卷枝毛霉生物合成番茄红素的研究

发布时间：2017-12-31 04:42

本文关键词：卷枝毛霉生物合成番茄红素的研究　出处：《江南大学》2017年博士论文　论文类型：学位论文

【摘要】：番茄红素是异戊二烯类化合物,属于类胡萝卜素的一种,是自然界最强的抗氧化剂之一,具有抗癌防癌、防治心血管疾病等多种生理功能。为了满足番茄红素在医药及营养领域的应用需求,通过生物技术大量生产高纯度、安全的番茄红素成为番茄红素合成技术的发展趋势。本论文在以类胡萝卜素为主要次级代谢产物的丝状真菌卷枝毛霉(Mucor circinelloides)基础上,挑选产量较高的突变株为出发菌株,从改造番茄红素合成途径及调控路径两方面着手,通过分子生物学等手段获得了一株番茄红素高产菌株,并对其发酵条件进行初探,进一步提高了番茄红素产量。论文的主要研究结果如下:(1)解除卷枝毛霉类胡萝卜素合成途径的负调控因子crg A,提高卷枝毛霉类胡萝卜素合成:通过敲除卷枝毛霉MU206和MU218中限制类胡萝卜素合成的调控基因crg A,共获得了5株Δcrg A菌株,类胡萝卜素的主要成分(β-胡萝卜素)含量提高了4.8~47.6倍。相较于MU206,以MU218为背景构建的Δcrg A菌株类胡萝卜素产量提高更为显著,其中含量最高的是MU606,其β-胡萝卜素含量在黑暗和光照条件下分别达到3.00 mg/g和4.01 mg/g,是初始菌株MU218的47.6倍和5.4倍,是用于构建卷枝毛霉番茄红素高产菌株的潜力菌株;同时对卷枝毛霉MU206和MU218中潜在的类胡萝卜素调控机理进行了初步探索,发现MU218中存在类似于Crg A途径的新调控路径。(2)阻断番茄红素环化为β-胡萝卜素的反应,构建番茄红素生产菌株:在上述重组菌株MU606基础上,同时采用定点突变和化学诱变两种方式使番茄红素环化酶失活,最终获得三株红色突变株。测序结果显示三株突变株中编码番茄红素环化酶的car RP基因均发生了突变。突变导致MUt1和MUt2番茄红素环化酶活力部分丧失,而突变株MUt3的番茄红素环化酶则完全失活,其番茄红素含量在黑暗与光照条件下分别达到1.90 mg/g和3.64 mg/g,与MU606相比提高了30.6倍和21.4倍。根据不同突变株的类胡萝卜组成及含量对卷枝毛霉番茄红素环化酶的催化机制进行了研究:其蛋白结构中两个重复的R domain结构域分别负责γ-胡萝卜素到β-胡萝卜素和番茄红素到γ-胡萝卜素的反应,且第1个R domain的功能依赖于第2个R domain的作用。此外,发现番茄红素含量较高的菌株中存在反馈抑制效应。(3)强化番茄红素合成途径限速步骤,增加目的产物代谢流:首先通过转录组分析推测HMG-Co A还原酶(HMGr)是番茄红素合成途径的限速酶,在突变株MUt3基础上成功同源过量表达编码HMGr的三个拷贝(hmgr1,hmgr2,hmgr3),揭示了HMGr在卷枝毛霉番茄红素合成过程中的作用:其中hmgr2、hmgr3的过量表达使番茄红素含量提高了2倍左右,说明其编码的HMGr是番茄红素合成途径的限速酶;而hmgr1的过表达并未造成番茄红素含量的变化,暗示其编码的HMGr可能参与了其他萜类物质的合成。本章获得了一株高产番茄红素的卷枝毛霉菌株MUhr3-1,在黑暗和光照条件下番茄红素含量分别达到3.75 mg/g和7.16 mg/g,是对照菌株的2倍。(4)抑制竞争代谢途径(脂肪酸合成途径),为番茄红素合成增加底物:首先通过氮限制培养基考察了卷枝毛霉中脂肪酸和类胡萝卜素积累的关系:脂肪酸和类胡萝卜素的合成均受氮源的调控,积累同步,存在竞争关系;并通过转录组分析发现在类胡萝卜素产量较高的卷枝毛霉菌株中,编码脂肪酸合酶和乙酰辅酶A羧化酶(FAS和ACC)的基因转录水平较低,进一步证明脂肪酸与类胡萝卜素合成的竞争关系;最后在MUhr3-1基础上分别构建了FAS和ACC的RNAi菌株,通过下调FAS和ACC的表达水平使番茄红素的含量分别提高了14%~43%和13%~46%,然而菌体生长受到影响;为避免抑制FAS和ACC的表达导致的菌体生长受阻,在菌体积累完成后添加FAS和ACC抑制剂(浅蓝菌素、棕榈油、红花籽油和橄榄油),进一步提高了番茄红素的含量。其中添加浅蓝菌素和棕榈油最高可使番茄红素含量提升至11.10和11.55 mg/g。(5)优化番茄红素合成发酵培养基碳源、氮源,提高番茄红素产量:以重组卷枝毛霉MUhr3-1为研究对象,考察碳源浓度、氮源的种类和浓度对番茄红素产量的影响,结果表明葡萄糖浓度为80 g/L、以大豆豆粕为氮源且浓度为25 g/L时番茄红素产量最高,与初始发酵条件相比提高了2.5倍。在发酵罐中进行扩大培养,发酵过程中添加棕榈油作为ACC抑制剂,发酵至120 h时番茄红素含量达到12.77±1.79 mg/g,与出发菌株MU218相比提高了386倍。同时番茄红素单位体积产量达到261.28±9.62 mg/L,是迄今为止报道的卷枝毛霉番茄红素最高产量的5倍。成功实现了在卷枝毛霉中番茄红素的高效合成。
[Abstract]:Lycopene is isoprenoid compounds, which belongs to a kind of carotenoids, is one of the strongest antioxidant in nature, with a variety of cardiovascular disease prevention and control of cancer prevention, and other physiological functions. In order to meet the application requirements of lycopene in medicine and nutrition in the field of biotechnology, through mass production of high purity and safety of lycopene into development the trend of lycopene synthesis technology. In this paper the filamentous fungi carotenoids as the main secondary metabolites of Mucor circinelloides (Mucor circinelloides) based on the selection of high yield strains of mutant strain, the transformation from two aspects of lycopene biosynthesis pathway and regulation pathway, through molecular biology methods obtained a strain of lycopene producing strain and its fermentation conditions were studied, and further improve the lycopene production. The main research results are as follows: (1) Lift the A CRG as a negative regulator of Mucor circinelloides carotenoid biosynthesis, improve Mucor circinelloides carotenoid synthesis by knockout of Mucor circinelloides MU206 and MU218 in the limit of carotenoid biosynthesis genes CRG A, received a total of 5 strains of CRG A strains, carotenoid pigment ingredients (beta carotene) content increased by 4.8~47.6 times. Compared with MU206, MU218 delta CRG A strain of carotenoids in the background of setting up the production increased significantly, the highest content is MU606, the beta carotene content in the dark and light conditions were respectively 3 mg/g and 4.01 mg/g, 47.6 times the initial strain MU218 and 5.4 times, is used to construct the potential strains of Mucor circinelloides lycopene high yield strains; at the same time to roll branches Mucor MU206 and MU218 potential of carotenoids in the regulation mechanism was explored, found that there are similar to the Crg A MU218. The new regulation path. (2) blocking the cyclization of lycopene beta carotene response, construct lycopene producing strains: in the recombinant strain MU606 on the basis of using site directed mutagenesis and chemical mutagenesis of two kinds of ways to lycopene cyclase inactivation, obtained three strains of red mutation strain. The sequencing result showed that three mutant car strain RP gene encoding lycopene cyclase were mutated. Mutations in the MUt1 and MUt2 of lycopene cyclase activity partially lost, and the mutant MUt3 of lycopene cyclase is completely inactivated, the lycopene content in the dark and light conditions were respectively 1.90 mg/g and 3.64 mg/g, compared with MU606 increased 30.6 times and 21.4 times. According to the catalytic mechanism of carotenoid composition and content of different mutant strains of Mucor circinelloides lycopene cyclase were studied: the protein structure in two replicates The R domain domain are responsible for gamma carotene to beta carotene and lycopene to gamma carotene response, and the first R domain function depends on second R domain. In addition, found that the feedback inhibition effect of lycopene (3) to strengthen the strain. Lycopene synthesis rate limiting step, increase the product metabolic flux: firstly, transcriptome analysis suggested that HMG-Co A reductase (HMGr) is the rate limiting enzyme of lycopene biosynthesis pathway, overexpression of three copies of successful homologous encoding HMGr in mutant MUt3 based on (hmgr1, hmgr2, hmgr3), revealing the role of HMGr in Mucor circinelloides lycopene synthesis process: hmgr2, hmgr3 overexpression of the lycopene content increased by about 2 times, its encoding HMGr is the rate limiting enzyme of lycopene biosynthesis; and the expression of hmgr1 did not cause tomato red The change of chlorophyll content, suggesting that the encoding of HMGr may be involved in the synthesis of other terpenoids. This chapter has a high yield of lycopene by Mucor circinelloides strains of MUhr3-1, in the dark and light conditions of lycopene content reached 3.75 mg/g and 7.16 mg/g, is 2 times of the control strains. (4) inhibition of competing metabolic pathways (fatty acid synthesis pathway), lycopene synthesis substrate: first through the nitrogen limited medium were investigated in volume of fatty acid and carotenoid accumulation in shoots of Mucor: fatty acid and carotenoid synthesis are regulated by the nitrogen accumulation, synchronization, existence the relationship between competition; and through the transcriptome analysis of carotenoids found in high yield of Mucor circinelloides strains, encoding fatty acid synthase and acetyl coenzyme A carboxylase (FAS and ACC) of the gene transcription level is low, further proof of fatty acid and carotenoid synthesis Competition on the basis of MUhr3-1; finally, we constructed RNAi strains FAS and ACC, the content of reduced expression levels of FAS and ACC for lycopene were enhanced by 14%~43% and 13%~46%, but the cell growth affected; in order to avoid the pathogen inhibited the expression of FAS and ACC resulted in the addition of FAS and ACC blocked the growth of after the completion of accumulation of inhibitors in bacteria (cerulenin, palm oil, safflower oil and olive oil), to further improve the lycopene content. The addition of cerulenin and palm oil can make the highest lycopene content increased to 11.10 and 11.55 mg/g. (5) to optimize the synthesis of lycopene fermentation medium carbon the source, nitrogen source, improve the lycopene yield with the recombinant Mucor circinelloides MUhr3-1 as the research object, the effects of carbon source concentration, influence the type and concentration of nitrogen source on Lycopene yield. The results showed that glucose concentration was 80 g/ L, from soybean meal Nitrogen source and concentration of 25 g/L when the highest lycopene yield, compared with the initial fermentation conditions increased by 2.5 times. The cultivation in the fermentation tank, the fermentation process to add palm oil as a ACC inhibitor, fermentation to 120 h lycopene content reached 12.77 + 1.79 mg/g, compared with the starting strain MU218 increased 386 times. At the same time the lycopene yield per unit volume reached 261.28 + 9.62 mg/L, 5 times of Mucor circinelloides lycopene reported to date in the highest yield. Success achieved in volume efficient synthesis of lycopene in the branches Mucor.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：TQ920.6

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