本文關(guān)鍵詞： 裂殖壺菌 DHA 甘油 FAS system 乙酰輔酶 A　出處：《華中科技大學》2016年博士論文　論文類型：學位論文

【摘要】：DHA (Docosahexaenoic acid,二十二碳六烯酸)是含有六個雙鍵的長鏈脂肪酸,屬于ω-3系列的。經(jīng)過多年研究發(fā)現(xiàn),DHA對人體的多種生理活動具有十分重要的調(diào)控和促進作用,DHA是人視網(wǎng)膜細胞和神經(jīng)系統(tǒng)組織中必需的主要成分之一,尤其對嬰幼兒大腦和視力發(fā)育方面起到非常重要的促進作用。裂殖壺菌(Schizochytrium),作為一種大型的海洋真菌,因為其生長快、油脂含量高,被廣泛的應(yīng)用于DHA油脂的生產(chǎn)研究中。但是,利用裂殖壺菌發(fā)酵生產(chǎn)DHA油脂過程中,存在培養(yǎng)基鹽離子濃度高,DHA含量偏低,生產(chǎn)成本過高等問題,從而嚴重制約著該產(chǎn)業(yè)的發(fā)展。該論文以本實驗室具有自主知識產(chǎn)權(quán)的裂殖壺菌S056 (Schizochytrium sp. S056)為出發(fā)菌株,建立了低成本、高強度DHA油脂的清潔生產(chǎn)新技術(shù),揭示了DHA大量合成的代謝機理,并通過對DHA中試條件的優(yōu)化及放大規(guī)律的研究,在10噸規(guī)模發(fā)酵罐上成功實現(xiàn)了DHA油脂的工業(yè)化生產(chǎn)。該研究解決了傳統(tǒng)培養(yǎng)基中高鹽的問題,并突破了DHA含量大幅提升的瓶頸,顯著提高了DHA含量和產(chǎn)品品質(zhì),具體研究內(nèi)容與結(jié)果如下：(1)建立了一種低鹽發(fā)酵新技術(shù),顯著降低了培養(yǎng)基中的鹽離子濃度。海水晶(NaCl)是裂殖壺菌發(fā)酵過程中為了模擬海水環(huán)境必須添加的培養(yǎng)基組分,但高濃度海鹽的存在,增加了發(fā)酵廢水的處理難度,且大量氯離子的存在對發(fā)酵罐腐蝕嚴重,增加了固定資產(chǎn)投入等。針對以上問題,本研究通過對海水晶在裂殖壺菌發(fā)酵過程中的生理作用開展了詳細研究分析,發(fā)現(xiàn)滲透壓的改變對裂殖壺菌S056的生長影響極小,且裂殖壺菌S056培養(yǎng)基中的高濃度海水晶(20 g/L)可被低濃度的硫酸鈉(2-10g/L)完全替換。該技術(shù)的建立顯著降低了培養(yǎng)基中鹽離子的濃度,使發(fā)酵廢水的處理難度下降明顯,也徹底解決了氯離子對發(fā)酵罐的腐蝕問題,降低了DHA生產(chǎn)的原料成本和固定資產(chǎn)投入成本。(2)建立了高含量DHA油脂發(fā)酵新技術(shù),顯著提高了DHA含量,初步揭示了DHA合成與初級代謝相關(guān)性。為了進一步提高裂殖壺菌所產(chǎn)油脂中DHA含量,本文針對影響油脂大量合成的關(guān)鍵因素進行了詳細分析,并對可影響DHA積累的發(fā)酵碳源開展了詳細研究,通過甘油對葡萄糖的替換,DHA含量顯著提高,由葡萄糖培養(yǎng)的39.68%提高到甘油培養(yǎng)時的52.28%,增幅達31.75%。進一步對甘油和葡萄糖參與裂殖壺菌的初級代謝途徑中的關(guān)鍵酶及關(guān)鍵代謝產(chǎn)物進行分析,發(fā)現(xiàn)甘油對菌體內(nèi)的磷酸戊糖路徑中葡萄糖-6-磷酸脫氫酶(G6PDH)有明顯的抑制作用,進而降低了菌體內(nèi)的還原型輔酶Ⅱ(NADPH)含量,DHA的合成量并沒有因NADPH的下降而下降,表明裂殖壺菌S056體內(nèi)DHA的合成路徑不止一條。(3)基于比較轉(zhuǎn)錄組學,對甘油和葡萄糖兩種碳源培養(yǎng)條件下的差異基因的表達進行全面分析,成功揭示了甘油促進DHA大量合成機理。與葡萄糖發(fā)酵相比,甘油培養(yǎng)時,裂殖壺菌S056體內(nèi)的糖異生路徑和磷酸戊糖路徑路徑均被明顯抑制,而與乙酰輔酶A合成相關(guān)的代謝路徑,包括糖酵解和支鏈氨基酸(Valine, Leucine and isoLeucine)代謝路徑均明顯增強。此外,甘油也顯著抑制了三羧酸循環(huán)路徑(TCA cycle),致使更多的乙酰輔酶A向油脂合成路徑轉(zhuǎn)化,顯著促進傳統(tǒng)脂肪酸合成路徑(fatty acid synthesis system, FAS system)的關(guān)鍵酶基因FAS基因表達量增強,使DHA含量得以大幅提高。該研究對促進DHA大量合成的調(diào)控提供了重要的理論指導(dǎo)。(4)建立了高品質(zhì)DHA油脂的中試培養(yǎng)技術(shù),并實現(xiàn)了該技術(shù)的工業(yè)化應(yīng)用。本研究基于以上研究成果,首先對影響DHA合成的關(guān)鍵因素一溶氧和溫度開展詳細研究,在100 L規(guī)模發(fā)酵罐上建立了分段溶氧控制和恒溫培養(yǎng)策略；其次,根據(jù)裂殖壺菌S056在100 L發(fā)酵罐上甘油的動態(tài)消耗曲線,在不延長發(fā)酵周期的前提條件下,確定了最佳補料方案,最終獲取的油脂中DHA含量高達52.61%,DHA產(chǎn)量達23.40g/L,顯著提高了DHA產(chǎn)量和生產(chǎn)效率；最后,以攪拌功率相等的準則為出發(fā)點,對裂殖壺菌S056的放大規(guī)律進行探索,通過對1000 L發(fā)酵罐中裂殖壺菌S056培養(yǎng)過程中的溶氧濃度進行調(diào)整,使其與100 L發(fā)酵罐上的溶氧濃度保持一致,從而很好的實現(xiàn)裂殖壺菌S056在1000 L發(fā)酵罐上的高密度培養(yǎng),并將該放大規(guī)律在10噸發(fā)酵罐上進行驗證,結(jié)果表明,DHA產(chǎn)量平均在22g/L以上,且油脂中DHA的含量維持在50%以上,在10噸規(guī)模發(fā)酵罐成功實現(xiàn)了高品質(zhì)DHA的高強度發(fā)酵生產(chǎn)。綜上所述,本文實現(xiàn)了裂殖壺菌的低鹽培養(yǎng),顯著提高了DHA的含量,成功揭示了甘油可促進DHA大量合成的代謝機理,并通過對溶氧和溫度的精準調(diào)控,實現(xiàn)了以上技術(shù)在10噸發(fā)酵罐上的集成應(yīng)用。
[Abstract]:DHA (Docosahexaenoic acid, twenty-two C six acid) long chain fatty acid containing six double bonds, belonging to the Omega -3 series. After many years of research found that the physiological activity of many DHA on the human body has a very important role in promoting and regulation, DHA is one of the main components of essential human retinal cells and nervous system tissues. In particular, a very important role in infant brain and eye development aspects. Schizochytrium (Schizochytrium), as a kind of large marine fungi, because of its fast growth, high oil content, is widely used in the production of DHA oil. However, the production of DHA oil cracking Schizochytrium sp. fermentation in the process, there are medium high salt concentration, DHA content is low, the production cost is too high, which seriously restricts the development of the industry. This thesis takes the laboratory with independent intellectual property rights of Schizochytrium S 056 (Schizochytrium sp. S056) as the starting strain, a low cost, the new technology of cleaner production of high strength DHA oil, DHA synthesis metabolic mechanism, and through the research of the optimization of test conditions of DHA and amplification of the law, the successful implementation of the industrial production scale of 10 tons of oil in DHA fermentor the study to solve the traditional medium high salt, and broke through the bottleneck of DHA content increased significantly, significantly improve the quality of products and the content of DHA, the specific research contents and results are as follows: (1) the establishment of a new low salt fermentation technique, significantly reduced the concentration of salt ions in the medium the sea crystal (NaCl) is Schizochytrium limacinum fermentation process in order to simulate the water environment must be added to the medium components, but high concentration of salt, increasing the difficulty of fermentation wastewater treatment, and a large number of chloride ions in the fermentation tank serious corrosion Weight, increase of fixed asset investment. To solve the above problems, this study based on the physiological role of sea crystal in Schizochytrium limacinum fermentation process carried out a detailed research and analysis, found that the osmotic pressure changes of Schizochytrium S056 growth is minimal, and Schizochytrium S056 medium with high concentration of sea crystal base in (20 g/L) can be a low concentration of sodium sulfate (2-10g/L) completely replaced. This technique can significantly reduce the concentration of salt ions based culture, the difficulty of fermentation wastewater treatment decreased significantly, but also completely solves the corrosion problem of chloride ions on the fermentation tank, reduce the production cost of raw materials and fixed DHA the asset investment cost. (2) a high content of DHA oil fermentation technology, significantly increased the content of DHA, revealed that the DHA synthesis and primary metabolism. In order to further improve the Schizochytrium DHA produced by the fat content in this paper, influence A detailed analysis of oil synthesis of a large number of key factors, and the influence of fermentable carbon source, the accumulation of DHA carried out a detailed study, through the replacement of glucose glycerol, DHA content increased significantly, increased by 39.68% glucose to glycerol when cultured in 52.28%, an increase of 31.75%. of glycerol and glucose in key enzymes and key the primary metabolites of metabolic pathways of Schizochytrium in analysis, found pentosephosphatepathway glycerol on bacteria in vivo glucose -6- phosphate dehydrogenase (G6PDH) significantly inhibited, thereby reducing the intracellular coenzyme II (NADPH) content, DHA synthesis was not decreased due to the decrease of NADPH that showed that the synthesis path of Schizochytrium S056 DHA in more than one. (3) comparative transcriptome based on glycerol and glucose of two kinds of carbon source of the culture difference in gene expression under the condition of Comprehensive analysis reveals a large number of DHA to promote the success of glycerol synthesis mechanism. Compared with the fermentation of glucose, glycerol fermentation, Schizochytrium S056 in gluconeogenesis and the pentose phosphate pathway path were significantly inhibited, and acetyl coenzyme A synthesis related metabolic pathways, including glycolysis and branched chain amino acids (Valine Leucine and isoLeucine) pathway was significantly enhanced. In addition, glycerol also inhibited the tricarboxylic acid cycle three (TCA cycle), the path leading to more acetyl coenzyme A into lipid synthesis route, significantly promote the synthesis of fatty acids (fatty acid the traditional path of synthesis system, FAS system) is the key enzyme gene FAS gene expression was enhanced the content of DHA can be greatly improved. The research provides important theoretical guidance to promote the DHA synthesis regulation. (4) established a high-quality DHA oil test technical training, and implementation of the technology The industrial application of this research. Based on the above research results, firstly, the key factors affecting the synthesis of DHA dissolved oxygen and temperature to carry out a detailed study, in 100 L fermentor scale a section dissolved oxygen control and constant temperature cultivation strategy; secondly, according to the consumption curve of Schizochytrium S056 glycerol in 100 L fermentor. In the premise of not prolonging the fermentation cycle, the optimum feeding scheme, up to 52.61% of the content of DHA obtained in oil production reached 23.40g/L, DHA, DHA significantly increased the yield and production efficiency; finally, the stirring power is the same criteria as the starting point, the scale-up of Schizochytrium was S056 to explore, through the 1000 L fermentor Schizochytrium S056 adjust the dissolved oxygen concentration in the process of training, which is consistent with the dissolved oxygen concentration in 100 L fermentation tank, so as to achieve good Schizochytrium S056 in 1000 L High density fermentation tank culture, and the scale-up of verification, in 10 ton fermentation tank. The results showed that the average yield of DHA was more than 22g/L, and the oil content in DHA remained above 50%, the successful implementation of the high strength and high quality DHA fermentation production scale of 10 tons in the fermentation tank. In conclusion, this paper realized the low salt Schizochytrium cultivation significantly increased the content of DHA, successfully reveals the glycerol synthesis of large amounts of DHA can promote the metabolism mechanism, and through the precise control of dissolved oxygen and temperature, realize the integrated application of these technologies in 10 ton fermentation tank.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：TQ920.1

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