【作者】 肖爱华；

【导师】 邓放明； 黄和；

【作者基本信息】 湖南农业大学 ， 食品工程， 2008， 硕士

【摘要】 花生四烯酸（ArachidonicAcid,简称AA,即5,8,11,14.二十碳四烯酸）属于ω-6系列长链多不饱和脂肪酸,是人体前列腺素、凝血噁烷、环前列腺素、白三烯合成的重要前体物质,具有多种生理活性。AA被广泛应用于医药、化妆品、食品、农业及其他领域。目前,高山被孢霉发酵法被公认为AA最好的工业化生产方法。但至今此生产方法发酵过程AA含量难以控制,饱和、单不饱和脂肪酸含量过高以及葡萄糖转化率低等关键问题没有得到解决。本文引入代谢工程的方法研究Mortierella alpina ME-1体内AA合成过程,采用多种策略提高AA单产水平。1.应用响应曲面法优化花生四烯酸发酵培养基通过响应曲面优化法优化了Mortierella alpina ME-1发酵生产生物质及AA的培养基组成。生成了2个经验多项式模型。根据模型优化,最优的生物质发酵培养基组成为:葡萄糖90.16g/L,酵母膏12.50g/L,KH₂PO₄ 3.80g/L,NaNO₃3.54 g/L。生物质单产水平预测为36.86 g/L。最优的AA发酵培养基为:葡萄糖103.16g/L,酵母膏11.66g/L,KU₂PO₄ 3.80g/L,NaNO₃ 3.43 g/L。该培养基下,AA单产水平预测为9.65 g/L。验证试验表明这两个模型较好的解释了生物质及AA的生成,预测值与实际值十分接近。同时发酵罐试验表明优化后的培养基同样提升了罐上的发酵水平:最大生物量34.21±1.01 g/L;最大AA单产水平9.86±0.45 g/L。2.花生四烯酸合成过程的代谢通量分析与调控代谢通量分布分析已经成为研究发酵过程特性的有效方法。今建立了花生四烯酸在高山被孢霉ME-1（Mortierella alpina ME-1）体内合成的代谢通量模型,求解不同氮源浓度下发酵各时期的碳流分布。充足氮源发酵时,指数生长期、减速期、稳定期流向AA的碳流分别占总碳流的3.28%,8.80%和6.97%。而通过限制性氮源发酵并在96 h补加0.05%的NaN03成功地引导了发酵碳流迁移,将各时期流向AA的碳流分别提高至3.95%,19.21%和39.29%,并最终实现AA单产水平从1.3 g/L提高到3.5 g/L。这些结果表明限制性氮源发酵并在稳定期补加低浓度的氮源能显著提高AA单产水平。3.基于动力学模型的动态代谢通量分析模型分析高山被孢霉发酵生产花生四烯酸动力学特征,建立了氮源与葡萄糖双底物限制动力学模型,在此基础上结合代谢流分析理论提出了花生四烯酸发酵动态代谢通量分析模型,通过非线性最小二乘优化拟合获得参数。进一步试验表明这个模型具有较高的精确度,能很好模拟发酵过程,并能很容易地获得不同发酵时间的代谢流分布。更多还原

【Abstract】 Arachidonic acid（AA;5,8,11,14-cis-eicosatetraenoic acid） belongs to omega-6 class polyunsaturated fatty acids（PUFAs）.As a precursor of prostaglandins,thromboxane,prostacyclin,and leucotrienes,it owns various physiological functions and has been found wide application in medicine, pharmacology,cosmetics,food industry,agriculture and other fields.Production of AA by cultivation of Mortierella fungi,which are thought to be the most prominent AA source,has been widely reported and industrialized.However,several key problems such as the hardness to control the AA percentage in lipids,high content of saturated and monounsaturated fatty acids and low conversion ratio of glucose to AA are not solved,which lead to high market price of AA.This maybe caused by the complicated fatty aicds biosynthesis mechanism and traditional methods’ lack of metabolic network analysis on AA fermentation.In the present work,metabolic engineering was introduced to analyze the mechanism of AA synthesis in Mortierella alpina ME-1,and several strategies were applied to enhance AA production.1.Optimization of media components for biomass and arachidonic acid production by Mortierella alpina ME-1 using response surface methodologyThe media for biomass and arachidonic acid production by Mortierella alpina ME-1 were optimized in shake flask cultures using response surface methodology （RSM）.Two imperial polynomial models were developed,and the maximum biomass yield of 36.86 g/L appeared at glucose,yeast extract（YE）,KH₂PO₄ and NaNO₃ of 90.16,12.50,3.80 and 3.54 g/L,respectively,while a maximum AA yield of 9.65 g/L appeared at glucose,YE,KH₂PO₄ and NaNO₃ of 103.16,11.66,3.80 and 3.43 g/L,respectively,in 6.5-day fermentation were predicted.These predicted values were also verified by validation experiments which showed excellent correlation between predicted and measured values.The results of bioreactor fermentation also illustrated that the optimized culture medium enhanced both biomass（34.21±1.01 g/L in 5-day fermentation） and AA（9.86±0.45 g/L in 6-day fermentation） production by Mortierella alpina ME-1 in a large-scale fermentation process.2.Metabolic flux analysis on arachidonic acid FermentationAnalysis of flux distributions in metabolic networks has become an important approach for understanding the fermentation characteristics of the process.A model of metabolic flux analysis of arachidonic acid（AA） synthesis in Mortierella alpina ME-1 was established and carbon flux distributions were estimated in different fermentation phases with different concentrations of N-source.During exponential, decelerating and stationary phase,carbon fluxes to AA were 3.28%,8.80%,6.97%, respectively,with sufficient N-source broth based on the flux of glucose uptake,and those were increased to 3.95%,19.21%and 39.29%,respectively,by regulating the shifts of carbon fluxes via fermentation with limited N-source broth and adding 0.05%NaNO₃ at 96h.Eventually AA yield was increased from 1.3 g/L to 3.5 g/L. These results suggest a way to improve AA fermentation,that is,fermentation with limited N-source broth and adding low concentration N-source during stationary phase.3.Dynamic flux analysis model for arachiconic acid fermentationKinetic models of arachidonic acid fermentation by Mortierella alpina ME-1 with limited N-source and glucose were established based on the experimental data. Then a dynamic flux analysis model based on the kinetic models was proposed according to the theory of metabolic flux analysis.And the optimal parameters were evaluated.Further experiment data verified the accuracy of the models and the results showed that the models appeared to provide a reasonable description for the fermentation,and the flux distributions in different time can be easily obtained.更多还原