基因工程蓝细菌生物合成脂肪酸及脂肪醇研究

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	基因工程蓝细菌生物合成脂肪酸及脂肪醇研究
	齐凤霞
导师	吕雪峰
	2013-07
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	蓝细菌脂肪酸硫脂酶脱饱和酶脂肪醇
其他摘要	当今世界化石资源日渐匮乏、能源结构不合理、消耗化石资源造成的环境污染以及温室气体排放等问题被高度关注。构建可再生能源新体系，发展生物燃料是解决这些问题的重要举措之一。蓝细菌作为一种能进行光合作用的原核微生物，可以直接利用二氧化碳和光能合成生物产品，其光合作用效率高，易于培养，并且遗传背景清晰，是一种理想的生物燃料合成系统。构建高效定向生物合成新型优质生物液体燃料的基因工程蓝细菌，对开发新一代光合能源微生物系统、解决生物质资源不足的瓶颈问题具有重要意义。脂肪酸是一种重要的生物燃料前体，可以通过生物或者化学途径转化为脂肪醇、脂肪烃及脂肪酸甲（乙）酯等具有优良燃料特性的燃料分子。通过对蓝细菌进行基因工程改造，可以实现利用二氧化碳生物合成生物燃料前体脂肪酸，也可以直接在蓝细菌中合成脂肪族生物燃料分子。本论文基于蓝细菌模式藻株集胞藻PCC6803和聚球藻PCC7942平台，分别围绕蓝细菌合成生物燃料前体脂肪酸以及在蓝细菌中生物合成燃料分子脂肪醇展开研究。论文成功构建了基于集胞藻 PCC6803的基因表达平台，分析了不同启动子的表达强度，为蓝细菌表达外源基因提供了重要支撑。通过表达硫酯酶以及对蓝细菌糖原合成途径进行基因工程改造，实现了在聚球藻PCC7942中合成中等链长的游离脂肪酸。通过表达脱饱和酶提高脂肪酸的不饱和程度，实现了对链长和饱和度的双控制，为分子尺度调控生物燃料性质提供了思路。在集胞藻 PCC6803中尝试了两个来自海洋细菌的脂肪酰-CoA还原酶（FAR）基因，最终筛选鉴定了来自海油水杆菌的maqu_2220的产脂肪醇能力，成功实现了蓝细菌直接生物合成脂肪醇，这是蓝细菌中继表达荷荷巴jojoba_far和拟南芥at3g11980基因产脂肪醇后的第二例报道。本研究具体的研究内容和结果如下：第一，基于基因工程改造蓝细菌合成生物燃料以及其他高价值产品的需求，成功构建了三个针对集胞藻PCC6803的外源基因的基因组整合表达平台（pFQ20、pFQ9F和pFQ9R）以及一个广宿主的穿梭表达平台（pFQ16）。并且以lacZ作为报告基因，鉴定了pFQ20表达平台的表达效率为109 Miller单位。第二，以lacZ作为报告基因，筛选和鉴定了7种来自集胞藻 PCC6803自身的启动子和1种来源于细菌诱导性启动子在集胞藻PCC6803中的表达效率；同时针对每个启动子的SD序列（Shine-Dalgarno sequence）与ATG之间的序列进行了突变，总计构建了22种不同的表达载体。通过以ONPG（o-nitrophenyl-p-D-galactopyranoside）为底物平行测定所得突变藻中的β-半乳糖苷酶活性，获得基因表达效率由弱至强的表达平台11个，其中包括2个较低效率的表达平台、2个中等强度表达平台及7个高效基因表达平台。第三，在集胞藻 PCC6803中分别用Prbc和PpetE启动子表达来自大肠杆菌的硫脂酶基因tesA’和来自樟树的CcFatB1，同时敲除脂肪酰基CoA合成酶基因slr1609，结果表明tesA’以及CcFatB1没有使集胞藻脂肪酸途径发生变化。在集胞藻PCC6803中表达密码子优化的tesA'和CcFatB1，结果表明硫脂酶对集胞藻脂肪酸途径同样没有产生影响。第四，在聚球藻 PCC7942在表达硫酯酶UcFatB1和来源于萼距花Cuphea hookeriana的硫酯酶ChFatB2，共表达了来自蓖麻Ricinus Communis的脂酰-ACP脱饱和酶，突变株成功分泌辛酸、癸酸和十二酸，同时外泌型游离脂肪酸的不饱和度得到提高，成功实现对脂肪酸链长和脂肪酸不饱和程度的双重调控。第五，在聚球藻PCC7942中过量表达自身的脂肪酸脱饱和酶desC，使BC028和BC029脂肪酸的不饱和度由39%（野生型）分别提高到65.2%和58.3%。共表达来自集胞藻6803的脱饱和酶基因desA、desB和desC，其中DesA没有发挥作用，但DesB和DesC成功将双不饱和脂肪酸及含有三个双键的不饱和脂肪酸引入聚球藻PCC7942，并使得突变株BC030和BC031脂肪酸的不饱和程度分别增加到47.6%和48.6%。第六，通过敲除聚球藻PCC7942中与糖原合成相关的基因glgC、Synpcc7942_1814和Synpcc7942_1725，研究了糖原合成途径对脂肪酸途径的影响。敲除糖原合成关键基因glgC，可以使细胞内总脂肪酸产量提高50%，其胞外分泌的游离脂肪酸是野生型菌株的4.6倍，然而敲除glgC同时表达硫酯酶UcFatB1后细胞不能存活。插入失活Synpcc7942_1648并表达硫酯酶UcFatB1使细胞内总脂肪酸含量增加78%，胞外分泌的游离脂肪酸含量达到128.9 μM/OD750。敲除Synpcc7942_1725并表达硫酯酶UcFatB1，细胞内总脂肪酸含量是野生型细胞的2倍，游离脂肪酸含量提高到217.1 μM/OD750。但仅敲除两个糖原合成调控因子并不能直接提高脂肪酸积累。第七，在集胞藻PCC6803中表达海洋放线菌 Marine actinobacterium PHSC20C1的脂酰-CoA还原酶基因A20C1_04336和水油海杆菌Marinobacter aquaeolei VT8的脂酰-CoA还原酶基因maqu_2220，GC-MS检测结果显示，表达A20C1_04336没有脂肪醇生成，而maqu_2220检测到了脂肪醇，其产量为5.6 ± 1.1 μg/L/OD730。 ; Diminishing fossil fuel reservoirs together with ever-deteriorating environment associated with the worldwide fossil-fuel-based industry make the use of sustainable alternative feedstocks imperative. Cyanobacteria, a type of photosynthetic prokaryote with high photosynthetic efficiency, rapid cell growth and clear genomic background, present a promising non-food biomass resource for supply of renewable biofuel molecules as the replacements or additives to the conventional fuels. The medium and long chain fatty acid derivate, such as fatty alcohols, fatty alka(e)nes and fatty acid methyl (ethyl) esters, are ideal alternatives for petroleum-derived fuel oils due to their similar molecular structures and fuel features to the fossil fuels. The metabolic engineering and synthetic biology enable us to manipulate the cyanobacteria metabolic pathways towards the production of these liquid biofuels by using light as the only energy source. In this thesis, the author investigated the production of fatty acids in the model cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC7942 for the production of precursors for fatty acid-derived biofuels. Firstly, three integrative expression plasmids and one broad-host conjugative plasmid have been constructed for the metabolic engineering of cyanobacteria. These expression platforms applied the native rbc (encodes Rubisco) promoter, rbc terminator of Synechocystis and a fusion expression strategy that enabled fusion expression of the target gene with rbcL. Meanwhile, the platform was evaluated using lacZ as a reporter gene and achieved a β-galactosidase expression of 109 Miller units. Secondly, a set of promoter parts were developed, modified and characterized by using the lacZ as the reporter gene. As a result, seven strong promoters (PrbcL-12, PpasD-12, PpsaD-12, PatpB-12, PpsbD-12, PpsbD-13, and PpsbD-14), two moderate promoters (PpetE, Plac) and two weak promoters (PpsbA1-12 and PpsbA1-13) were screened as molecular tools for metabolic engineering of cyanobacteria.Thirdly, the E.coli thioesterase gene tesA' and the Cinamomus camphorum thioesterase gene C.c FatB1 were separately expressed in Synechocystis sp strain PCC6803, along with knocking out the fatty acyl-CoA synthase gene slr1609. However, the fatty acids profiles and the fatty acid productivity in the mutants did not change. Expression of the codon-optimized tesA' and C.c FatB1 had no effects on fatty acid synthesis in Synechocystis sp strain PCC6803.Fourthly, co-expression of the Ricinus Communis stearoyl fatty acyl-ACP desaturase, the thioesterase (the Umbellularia californica thioesterase UcFatB1 and the Cuphea hookeriana thioesterase ChFatB2) generated unsaturated, octenoic acid (C8:1), decenoic acid (C10:1） and dodecenoic acid (C12:1) in the secreted free fatty acids in Synechococcus sp. PCC7942. By over-expressing the native fatty acyl lipid desaturase DesC, the degree of unsaturation (DU) of the fatty acids was enhanced from 39% ( wild type strain) to 65.2% and 58.3% in BC028 and BC029, respectively. Heterologously co-expression the Synechocystis lipid desaturase DesA, DesB and DesC introduced polyunsaturated fatty acids into Synechococcus and increased the DU to 47.6% and 48.6% in the mutant strain BC030 and BC031, respectively. Fifthly, the effects of the glycogen synthesis on fatty acid accumulation were investigated in Synechococcus sp strain PCC7942. Disruption of glgC (encodes the glucose-1-phosphate adenylyl-transferase) increased the intracellular fatty acids content by 50% and the secreted free fatty acids was enhanced by 4.6 fold. However, the mutant strain with glgC disruption and expression of UcFatB1 failed to survive. Disruption of two putative regulators for glycogen synthesis (Synpcc7942_1648, Synpcc7942_1725) did not affect the fatty acid accumulation, while expressing UcFatB1 along with the disruption dramatically increased the fatty acid production. Disruption of Synpcc7942_1648, along with UcFatB1 expression enhanced the intracellular fatty acid by 78% and secreted 128.9 μM/OD750 free fatty acids out of cells. Similarly, the strain with Synpcc7942_1725 disruption and UcFatB1 expression generated 2-fold of intracellular fatty acids, compared to the wild type strain. The secreted free fatty acids accumulated to 128.9 μM/OD750. Sixthly, this thesis also investigated the production of fatty alcohol in Synechocystis PCC 6803 via expression of two fatty acyl-CoA (ACP) synthases, the maqu_2220 from Marinobacter aquaeolei VT8 and the A20C1_04336 from Marine actinobacterium PHSC20C1. The expression of maqu_2220 generated fatty alcohols in Synechocystis, while A20C1_04336 did not produce any fatty alcohols.
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/337004/1502
专题	微生物代谢工程研究组
推荐引用方式 GB/T 7714	齐凤霞. 基因工程蓝细菌生物合成脂肪酸及脂肪醇研究[D]. 北京. 中国科学院研究生院,2013.

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