通過對菌種合成霉酚酸代謝途徑的定向遺傳改造,提高工業(yè)菌種的生產(chǎn)性能,并對絲狀菌的發(fā)酵放大規(guī)律進行研究。(1)短密青霉發(fā)酵生產(chǎn)霉酚酸初始培養(yǎng)基配方優(yōu)化運用Plackett-Burman設(shè)計法和響應(yīng)面分析法,優(yōu)化霉酚酸初始發(fā)酵培養(yǎng)基組分,最終確定優(yōu)化后的發(fā)酵培養(yǎng)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發(fā)酵罐中發(fā)酵驗證,霉酚酸產(chǎn)量達到1.72 g/L,實現(xiàn)了響應(yīng)面優(yōu)化預(yù)期效果,比初始培養(yǎng)基發(fā)酵產(chǎn)量提高了63.8%。(2)組合補料策略提高短密青霉發(fā)酵生產(chǎn)霉酚酸的產(chǎn)量對短密青霉ATCC16024生產(chǎn)霉酚酸發(fā)酵策略進行了優(yōu)化。在7L發(fā)酵罐中,在發(fā)酵120 h 后即霉酚酸生產(chǎn)合成期,分別設(shè)計 CN-feeding strategy(F-CN)、pH-controlled strategy(F-pH)、Met-feeding strategy(F-Met)策略并對相關(guān)參數(shù)進行了考察分析。實驗結(jié)果顯示,三種策略控制下霉酚酸產(chǎn)量分別達到2.23 g/L、1.92g/L和2.03g/L,比分批發(fā)酵實驗分別提高了29.65%、11.63%、18.02%。在上述基礎(chǔ)上,建立了一種組合補料策略(F-CPM)。發(fā)酵120h后,通過三種方式控制發(fā)酵過程:1)補加質(zhì)量比C/N=7:1的葡萄糖-甘氨酸;2)控制pH維持在6.5;3)補加蛋氨酸,使其濃度達到1.0 g/L。在本組合補料策略控制下,發(fā)酵240h時菌體最大干重(DCWmax)達到45.77g/L,312h時霉酚酸最大產(chǎn)量(Pmax)達到2.68 g/L,比分批發(fā)酵提高了55.81%。(3)透明顫菌血紅蛋白基因在短密青霉菌中的克隆轉(zhuǎn)化與表達通過根癌農(nóng)桿菌LBA4404介導轉(zhuǎn)化,編碼透明顫菌血紅蛋白的結(jié)構(gòu)基因(Vitreoscilla Hemoglobingene,vgb)在短密青霉ATCC16024中克隆,從而表達透明顫菌血紅蛋白(VHb),有效的改善菌體對氧的攝取能力,提高了霉酚酸的發(fā)酵產(chǎn)量。通過潮霉素(Hygromycin B)抗性篩選、基因鑒定和7L反應(yīng)器中發(fā)酵驗證得出,vgb基因在短密青霉中成功獲得了克隆;VHb的表達提高了短密青霉的菌體密度,轉(zhuǎn)化子霉酚酸產(chǎn)量達到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發(fā)酵產(chǎn)量運用巢式PCR和基因步移技術(shù),首次獲得了霉酚酸途徑中的一個關(guān)鍵酶HMG-CoA裂解酶的全酶基因序列。同時設(shè)計對應(yīng)簡并引物獲得了霉酚酸生物合成途徑中其余4個關(guān)鍵酶的基因片段序列。利用根癌農(nóng)桿菌LBA4404轉(zhuǎn)化系統(tǒng)(ATMT),將HMG-CoA裂解酶基因進行了定向插入失活。潮霉素抗性篩選、基因鑒定等結(jié)果表明:HMG-CoA裂解酶基因定向敲除成功,霉酚酸產(chǎn)量達到2.94 g/L,較初始菌株提高了70.9%。ATMT轉(zhuǎn)化系統(tǒng)在短密青霉發(fā)酵生產(chǎn)霉酚酸的遺傳改造,為該菌通過基因工程方法提高霉酚酸發(fā)酵水平提供了強有力的工具。本課題闡述了提高短密青霉發(fā)酵生產(chǎn)霉酚酸產(chǎn)量的一系列方法。通過設(shè)計基于碳氮源配比分段式流加發(fā)酵策略,對于碳氮源的配比在菌體生長和霉酚酸生產(chǎn)階段進行優(yōu)化。深入探索霉酚酸合成代謝機理、發(fā)酵調(diào)控機理,跟蹤菌絲形態(tài)、發(fā)酵液顏色、溶氧等參數(shù),優(yōu)化霉酚酸發(fā)酵策略。開展基于代謝工程技術(shù)的代謝網(wǎng)絡(luò)關(guān)鍵酶研究,采用巢式PCR和基因步移等技術(shù)手段分析霉酚酸合成途徑中基因簇和關(guān)鍵酶基因,通過對代謝途徑中關(guān)鍵酶進行基因水平研究,進而對霉酚酸的發(fā)酵生產(chǎn)進行調(diào)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.

【學位授予單位】：華東理工大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：TQ927

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