發(fā)布時(shí)間：2018-01-17 21:01

  本文關(guān)鍵詞：產(chǎn)油真菌卷枝毛霉WJ11高產(chǎn)脂質(zhì)的分子機(jī)制　出處：《江南大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 卷枝毛霉 脂質(zhì)積累 基因組學(xué) 蛋白質(zhì)組學(xué) 酶活 氨基酸

【摘要】：產(chǎn)油微生物能夠在細(xì)胞內(nèi)積累大量脂肪酸并以甘油三酯的形式儲(chǔ)存,是傳統(tǒng)動(dòng)植物油脂的重要替代資源。某些微生物積累的油脂還富含高價(jià)值的多不飽和脂肪酸,具有重要的應(yīng)用價(jià)值和商業(yè)價(jià)值。在產(chǎn)油微生物中,不同物種的微生物油脂產(chǎn)量相差巨大,其油脂積累量從細(xì)胞干重的20%到80%以上不等。由于不同物種間生理生化特征差異較大,導(dǎo)致不同微生物之間脂質(zhì)積累差異的機(jī)制難于解析,而對(duì)同一物種產(chǎn)脂差異顯著的不同菌株的研究能夠很好地解決這一問題。本文通過基因組學(xué)、蛋白質(zhì)組學(xué)以及關(guān)鍵酶的活性分析系統(tǒng)研究了卷枝毛霉高產(chǎn)脂菌株WJ11和低產(chǎn)脂菌株CBS 277.49在脂質(zhì)代謝調(diào)控上的差異;結(jié)合轉(zhuǎn)錄分析找出了一些關(guān)鍵調(diào)控基因;通過基因工程過表達(dá)這些關(guān)鍵基因,有效提高了脂肪酸的積累;還研究了氨基酸氮源對(duì)卷枝毛霉γ-亞麻酸(GLA)產(chǎn)量的影響。論文的主要研究?jī)?nèi)容和實(shí)驗(yàn)結(jié)果如下:通過對(duì)卷枝毛霉WJ11和CBS 277.49生長(zhǎng)和產(chǎn)脂的分析,發(fā)現(xiàn)WJ11脂肪酸含量高達(dá)36%,而CBS 277.49則不超過15%。WJ11全基因組的測(cè)序結(jié)果顯示其基因組大小為35.4 Mb,包含827個(gè)scaffold,由2530個(gè)contig組成,GC含量為39.7%,有10973個(gè)預(yù)測(cè)的基因和177個(gè)t RNA基因。通過比較WJ11(本實(shí)驗(yàn)室測(cè)序)和CBS 277.49(美國(guó)聯(lián)合基因研究所測(cè)序)的基因組發(fā)現(xiàn),兩株菌的基因組特征總體相似度較高,MAUVE分析顯示兩株菌的基因組序列擁有很多的同源區(qū)域,MUMmer分析也表明兩株菌的基因組整體共線性良好。根據(jù)基因組信息,構(gòu)建了WJ11和CBS 277.49中心碳代謝和脂代謝網(wǎng)絡(luò),包括葡萄糖的代謝,脂肪酸的合成,甘油三酯、磷脂、類固醇和類胡蘿卜素的合成以及脂肪的β氧化。通過比較兩株菌中編碼這些代謝途徑中相關(guān)酶的基因個(gè)數(shù),發(fā)現(xiàn)基因個(gè)數(shù)的差異可能對(duì)兩株菌的脂質(zhì)積累產(chǎn)生影響。另外,通過Ortho MCL聚類分析找出了WJ11和CBS 277.49之間的特異基因,這些特異基因可能與兩株菌的細(xì)胞生長(zhǎng)和脂肪積累差異相關(guān)。利用蛋白質(zhì)組學(xué)分析了高產(chǎn)脂菌株WJ11在脂質(zhì)積累期(氮缺乏)和生長(zhǎng)平衡期全蛋白的表達(dá)差異情況,結(jié)果發(fā)現(xiàn)氮缺乏促進(jìn)了氮的同化,減緩了氨基酸的代謝;上調(diào)了糖酵解相關(guān)蛋白的表達(dá)并下調(diào)了三羧酸循環(huán)相關(guān)蛋白的表達(dá),使更多的碳流向脂肪酸合成;氮缺乏提高了磷酸戊糖途徑相關(guān)蛋白的表達(dá),為脂肪酸合成提供更多的還原力NADPH;在氮缺乏時(shí),蛋白質(zhì)和核酸的代謝受到抑制,細(xì)胞主要進(jìn)行脂質(zhì)合成和積累;氮缺乏還上調(diào)了一些與能量代謝、信號(hào)傳導(dǎo)、分子伴侶和氧化還原相關(guān)蛋白的表達(dá),以應(yīng)對(duì)氮限制這種環(huán)境壓力。另一方面,通過比較高產(chǎn)脂菌株WJ11和低產(chǎn)脂菌株CBS277.49在脂質(zhì)快速積累期全蛋白的表達(dá)差異,發(fā)現(xiàn)WJ11中與支鏈氨基酸代謝相關(guān)蛋白的表達(dá)顯著上調(diào),這可能為脂肪酸合成提供更多的底物乙酰輔酶A;與CBS 277.49相比,WJ11中糖酵解相關(guān)蛋白的表達(dá)量更高,而三羧酸循環(huán)相關(guān)蛋白的表達(dá)量則較低,這表明WJ11中碳更多流向脂肪酸的合成,而非參與三羧酸循環(huán);WJ11中磷酸戊糖途徑相關(guān)蛋白的表達(dá)量也較CBS 277.49要高,這表明WJ11中會(huì)產(chǎn)生更多的還原力NADPH。通過對(duì)卷枝毛霉WJ11和CBS 277.49脂質(zhì)合成關(guān)鍵酶的活力測(cè)定發(fā)現(xiàn),與CBS277.49相比,WJ11中果糖磷酸激酶(PFK)的酶活較低,而磷酸戊糖途徑中葡萄糖-6-磷酸脫氫酶(G6PDH)和6-磷酸葡萄糖酸脫氫酶(6PGDH)的酶活較高,這將導(dǎo)致更多的碳流向磷酸戊糖途徑,從而為脂肪酸合成提供更多的還原力NADPH;對(duì)于脂質(zhì)合成過程中還原力的重要提供者蘋果酸酶(ME),其在WJ11脂質(zhì)積累初期的酶活也略微高出CBS 277.49;WJ11中調(diào)控三羧酸循環(huán)的異檸檬酸脫氫酶(ICDH)的酶活顯著低于CBS 277.49,這表明WJ11中三羧酸循環(huán)更為緩慢,將會(huì)有更多的檸檬酸轉(zhuǎn)運(yùn)到線粒體外;同時(shí)WJ11中ATP:檸檬酸裂解酶(ACL)的酶活相比較CBS 277.49又有所提高,這將增加脂肪酸合成的前體乙酰輔酶A的供應(yīng);WJ11中脂肪酸合酶(FAS)的活力也明顯高于CBS 277.49。這些酶活差異較為清晰地解析了WJ11高產(chǎn)脂質(zhì)的調(diào)控機(jī)制。針對(duì)脂質(zhì)合成中的關(guān)鍵酶以及蛋白質(zhì)組分析得到的重要差異蛋白,利用RT-q PCR研究它們基因的轉(zhuǎn)錄水平,結(jié)果顯示轉(zhuǎn)錄分析數(shù)據(jù)能夠與酶活及蛋白質(zhì)組數(shù)據(jù)較好的對(duì)應(yīng);并發(fā)現(xiàn)與生長(zhǎng)平衡期相比,WJ11中的g6pdh1和g6pdh2基因的表達(dá)在脂質(zhì)積累期提高幅度較大,推測(cè)這兩個(gè)基因可能與脂質(zhì)積累密切相關(guān);同時(shí)還發(fā)現(xiàn)脂質(zhì)積累期WJ11中與支鏈氨基酸代謝相關(guān)基因(如leu B)的轉(zhuǎn)錄水平下調(diào)幅度要顯著小于CBS 277.49,這些基因也可能通過某種機(jī)制參與卷枝毛霉脂質(zhì)合成的調(diào)控�；蚪M、蛋白質(zhì)組、酶活以及轉(zhuǎn)錄分析的數(shù)據(jù)顯示,WJ11中g(shù)6pdh1、g6pdh2及l(fā)eu B基因可能對(duì)脂質(zhì)積累有重要的調(diào)控作用。通過在卷枝毛霉CBS 277.49缺陷型菌株中過表達(dá)WJ11中的g6pdh1、g6pdh2及l(fā)eu B基因,發(fā)現(xiàn)這些基因?qū)碇γ沟纳L(zhǎng)沒有明顯影響,但均顯著提高了脂肪酸的積累:其中過表達(dá)g6pdh1的重組菌株脂肪酸含量提高了23~28%,過表達(dá)g6pdh2的重組菌株脂肪酸含量提高了41~47%,這表明g6pdh1和g6pdh2均能夠調(diào)控卷枝毛霉的脂質(zhì)積累,且g6pdh2的效果更為顯著;另外,leu B基因的過表達(dá)使重組菌株脂肪酸含量提高了67~73%,表明leu B基因在脂肪酸積累過程中起到重要的調(diào)控作用。針對(duì)氨基酸對(duì)于脂肪酸積累可能的調(diào)控作用,分別以二十種標(biāo)準(zhǔn)氨基酸為氮源培養(yǎng)卷枝毛霉,分析其細(xì)胞生長(zhǎng)、脂質(zhì)積累及GLA產(chǎn)量。首先比較本實(shí)驗(yàn)室三株卷枝毛霉(WJ11、CBS 108.16和CBS 277.49)GLA的積累能力,發(fā)現(xiàn)WJ11總脂肪酸含量最高,但由于脂肪酸組分中GLA比例較低,使GLA產(chǎn)量顯著小于CBS 108.16,因此選取CBS108.16作為研究菌株。利用不同氨基酸為氮源培養(yǎng)卷枝毛霉CBS 108.16,發(fā)現(xiàn)其菌體生長(zhǎng)、脂肪酸積累以及GLA產(chǎn)量會(huì)有顯著差別,其中,酪氨酸為最適合生長(zhǎng)和脂質(zhì)積累的氮源,且GLA產(chǎn)量也最高(0.81 g/L)。通過對(duì)脂肪酸合成相關(guān)酶活力的測(cè)定,發(fā)現(xiàn)酪氨酸顯著提高了G6PDH和6PGDH的活力,為脂肪酸合成提供更多的還原力;酪氨酸也提高了ACL的活力,為脂肪酸合成提供更多的乙酰輔酶A,同時(shí),ICDH活力的下調(diào)也可能減緩了三羧酸循環(huán),促進(jìn)脂肪酸的合成。這與蛋白質(zhì)組學(xué)及WJ11與CBS 277.49脂質(zhì)合成相關(guān)酶的活力分析結(jié)果相一致。
[Abstract]:Oleaginous microorganisms can accumulate large amounts of fatty acids in cells and stored in the form of triglycerides, is an important substitute resource of traditional animal fats and vegetable oils. Some microorganisms accumulated oil is also rich in high value of polyunsaturated fatty acid has important application value and commercial value. In the oil producing microorganisms, microbial oil production between different species huge accumulation from 20% of the cell dry weight more than 80% of its oil. Due to different physiological and biochemical characteristics of different species, different mechanisms leading to lipid accumulation difference between microorganisms is difficult to parse, and study different strains of the same species significantly differences in lipid production can solve this problem in this paper. Genomics, proteomics and key enzyme activity analysis of Mucor circinelloides high fat and low fat strain WJ11 strain CBS 277.49 in lipid metabolism Differences in Xie regulation; binding transcription analysis to identify the key regulatory genes by gene engineering; over expression of these genes, effectively improve the accumulation of fatty acids; of amino acid nitrogen source on Mucor circinelloides gamma linolenic acid (GLA) yield. The main research contents and results are as follows through analysis of Mucor circinelloides WJ11 and CBS 277.49 growth and lipid production, found that WJ11 fatty acid content as high as 36%, while the CBS is not more than 277.49 sequencing of 15%.WJ11 genome showed that the genome size is 35.4 Mb, containing 827 scaffold, consisting of 2530 contig, the content of GC is 39.7%, 10973 a prediction of T genes and 177 RNA genes. Through the comparison of the WJ11 (the laboratory sequencing) and CBS 277.49 (United States Institute of gene sequencing) genome revealed genomic characteristics of two strains were similar to the overall high degree, MAUVE analysis The genome sequences of two strains have many homologous regions, MUMmer analysis showed that two strains of the whole genome were linear. Based on the genomic information, construct WJ11 and CBS 277.49 central carbon metabolism and lipid metabolism, including glucose metabolism, fatty acid synthesis, triglycerides, phospholipids, steroids and carotenoids the synthesis of fat and beta oxidation. Through the comparison of two strains encoding enzymes in the metabolic pathway of gene number, found differences in the gene number of two strains of bacteria may lipid accumulation effect. In addition, through the analysis of the Ortho MCL clustering to find specific gene WJ11 and CBS 277.49, these specific genes may the cell growth and the accumulation of fat and two strains. Differences using proteomic analysis of high yield strains of WJ11 lipid accumulation period in lipid (nitrogen deficiency) expression and growth of the whole protein balance period The differences, results showed that nitrogen deficiency promoted nitrogen assimilation, slowing down the metabolism of amino acid; expression of glycolytic protein expression and down regulated three tricarboxylic acid cycle related protein, so that more carbon flow to the synthesis of fatty acids; nitrogen deficiency increased the expression of the pentose phosphate pathway related protein, provide more reduction NADPH for fatty acid synthesis; in the lack of nitrogen, protein and nucleic acid metabolism is inhibited, cell mainly lipid synthesis and accumulation; nitrogen deficiency also raised some of the energy metabolism, signal transduction, protein expression of molecular chaperones and the reduction of oxygen and nitrogen limitation in response to this environmental stress. On the other hand, the rapid accumulation of differentially expressed proteins in the lipid phase through the comparison of high fat and low fat strain WJ11 strain CBS277.49 showed the expression of WJ 11 and branched chain amino acid metabolism protein significantly increased, the May provide a substrate of acetyl coenzyme A more for fatty acid synthesis; compared with CBS 277.49, WJ11 expression of glycolysis proteins related to higher expression of three tricarboxylic acid cycle related protein is low, which indicates that WJ 11 more carbon to fatty acid synthesis, rather than participate in the three tricarboxylic acid cycle; expression WJ 11 the pentose phosphate pathway related protein is 277.49 CBS higher, suggesting that WJ 11 will produce more reducing power NADPH. based on WJ11 and CBS of Mucor circinelloides 277.49 lipid synthesis key enzyme activity assay showed that compared with CBS277.49, WJ 11 phosphofructokinase (PFK) enzyme activity in low phosphate the pentose pathway of glucose -6- phosphate dehydrogenase (G6PDH) and 6- phosphogluconate dehydrogenase (6PGDH) activity is high, which will lead to more carbon in the pentose phosphate pathway, thus for fatty acid synthesis provides more power for lipid NADPH; An important provider of quality apple acid enzyme synthesis process is the force (ME), the WJ11 in the initial activity of lipid accumulation is also slightly higher than CBS 277.49; isocitrate dehydrogenase three tricarboxylic acid cycle WJ regulation 11 (ICDH) was significantly lower than that of CBS 277.49 of the enzyme, suggesting that WJ 11 three tricarboxylic acid cycle more slowly and there will be more to the mitochondrial citrate transport; at the same time WJ11 ATP: citrate lyase (ACL) activity compared to CBS 277.49 has improved, which will increase the fatty acid precursors for the synthesis of acetyl coenzyme A supply; WJ 11 fatty acid synthase (FAS) activity was significantly higher than that of CBS the 277.49. enzyme activity difference is clearly the regulation mechanism of WJ11 high yield lipid. Important differences protein analysis for the key enzyme in lipid synthesis and protein group, the transcription level of RT-q gene by PCR are studied, the results showed the number of transcription analysis According to the can and the enzyme activity and protein data correspond well; and found that compared with the balance of growth period, the expression of g6pdh1 and g6pdh2 gene WJ11 in lipid accumulation period increased greatly, that these two genes may be closely related to lipid accumulation; it was also found that the lipid accumulation period WJ 11 and branched chain amino acid metabolism (Leu B) of the gene transcription level were significantly less than 277.49 CBS, these genes may also be involved in the regulation of lipid synthesis volume branches Mucor through a mechanism. The genome, proteome, enzyme activity and transcription analysis of the data showed that WJ11 in g6pdh1, g6pdh2 and Leu of B gene may play a role in regulating the lipid accumulation important. By over expression of WJ11 g6pdh1 in Mucor circinelloides 277.49 CBS deficient strains, g6pdh2 and Leu B gene showed that these genes had no significant influence on the growth of Mucor circinelloides, but significantly To improve the accumulation of fatty acids: the overexpression of recombinant strains of fatty acid content of g6pdh1 was increased by 23~28%, over expression of recombinant strains of fatty acid content of g6pdh2 was increased by 41~47%, indicating that g6pdh1 and g6pdh2 were able to accumulate lipid regulation of Mucor circinelloides, and g6pdh2 effect is more significant; in addition, over expression of Leu B gene the recombinant strain of fatty acid content increased by 67~73%, Leu showed that B gene plays an important role in the regulation of fatty acid accumulation. The amino acid accumulation potential regulatory roles for fatty acids, respectively, with twenty standard amino acids as nitrogen source culture of Mucor circinelloides, cell growth analysis, lipid accumulation and the yield of GLA. The first laboratory of three strains of Mucor circinelloides (WJ11, CBS 108.16 and CBS 277.49) ability to accumulate GLA, found WJ11 the total fatty acid content is the highest, but because of the fatty acid composition of GLA is relatively low, the yield of GLA CBS was significantly less than 108.16, so the CBS108.16 is chosen as the research strain. Mucor circinelloides CBS 108.16 as nitrogen source culture by using different amino acids, found that the cell growth, fatty acid accumulation and GLA yield will be significantly different, among them, tyrosine was the best nitrogen source on growth and lipid accumulation, and the yield of GLA was the highest (0.81 g/L) through the determination of fatty acid synthesis related enzymes, tyrosine found significantly increased G6PDH and 6PGDH activity, provide more reducing power for fatty acid synthesis; tyrosine also increased the activity of ACL, providing more acetyl coenzyme A for fatty acid synthesis and down-regulation of ICDH activity may also slow down three tricarboxylic acid cycle, promote the synthesis of fatty acids. The dynamic analysis and proteomics and WJ11 and CBS 277.49 lipogenic enzyme results are consistent.

【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】：2016
【分類號(hào)】：Q93;TQ645

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