本文關(guān)鍵詞：卷枝毛霉生物合成番茄紅素的研究　出處：《江南大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： 卷枝毛霉 番茄紅素 crgA 番茄紅素環(huán)化酶 甲羥戊酸途徑 脂質(zhì)合成

【摘要】：番茄紅素是異戊二烯類(lèi)化合物,屬于類(lèi)胡蘿卜素的一種,是自然界最強(qiáng)的抗氧化劑之一,具有抗癌防癌、防治心血管疾病等多種生理功能。為了滿足番茄紅素在醫(yī)藥及營(yíng)養(yǎng)領(lǐng)域的應(yīng)用需求,通過(guò)生物技術(shù)大量生產(chǎn)高純度、安全的番茄紅素成為番茄紅素合成技術(shù)的發(fā)展趨勢(shì)。本論文在以類(lèi)胡蘿卜素為主要次級(jí)代謝產(chǎn)物的絲狀真菌卷枝毛霉(Mucor circinelloides)基礎(chǔ)上,挑選產(chǎn)量較高的突變株為出發(fā)菌株,從改造番茄紅素合成途徑及調(diào)控路徑兩方面著手,通過(guò)分子生物學(xué)等手段獲得了一株番茄紅素高產(chǎn)菌株,并對(duì)其發(fā)酵條件進(jìn)行初探,進(jìn)一步提高了番茄紅素產(chǎn)量。論文的主要研究結(jié)果如下:(1)解除卷枝毛霉類(lèi)胡蘿卜素合成途徑的負(fù)調(diào)控因子crg A,提高卷枝毛霉類(lèi)胡蘿卜素合成:通過(guò)敲除卷枝毛霉MU206和MU218中限制類(lèi)胡蘿卜素合成的調(diào)控基因crg A,共獲得了5株Δcrg A菌株,類(lèi)胡蘿卜素的主要成分(β-胡蘿卜素)含量提高了4.8~47.6倍。相較于MU206,以MU218為背景構(gòu)建的Δcrg A菌株類(lèi)胡蘿卜素產(chǎn)量提高更為顯著,其中含量最高的是MU606,其β-胡蘿卜素含量在黑暗和光照條件下分別達(dá)到3.00 mg/g和4.01 mg/g,是初始菌株MU218的47.6倍和5.4倍,是用于構(gòu)建卷枝毛霉番茄紅素高產(chǎn)菌株的潛力菌株;同時(shí)對(duì)卷枝毛霉MU206和MU218中潛在的類(lèi)胡蘿卜素調(diào)控機(jī)理進(jìn)行了初步探索,發(fā)現(xiàn)MU218中存在類(lèi)似于Crg A途徑的新調(diào)控路徑。(2)阻斷番茄紅素環(huán)化為β-胡蘿卜素的反應(yīng),構(gòu)建番茄紅素生產(chǎn)菌株:在上述重組菌株MU606基礎(chǔ)上,同時(shí)采用定點(diǎn)突變和化學(xué)誘變兩種方式使番茄紅素環(huán)化酶失活,最終獲得三株紅色突變株。測(cè)序結(jié)果顯示三株突變株中編碼番茄紅素環(huán)化酶的car RP基因均發(fā)生了突變。突變導(dǎo)致MUt1和MUt2番茄紅素環(huán)化酶活力部分喪失,而突變株MUt3的番茄紅素環(huán)化酶則完全失活,其番茄紅素含量在黑暗與光照條件下分別達(dá)到1.90 mg/g和3.64 mg/g,與MU606相比提高了30.6倍和21.4倍。根據(jù)不同突變株的類(lèi)胡蘿卜組成及含量對(duì)卷枝毛霉番茄紅素環(huán)化酶的催化機(jī)制進(jìn)行了研究:其蛋白結(jié)構(gòu)中兩個(gè)重復(fù)的R domain結(jié)構(gòu)域分別負(fù)責(zé)γ-胡蘿卜素到β-胡蘿卜素和番茄紅素到γ-胡蘿卜素的反應(yīng),且第1個(gè)R domain的功能依賴于第2個(gè)R domain的作用。此外,發(fā)現(xiàn)番茄紅素含量較高的菌株中存在反饋抑制效應(yīng)。(3)強(qiáng)化番茄紅素合成途徑限速步驟,增加目的產(chǎn)物代謝流:首先通過(guò)轉(zhuǎn)錄組分析推測(cè)HMG-Co A還原酶(HMGr)是番茄紅素合成途徑的限速酶,在突變株MUt3基礎(chǔ)上成功同源過(guò)量表達(dá)編碼HMGr的三個(gè)拷貝(hmgr1,hmgr2,hmgr3),揭示了HMGr在卷枝毛霉番茄紅素合成過(guò)程中的作用:其中hmgr2、hmgr3的過(guò)量表達(dá)使番茄紅素含量提高了2倍左右,說(shuō)明其編碼的HMGr是番茄紅素合成途徑的限速酶;而hmgr1的過(guò)表達(dá)并未造成番茄紅素含量的變化,暗示其編碼的HMGr可能參與了其他萜類(lèi)物質(zhì)的合成。本章獲得了一株高產(chǎn)番茄紅素的卷枝毛霉菌株MUhr3-1,在黑暗和光照條件下番茄紅素含量分別達(dá)到3.75 mg/g和7.16 mg/g,是對(duì)照菌株的2倍。(4)抑制競(jìng)爭(zhēng)代謝途徑(脂肪酸合成途徑),為番茄紅素合成增加底物:首先通過(guò)氮限制培養(yǎng)基考察了卷枝毛霉中脂肪酸和類(lèi)胡蘿卜素積累的關(guān)系:脂肪酸和類(lèi)胡蘿卜素的合成均受氮源的調(diào)控,積累同步,存在競(jìng)爭(zhēng)關(guān)系;并通過(guò)轉(zhuǎn)錄組分析發(fā)現(xiàn)在類(lèi)胡蘿卜素產(chǎn)量較高的卷枝毛霉菌株中,編碼脂肪酸合酶和乙酰輔酶A羧化酶(FAS和ACC)的基因轉(zhuǎn)錄水平較低,進(jìn)一步證明脂肪酸與類(lèi)胡蘿卜素合成的競(jìng)爭(zhēng)關(guān)系;最后在MUhr3-1基礎(chǔ)上分別構(gòu)建了FAS和ACC的RNAi菌株,通過(guò)下調(diào)FAS和ACC的表達(dá)水平使番茄紅素的含量分別提高了14%~43%和13%~46%,然而菌體生長(zhǎng)受到影響;為避免抑制FAS和ACC的表達(dá)導(dǎo)致的菌體生長(zhǎng)受阻,在菌體積累完成后添加FAS和ACC抑制劑(淺藍(lán)菌素、棕櫚油、紅花籽油和橄欖油),進(jìn)一步提高了番茄紅素的含量。其中添加淺藍(lán)菌素和棕櫚油最高可使番茄紅素含量提升至11.10和11.55 mg/g。(5)優(yōu)化番茄紅素合成發(fā)酵培養(yǎng)基碳源、氮源,提高番茄紅素產(chǎn)量:以重組卷枝毛霉MUhr3-1為研究對(duì)象,考察碳源濃度、氮源的種類(lèi)和濃度對(duì)番茄紅素產(chǎn)量的影響,結(jié)果表明葡萄糖濃度為80 g/L、以大豆豆粕為氮源且濃度為25 g/L時(shí)番茄紅素產(chǎn)量最高,與初始發(fā)酵條件相比提高了2.5倍。在發(fā)酵罐中進(jìn)行擴(kuò)大培養(yǎng),發(fā)酵過(guò)程中添加棕櫚油作為ACC抑制劑,發(fā)酵至120 h時(shí)番茄紅素含量達(dá)到12.77±1.79 mg/g,與出發(fā)菌株MU218相比提高了386倍。同時(shí)番茄紅素單位體積產(chǎn)量達(dá)到261.28±9.62 mg/L,是迄今為止報(bào)道的卷枝毛霉番茄紅素最高產(chǎn)量的5倍。成功實(shí)現(xiàn)了在卷枝毛霉中番茄紅素的高效合成。
[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.

【學(xué)位授予單位】：江南大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：TQ920.6

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