产油真菌卷枝毛霉WJ11高产脂质的分子机制

发布时间：2018-01-17 21:01

本文关键词：产油真菌卷枝毛霉WJ11高产脂质的分子机制　出处：《江南大学》2016年博士论文　论文类型：学位论文

【摘要】：产油微生物能够在细胞内积累大量脂肪酸并以甘油三酯的形式储存,是传统动植物油脂的重要替代资源。某些微生物积累的油脂还富含高价值的多不饱和脂肪酸,具有重要的应用价值和商业价值。在产油微生物中,不同物种的微生物油脂产量相差巨大,其油脂积累量从细胞干重的20%到80%以上不等。由于不同物种间生理生化特征差异较大,导致不同微生物之间脂质积累差异的机制难于解析,而对同一物种产脂差异显著的不同菌株的研究能够很好地解决这一问题。本文通过基因组学、蛋白质组学以及关键酶的活性分析系统研究了卷枝毛霉高产脂菌株WJ11和低产脂菌株CBS 277.49在脂质代谢调控上的差异;结合转录分析找出了一些关键调控基因;通过基因工程过表达这些关键基因,有效提高了脂肪酸的积累;还研究了氨基酸氮源对卷枝毛霉γ-亚麻酸(GLA)产量的影响。论文的主要研究内容和实验结果如下:通过对卷枝毛霉WJ11和CBS 277.49生长和产脂的分析,发现WJ11脂肪酸含量高达36%,而CBS 277.49则不超过15%。WJ11全基因组的测序结果显示其基因组大小为35.4 Mb,包含827个scaffold,由2530个contig组成,GC含量为39.7%,有10973个预测的基因和177个t RNA基因。通过比较WJ11(本实验室测序)和CBS 277.49(美国联合基因研究所测序)的基因组发现,两株菌的基因组特征总体相似度较高,MAUVE分析显示两株菌的基因组序列拥有很多的同源区域,MUMmer分析也表明两株菌的基因组整体共线性良好。根据基因组信息,构建了WJ11和CBS 277.49中心碳代谢和脂代谢网络,包括葡萄糖的代谢,脂肪酸的合成,甘油三酯、磷脂、类固醇和类胡萝卜素的合成以及脂肪的β氧化。通过比较两株菌中编码这些代谢途径中相关酶的基因个数,发现基因个数的差异可能对两株菌的脂质积累产生影响。另外,通过Ortho MCL聚类分析找出了WJ11和CBS 277.49之间的特异基因,这些特异基因可能与两株菌的细胞生长和脂肪积累差异相关。利用蛋白质组学分析了高产脂菌株WJ11在脂质积累期(氮缺乏)和生长平衡期全蛋白的表达差异情况,结果发现氮缺乏促进了氮的同化,减缓了氨基酸的代谢;上调了糖酵解相关蛋白的表达并下调了三羧酸循环相关蛋白的表达,使更多的碳流向脂肪酸合成;氮缺乏提高了磷酸戊糖途径相关蛋白的表达,为脂肪酸合成提供更多的还原力NADPH;在氮缺乏时,蛋白质和核酸的代谢受到抑制,细胞主要进行脂质合成和积累;氮缺乏还上调了一些与能量代谢、信号传导、分子伴侣和氧化还原相关蛋白的表达,以应对氮限制这种环境压力。另一方面,通过比较高产脂菌株WJ11和低产脂菌株CBS277.49在脂质快速积累期全蛋白的表达差异,发现WJ11中与支链氨基酸代谢相关蛋白的表达显著上调,这可能为脂肪酸合成提供更多的底物乙酰辅酶A;与CBS 277.49相比,WJ11中糖酵解相关蛋白的表达量更高,而三羧酸循环相关蛋白的表达量则较低,这表明WJ11中碳更多流向脂肪酸的合成,而非参与三羧酸循环;WJ11中磷酸戊糖途径相关蛋白的表达量也较CBS 277.49要高,这表明WJ11中会产生更多的还原力NADPH。通过对卷枝毛霉WJ11和CBS 277.49脂质合成关键酶的活力测定发现,与CBS277.49相比,WJ11中果糖磷酸激酶(PFK)的酶活较低,而磷酸戊糖途径中葡萄糖-6-磷酸脱氢酶(G6PDH)和6-磷酸葡萄糖酸脱氢酶(6PGDH)的酶活较高,这将导致更多的碳流向磷酸戊糖途径,从而为脂肪酸合成提供更多的还原力NADPH;对于脂质合成过程中还原力的重要提供者苹果酸酶(ME),其在WJ11脂质积累初期的酶活也略微高出CBS 277.49;WJ11中调控三羧酸循环的异柠檬酸脱氢酶(ICDH)的酶活显著低于CBS 277.49,这表明WJ11中三羧酸循环更为缓慢,将会有更多的柠檬酸转运到线粒体外;同时WJ11中ATP:柠檬酸裂解酶(ACL)的酶活相比较CBS 277.49又有所提高,这将增加脂肪酸合成的前体乙酰辅酶A的供应;WJ11中脂肪酸合酶(FAS)的活力也明显高于CBS 277.49。这些酶活差异较为清晰地解析了WJ11高产脂质的调控机制。针对脂质合成中的关键酶以及蛋白质组分析得到的重要差异蛋白,利用RT-q PCR研究它们基因的转录水平,结果显示转录分析数据能够与酶活及蛋白质组数据较好的对应;并发现与生长平衡期相比,WJ11中的g6pdh1和g6pdh2基因的表达在脂质积累期提高幅度较大,推测这两个基因可能与脂质积累密切相关;同时还发现脂质积累期WJ11中与支链氨基酸代谢相关基因(如leu B)的转录水平下调幅度要显著小于CBS 277.49,这些基因也可能通过某种机制参与卷枝毛霉脂质合成的调控。基因组、蛋白质组、酶活以及转录分析的数据显示,WJ11中g6pdh1、g6pdh2及leu B基因可能对脂质积累有重要的调控作用。通过在卷枝毛霉CBS 277.49缺陷型菌株中过表达WJ11中的g6pdh1、g6pdh2及leu B基因,发现这些基因对卷枝毛霉的生长没有明显影响,但均显著提高了脂肪酸的积累:其中过表达g6pdh1的重组菌株脂肪酸含量提高了23~28%,过表达g6pdh2的重组菌株脂肪酸含量提高了41~47%,这表明g6pdh1和g6pdh2均能够调控卷枝毛霉的脂质积累,且g6pdh2的效果更为显著;另外,leu B基因的过表达使重组菌株脂肪酸含量提高了67~73%,表明leu B基因在脂肪酸积累过程中起到重要的调控作用。针对氨基酸对于脂肪酸积累可能的调控作用,分别以二十种标准氨基酸为氮源培养卷枝毛霉,分析其细胞生长、脂质积累及GLA产量。首先比较本实验室三株卷枝毛霉(WJ11、CBS 108.16和CBS 277.49)GLA的积累能力,发现WJ11总脂肪酸含量最高,但由于脂肪酸组分中GLA比例较低,使GLA产量显著小于CBS 108.16,因此选取CBS108.16作为研究菌株。利用不同氨基酸为氮源培养卷枝毛霉CBS 108.16,发现其菌体生长、脂肪酸积累以及GLA产量会有显著差别,其中,酪氨酸为最适合生长和脂质积累的氮源,且GLA产量也最高(0.81 g/L)。通过对脂肪酸合成相关酶活力的测定,发现酪氨酸显著提高了G6PDH和6PGDH的活力,为脂肪酸合成提供更多的还原力;酪氨酸也提高了ACL的活力,为脂肪酸合成提供更多的乙酰辅酶A,同时,ICDH活力的下调也可能减缓了三羧酸循环,促进脂肪酸的合成。这与蛋白质组学及WJ11与CBS 277.49脂质合成相关酶的活力分析结果相一致。
[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.

【学位授予单位】：江南大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q93;TQ645

【相似文献】