集胞藻PCC6803中表达脂酰辅酶A还原酶生物合成脂肪醇研究
文献类型：学位论文
| 作者 | 姚 伦 |
| 学位类别 | 博士 |
| 答辩日期 | 2013-07 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 吕雪峰 |
| 关键词 | 脂肪醇 蓝细菌 集胞藻PCC6803 脂酰辅酶A还原酶 |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 生物燃料是解决由于传统化石燃料使用带来的能源、环境、资源等一系列问题的有效途径，近年来已经取得快速发展。第一代生物燃料以糖类、淀粉、油料作物等为原料，主要有生物乙醇和生物柴油，但存在与人争粮、与粮争地等诸多问题。以非粮生物质为原料的第二代生物燃料也面临一系列瓶颈问题，如原料的收集、运输、储存与预处理、纤维素降解、五碳糖转化等。第三代生物燃料以薇藻为主要原料，同样面临薇藻的规模培养、细胞收集等瓶颈问题。因此，研发建立生物燃料的新型转化技术路线成为当前的研究热点。近年来，蓝细菌由于具有能够进行光合作用、易于遗传改造等优点，以其作为新一代能源微生物系统进行生物燃料的研究越来越受到关注。其中，集胞藻PCC6803（Synechocystis sp. PCC6803）作为蓝细菌的代表物种，是第一个完成基因组测序的光能自养微生物，被作为模式生物受到广泛研究。本研究以集胞藻PCC6803为基因工程宿主菌，通过表达外源脂酰辅酶A还原酶（fatty acyl-CoA reductase，FAR）基因，实现了利用光合作用从二氧化碳到脂肪醇的直接生物合成。并通过相关代谢工程改造，进一步提高了脂肪醇产量，同时，研究了不同培养条件对于脂肪醇产量的影响。主要研究内容及结果如下：（一）分别构建了表达来源于小鼠（Mus musculus）、拟南芥（Arabidopsis thaliana）、荷荷芭（Simmondsia chinensis）和水油海杆菌（Marinobacter aquaeolei VT8）的脂酰辅酶A还原酶基因的集胞藻PCC6803基因工程藻株，分析了脂肪醇的生物合成。结果显示，表达拟南芥的at3g11980、at3g11980-trunc、荷荷芭的far_jojoba和海杆菌的maqu_2220基因的藻株中检测到脂肪醇的积累，其中藻株Syn-LY43（表达来源于拟南芥的at3g11980-trunc）产量最高，为85.2 μg OD-1 L-1，脂肪醇的主要组成为正十六烷醇和正十八烷醇。（二）研究了不同培养条件对集胞藻PCC6803基因工程藻株脂肪醇产量的影响。以藻株Syn-XT14为例研究了培养方式、培养时间、光照强度和环境胁迫（高光照、缺氮、缺磷）对于脂肪醇产量的影响，结果表明：（1）用柱式光反应器培养时，达到生长稳定期后的细胞浓度和单位产量与普通三角瓶培养相比均显著提高；（2）当藻株培养至稳定期后，细胞内的脂肪醇随培养时间延长而不断积累；（3）在一定光照范围之内，增加光照强度能够有助于提高脂肪醇产量，高光照胁迫条件下（250 µE m-2 s-1）脂肪醇产量提高了约1倍；（4）藻株的缺氮和缺磷胁迫对于脂肪醇产量没有显著影响。（三）脂酰辅酶A还原酶的多拷贝表达：（1）对不同来源的脂酰辅酶A还原酶基因在slr0168位点进行了串联表达；（2）在slr0168、agp（编码ADP-葡萄糖焦磷酸化酶）和phaAB （phaA编码β-酮硫解酶，phaB编码乙酰乙酰辅酶A还原酶）位点表达三份拷贝的脂酰辅酶A还原酶。同时，研究了agp和phaAB的敲除对脂肪醇产量的影响。结果表明，（1）在slr0168位点串联表达at3g11980和far_jojoba时，由于这两个脂酰辅酶A还原酶的底物选择性差异，不同碳链长度的脂肪醇的比例与分别进行单独基因表达时相比发生改变；（2）在slr0168、agp和phaAB位点表达三份拷贝的脂酰辅酶A还原酶时，脂肪醇产量比Syn-XT14提高1.4倍，达到152.2 μg OD-1 L-1；（3）敲除phaAB时脂肪醇产量均没有显著变化，而agp基因在集胞藻PCC6803中不能被完全敲除。（四）研究了sll0208基因（编码集胞藻PCC6803脂肪醛脱甲酰加氧酶）的敲除以及slr1609（编码集胞藻PCC6803脂酰辅酶A合成酶和脂酰-ACP合成酶）、sigE（编码sigma转录调节因子）、acc（编码集胞藻PCC6803乙酰辅酶A羧化酶）、fadD（编码E. coli脂酰辅酶A合成酶）、acrM（编码不动杆菌脂酰辅酶A还原酶）和cer5（编码拟南芥ABC转运蛋白）的过表达及其对脂肪醇产量的影响。结果表明：（1） sll0208敲除之后，脂肪醇产量显著提高，其中，Syn-FQ52D08比Syn-FQ52脂肪醇产量提高42倍； Syn-XT14D08比 Syn-XT14提高约2倍；（2）fadD、slr1609、acc、sigE和acrM的过表达对脂肪醇产量没有显著影响；（3）藻株Syn-LY198中表达cer5转运蛋白之后，细胞内的脂肪醇没有被有效地转运到细胞外，但细胞外游离脂肪酸的含量显著增加。 |
| 英文摘要 | Biofuels are thought to be one of strategies to solve the energy, resource and environment problems caused by the utilization of fossil fuels. First generation biofuels, mainly refer as bioethanol and biodiesel, are made from starch or vegetable oil-based plant biomass and food-competitive. Second generation biofuels are produced from non-food crop feedstock, cellulosic biomass, and are facing major constraints such as collection, transportation, storage, pretreatment and degradation of cellulosic biomass. Microalgae derived biofuels are considered to be the third generation biofuels. However, several important technical barriers remain to be overcome including mass cultivation, biomass harvesting and downstream processing, etc. Therefore the development of novel biofuel-producing technical routes is in great demand and has been extensively explored. Cyanobacteria, the promising candidates for biofuel-producing microbial system, have received increasing public and scientific attention in recent years because of their photosynthetic capability and capacity for genetic engineering. Synechocystis sp. PCC6803, a unicellular cyanobacterium, is the first phototrophic organism with its genome sequenced and has been widely studied as a model organism. In this study, biosynthesis of fatty alcohols was achieved in a genetically engineered cyanobacterial system through heterologously expressing fatty acyl-CoA reductase (FAR) in Synechocystis sp. PCC6803. Potential methods to improve productivity of fatty alcohol through genetic modifications were demonstrated and the effect of environmental stresses on the production of fatty alcohols in the mutant strains was also characterized. The main results of this study are as follows:(1) Fatty acyl-CoA reductase genes from mouse (Mus musculus), Arabidopsis (Arabidopsis thaliana), jojoba (Simmondsia chinensis) and marine bacterium (Marinobacter aquaeolei VT8) were cloned and introduced into Synechocystis sp. PCC6803, and the production of fatty alcohols in cultured Synechocystis sp. PCC6803 mutant strains was analyzed. The results show that fatty alcohols are accumulated in Synechocystis mutant strains expressing fatty acyl-CoA reductases from Arabidopsis (at3g11980 and at3g11980-trunc), jojoba (far_jojoba) and the marine bacterium (maqu_2220). The greatest yield of total fatty alcohols, 85.2 μg OD-1 L-1 was observed for the strain Syn-LY43 that contained the Arabidopsis FAR gene and the main components of fatty alcohols were hexadecanol and octadecanol.(2) The impact of various culture conditions including cultivation systems, culturing time, light intensity and environmental stresses (high light, nitrogen deprivation and phosphorus deprivation) on the production of fatty alcohols in Synechocystis mutant strains was investigated. The results showed that: (i) Both the cell density and fatty alcohol yields increased significantly in Synechocystis mutant strains grown in bubble column photo-bioreactors; (ii) The production of fatty alcohols depends on culturing time in the stationary phase; (iii) The fatty alcohol yields can be improved with the increase of light intensity within a certain range and is approximately doubled under high light condition; (iv) No significant change takes place in the production of fatty alcohols under nitrogen or phosphorus deprivation conditions.(3) Fatty acyl-CoA reductases from Arabidopsis (at3g11980) and jojoba (far_jojoba) were coexpressed at slr0168 locus and no increase of fatty alcohol yield was observed. However, the composition of fatty alcohols changed due to different substrate specificities of FAR enzymes. Three copies of fatty acyl-CoA reductases were expressed at slr0168, agp and phaAB locus and the yield of fatty alcohol showed an increase of 1.4 fold compared to that of Syn-XT14, reaching up to 152.2 μg OD-1 L-1. The partial deletion of agp gene encoding the ADP-glucose pyrophosphorylase and deletion of phaAB gene (encoding β-ketothiolase and acetoacetyl-CoA reductase) has no significant effect on the yield of fatty alcohols respectively. (4) Knocking-out of sll0208 gene encoding fatty aldehyde deformylating oxygenase and over-expression of slr1609 gene encoding acyl-ACP synthetase, sigE gene encoding a group 2 sigma factor, acc gene encoding acetyl-coA carboxylase, fadD gene encoding acyl-CoA synthetase of E. coli, acrM gene encoding fatty acyl-CoA reductase of Acinetobacter and cer5 gene encoding an ABC transporter of Arabidopsis in fatty alcohol producing strains were investigated. The results show that: (i) Knocking-out of sll0208 gene leads to a significant increase of the yield of fatty alcohols. The content of fatty alcohols in Syn-FQ52D08 increases by 42 times compared to the yield of starting strain Syn-FQ52 and the deletion of sll0208 also results in a 2-fold increased yield of fatty alcohols in Syn-XT14D08 compared that in Syn-XT14. (ii) No significant change takes place in the production of fatty alcohols by over-expressing slr1609, sigE, acc, fadD and acrM respectively. (iii) By expressing cer5 in Syn-LY198, the production of extracellular free fatty acids increases, but no significant amount of intracellular fatty alcohols is secreted. |
| 学科主题 | 生物代谢工程 |
| 语种 | 中文 |
| 公开日期 | 2013-07-13 |
| 源URL | [http://ir.qibebt.ac.cn:8080/handle/337004/1501]  |
| 专题 | 青岛生物能源与过程研究所_微生物代谢工程团队 |
| 推荐引用方式 GB/T 7714 | 姚 伦. 集胞藻PCC6803中表达脂酰辅酶A还原酶生物合成脂肪醇研究[D]. 北京. 中国科学院研究生院. 2013. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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