裂殖壶菌高产DHA的发酵技术研究及其代谢机理分析

发布时间：2018-03-01 11:04

本文关键词： 裂殖壶菌 DHA 甘油 FAS system 乙酰辅酶 A　出处：《华中科技大学》2016年博士论文　论文类型：学位论文

【摘要】：DHA (Docosahexaenoic acid,二十二碳六烯酸)是含有六个双键的长链脂肪酸,属于ω-3系列的。经过多年研究发现,DHA对人体的多种生理活动具有十分重要的调控和促进作用,DHA是人视网膜细胞和神经系统组织中必需的主要成分之一,尤其对婴幼儿大脑和视力发育方面起到非常重要的促进作用。裂殖壶菌(Schizochytrium),作为一种大型的海洋真菌,因为其生长快、油脂含量高,被广泛的应用于DHA油脂的生产研究中。但是,利用裂殖壶菌发酵生产DHA油脂过程中,存在培养基盐离子浓度高,DHA含量偏低,生产成本过高等问题,从而严重制约着该产业的发展。该论文以本实验室具有自主知识产权的裂殖壶菌S056 (Schizochytrium sp. S056)为出发菌株,建立了低成本、高强度DHA油脂的清洁生产新技术,揭示了DHA大量合成的代谢机理,并通过对DHA中试条件的优化及放大规律的研究,在10吨规模发酵罐上成功实现了DHA油脂的工业化生产。该研究解决了传统培养基中高盐的问题,并突破了DHA含量大幅提升的瓶颈,显著提高了DHA含量和产品品质,具体研究内容与结果如下：(1)建立了一种低盐发酵新技术,显著降低了培养基中的盐离子浓度。海水晶(NaCl)是裂殖壶菌发酵过程中为了模拟海水环境必须添加的培养基组分,但高浓度海盐的存在,增加了发酵废水的处理难度,且大量氯离子的存在对发酵罐腐蚀严重,增加了固定资产投入等。针对以上问题,本研究通过对海水晶在裂殖壶菌发酵过程中的生理作用开展了详细研究分析,发现渗透压的改变对裂殖壶菌S056的生长影响极小,且裂殖壶菌S056培养基中的高浓度海水晶(20 g/L)可被低浓度的硫酸钠(2-10g/L)完全替换。该技术的建立显著降低了培养基中盐离子的浓度,使发酵废水的处理难度下降明显,也彻底解决了氯离子对发酵罐的腐蚀问题,降低了DHA生产的原料成本和固定资产投入成本。(2)建立了高含量DHA油脂发酵新技术,显著提高了DHA含量,初步揭示了DHA合成与初级代谢相关性。为了进一步提高裂殖壶菌所产油脂中DHA含量,本文针对影响油脂大量合成的关键因素进行了详细分析,并对可影响DHA积累的发酵碳源开展了详细研究,通过甘油对葡萄糖的替换,DHA含量显著提高,由葡萄糖培养的39.68%提高到甘油培养时的52.28%,增幅达31.75%。进一步对甘油和葡萄糖参与裂殖壶菌的初级代谢途径中的关键酶及关键代谢产物进行分析,发现甘油对菌体内的磷酸戊糖路径中葡萄糖-6-磷酸脱氢酶(G6PDH)有明显的抑制作用,进而降低了菌体内的还原型辅酶Ⅱ(NADPH)含量,DHA的合成量并没有因NADPH的下降而下降,表明裂殖壶菌S056体内DHA的合成路径不止一条。(3)基于比较转录组学,对甘油和葡萄糖两种碳源培养条件下的差异基因的表达进行全面分析,成功揭示了甘油促进DHA大量合成机理。与葡萄糖发酵相比,甘油培养时,裂殖壶菌S056体内的糖异生路径和磷酸戊糖路径路径均被明显抑制,而与乙酰辅酶A合成相关的代谢路径,包括糖酵解和支链氨基酸(Valine, Leucine and isoLeucine)代谢路径均明显增强。此外,甘油也显著抑制了三羧酸循环路径(TCA cycle),致使更多的乙酰辅酶A向油脂合成路径转化,显著促进传统脂肪酸合成路径(fatty acid synthesis system, FAS system)的关键酶基因FAS基因表达量增强,使DHA含量得以大幅提高。该研究对促进DHA大量合成的调控提供了重要的理论指导。(4)建立了高品质DHA油脂的中试培养技术,并实现了该技术的工业化应用。本研究基于以上研究成果,首先对影响DHA合成的关键因素一溶氧和温度开展详细研究,在100 L规模发酵罐上建立了分段溶氧控制和恒温培养策略；其次,根据裂殖壶菌S056在100 L发酵罐上甘油的动态消耗曲线,在不延长发酵周期的前提条件下,确定了最佳补料方案,最终获取的油脂中DHA含量高达52.61%,DHA产量达23.40g/L,显著提高了DHA产量和生产效率；最后,以搅拌功率相等的准则为出发点,对裂殖壶菌S056的放大规律进行探索,通过对1000 L发酵罐中裂殖壶菌S056培养过程中的溶氧浓度进行调整,使其与100 L发酵罐上的溶氧浓度保持一致,从而很好的实现裂殖壶菌S056在1000 L发酵罐上的高密度培养,并将该放大规律在10吨发酵罐上进行验证,结果表明,DHA产量平均在22g/L以上,且油脂中DHA的含量维持在50%以上,在10吨规模发酵罐成功实现了高品质DHA的高强度发酵生产。综上所述,本文实现了裂殖壶菌的低盐培养,显著提高了DHA的含量,成功揭示了甘油可促进DHA大量合成的代谢机理,并通过对溶氧和温度的精准调控,实现了以上技术在10吨发酵罐上的集成应用。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：TQ920.1

【相似文献】