蓝细菌与大肠杆菌中脂肪族生物燃料代谢工程研究

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	蓝细菌与大肠杆菌中脂肪族生物燃料代谢工程研究
	刘爱秋
导师	吕雪峰
	2013-06
学位授予单位	中国科学院研究生院
学位授予地点	北京
学位专业	生物化学与分子生物学
关键词	蓝细菌大肠杆菌脂肪族生物燃料脂酰acp还原酶脂肪醛去甲酰基加氧酶脂酰coa还原酶
摘要	随着世界对能源需求量的日益增加和石化燃料燃烧排放的二氧化碳等温室气体而导致的环境问题日益严重，可再生生物燃料的发展逐渐引起了人们的重视。从燃料性能角度考虑，可再生石油是石油的最佳替代品。由脂肪酸衍生的脂肪烃、脂肪醇等具有高能量密度、低吸湿性、低挥发性等优点，是一类优质液体燃料。脂肪酸衍生物的碳链长度、分支、饱和度等直接影响其作为生物燃料的性能。合成生物学和代谢工程的发展使得人们可以通过改造微生物代谢途径生产生物燃料。研究脂肪酸衍生物合成途径中关键酶的性质，对于发展新型优质生物液体燃料产品、解决传统生物液体燃料产品品质不佳的瓶颈问题具有重要意义。本研究一方面从种质水平上对产烃蓝细菌进行株系筛选和克隆产烃关键基因，分析蓝细菌菌株进化和产烃特性及产烃关键基因之间的关系；另一方面，分析大肠杆菌中影响脂肪烃产量和链长的关键因素；同时在大肠杆菌中通过检测脂肪醇的生成来筛选优质产脂肪醇基因，为脂肪族生物燃料的生产提供基因优质资源。通过实验，本研究得出如下结果和结论：（1）通过对19株淡水蓝细菌产脂肪烃的情况进行分析，根据其产烃特征将蓝细菌分为5类，结合已报道蓝细菌的产烃情况，我们发现支链烃主要存在于丝状蓝细菌中，而很少存在于单细胞蓝细菌中。根据产烃关键基因的保守序列，扩增得到了19株蓝细菌的脂肪醛去甲酰基加氧酶（Aldehyde Deformylating Oxygenase，ADO)基因和脂酰ACP还原酶（Acyl-acyl carrier protein reductase，AAR）基因的部分序列。研究发现所检测蓝细菌脂肪烃的分布特征与基于蓝细菌16S rDNA序列和AAR蛋白序列的聚类分析相一致，而与基于ADO蛋白序列的聚类分析不一致。（2）来源于蓝细菌的脂肪烃合成途径中的关键酶—脂肪醛去甲酰基加氧酶基因sll0208、npun_R1711和脂酰ACP还原酶基因sll0209、orf1594在大肠杆菌中能够以可溶性形式表达，初步酶活检测表明这两种酶的活性均比较低。（3）在大肠杆菌中过表达硫酯酶CcTE使自由脂肪酸特别是C14和C14:1中等链长脂肪酸含量分别提高了28倍和59倍；脂酰CoA合成酶FadD、Aas PCC7942和Slr1609分别与硫酯酶CCTE共表达，细胞内自由脂肪酸含量明显下降，表明FadD、Aas PCC7942和Slr1609能够将脂肪酸有效地转化为脂酰CoA或脂酰ACP；过表达CcTE能够使大肠杆菌中脂肪醇和脂肪烃的产量分别提高74%和47%，但并没有提高C14链长脂肪醇和脂肪烃的产量。通过体外添加等摩尔浓度脂肪酸培养产脂肪醇大肠杆菌，发现过表达Orf1594的菌株中C16和C18链长脂肪醇占脂肪醇总量的89.5%，表明Orf1594具有链长选择性和饱和度选择性，对C16和C18:1脂酰CoA或脂酰ACP具有较高的活性。（4）来源于海杆菌Marinobacter aquaeolei VT8的脂酰CoA还原酶Maqu_2507、Maqu_2220与不同来源控制形成不同链长脂肪酸的硫酯酶共表达，能够有效地产生相应链长的脂肪醇，表现出较宽的底物链长选择性和较高的产脂肪醇能力。因此，通过硫酯酶控制脂肪酸的碳链链长，可以控制脂肪醇的碳链长度。在摇瓶发酵培养条件下，菌株AL322（过表达硫酯酶TesA和Maqu_2220)脂肪醇的产量达到422 mg/L。利用发酵罐对菌株AL322进行发酵，在发酵培养条件未进行优化的情况下，脂肪醇的最高产量达到1.725 g/L。
其他摘要	Rapidly growing demand for energy and environmental concerns about carbon dioxide emissions have sparked interest in renewable biofuels. Products derived from fatty acids such as fatty alkanes and fatty alcohols have the merits of high energy density, low hygroscopicity, low volatility, etc. Synthetic biology and metabolic engineering make it possible to produce biofuels with high efficiency in microorganism. The quality of biofuels depends on the carbon chain length, branching and degree of saturation of the molecules. It is important to study the specificity of the key enzymes in the pathway of synthesizing the fatty acids derived products for developing the new liquid biofuels. In this study, on the one hand the relationship between hydrocarbon profiles of the cyanobateria and the key genes for the alkane biosynthesis was analyzed. On the other hand, the limiting factors of affecting carbon chain length of fatty alkanes were explored and screening candidate fatty acyl-CoA reductase genes for the fatty alcohol production in E.coli was conducted. Some results and conclusions are summarized as follows:First, hydrocarbon compositions of 19 fresh-water cyanobacterial strains belonging to 13 genera were analyzed. Based on their hydrocarbon compositions, these cyanobacterial strains were classified into 5 major subgroups. Combined with previously reported hydrocarbon production profiles in different cyanobacterial strains, we found that branched-chain alkanes were limited predominantly in filamentous but rarely in unicellular strains. Phylogenetic analysis using traditional small-subunit ribosomal RNA (16S rDNA) of these strains presented similar clustering to their hydrocarbon production profiles. Comparison of phylogenies revealed that the topology of 16S rDNA showed a general congruence with that of AAR （fatty acyl-ACP reductase） but not with that of ADO (Aldehyde deformylating oxygenase). Second, the sll0208 and npun_R1711 genes encoding aldehyde-deformylating oxygenase and sll0209 and orf1594 encoding fatty acyl-ACP reductase were over-expressed in E.coli as soluble forms. The preliminary results show that the activity of these enzymes were low.Third, over-expression of CCTE (thioesterase from Cinnamomum camphorum) lead to an increase of the levels of free fatty acids, especially the C14 and C14:1 fatty acids were 28 fold and 59 fold higher than the control strain respectively. When fatty acyl-CoA synthases FadD, Aas PCC7942 and Slr1609 were co-expressed with CCTE separately, the level of free fatty acids decreased sharply. These results show that free fatty acids can be efficiently converted into fatty acyl-CoA or fatty acyl-ACP. The production of fatty alkanes (or alkenes) and fatty alcohols was improved 47% and 74% by over-expressing CCTE respectively. But we failed to see the desired carbon chain length of C14 fatty alkanes (or alkenes) and fatty alcohols being improved. Fatty aochols produced by strain over-expressing orf1594 by feeding an equivalent free fatty acidis were maily C16 and C18:1 carbon chain length, which constitute 89.5% of totoal fatty alcohol. These results show that Orf1594 prefers for C16 and C18:1 fatty acyl chains as its substrate. Fourth, we examined the characteristics of two fatty acyl-CoA reductases encoded by maqu_2220 and maqu_2507 genes from Marinobacter aquaeolei VT8 on fatty alcohol production in Escherichia coli. Fatty alcohols with diversified carbon chain length were obtained by co-expressing Maqu_2220 with different carbon chain length-specific acyl-ACP thioesterases. Both fatty acyl-CoA reductases displayed broad substrate specificities for C12-C18 fatty acyl chains in vivo. The optimized mutant strain of E. coli carrying the modified tesA gene and fadD gene from E. coli and maqu_2220 gene from Marinobacter aquaeolei VT8 produced fatty alcohols at a remarkable level of 422mg/L in the shake flasks and 1.725 g/L under the fermentation condition.
作者部门	生物代谢工程
学科领域	生物代谢工程
公开日期	2013-07-13
学位类型	博士
语种	中文
文献类型	学位论文
条目标识符	http://ir.qibebt.ac.cn/handle/337004/1489
专题	微生物代谢工程研究组
推荐引用方式 GB/T 7714	刘爱秋. 蓝细菌与大肠杆菌中脂肪族生物燃料代谢工程研究[D]. 北京. 中国科学院研究生院,2013.

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