用于纤维素制液体燃料的嗜热厌氧菌产能遗传机制研究

QIBEBT-IR > 单细胞中心组群

	用于纤维素制液体燃料的嗜热厌氧菌产能遗传机制研究
	林璐
导师	徐健
	2011
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	：纤维素制乙醇嗜热厌氧产乙醇杆菌（tgpas）糖代谢组学基因共表达网络乙醇耐受超声转化
摘要	纤维素液体燃料是目前最有希望取代石油能源的一种可再生的清洁能源。在纤维素液体燃料生产中CBP是最高产、节能以及低成本的一种工艺流程，但是对其发酵菌株有一系列苛刻的要求。嗜热厌氧革兰氏阳性菌（TGPAs）由于其高温的生长环境、高效的纤维素降解能力、广谱的碳源利用及独特的五碳糖和六碳糖共利用等特点使其成为最有希望满足CBP要求的菌株。然而由于TGPAs对高浓度碳源敏感、较低的乙醇耐受性以及低乙醇产量等一系列问题大大限制了其直接用于工业生产。本论文以Thermoanaerobacter sp. X514为模型，围绕这些问题展开了一系列研究。首先，理解TGPAs碳源分解代谢网络是解决上述问题的第一步。因此，我们基于实验数据重构了第一个嗜热厌氧菌的碳源代谢网络。该网络提供了许多新的代谢途径，并且精确的指出了以前未认知的，而又十分关键的途径之间的相互作用及其关键的节点。第一，葡萄糖、木糖、果糖和纤维二糖的分解代谢基因簇分别位于不同的功能单元内，并且己糖和戊糖的转运系统都受bglG调控，这合理的解释了己糖和戊糖共代谢的机制。第二，葡萄糖和木糖的功能单元能相互作用，因为前者通过促进后者的转运与分解代谢来加速后者的利用，而后者通过维持辅酶和离子的代谢来推迟细胞裂解。第三，维他命B12通过介导乙醇胺和丙二醇降解来提高乙醇产量，而精氨酸分解途径则有助于细胞在稳定期的存活。最后，我们实验验证了葡萄糖和木糖不同功能又相互合作的特性，并且发现当优化加入碳源的时间和比例能够提高乙醇产量。总之，该模型揭示出的新的碳源分解代谢特点能直接应用于工业生产，并为发酵和基因工程改造提供了新的思路和目标。其次，我们通过揭示单碱基水平和全基因组水平（DNA水平和转录水平）的多样性和差异性，系统的监控、比较和建模了嗜热厌氧菌获得溶剂耐受性的全过程。结果显示：第一，乙醇压力重塑了碳代谢模式。其中Teth5140145 (9个adh之一) 和乳酸脱氢酶（ldh）过量表达使来自乙醇降解的碳流量流入乳酸发酵途径。这个机制是嗜热厌氧菌乙醇压力应答各个阶段都使用的但又有别于E.coli的应答机制。第二，在乙醇压力下，细胞膜的代谢减缓，并且转运离子和碳源的转运子基因的表达水平也受到抑制，然而维他命B的合成途径则被激活，这是嗜热厌氧菌独特的应答机制。此外，乙醇压力诱导了毒素排出系统基因的表达并开启了压力应答途径来保护细胞免受溶剂伤害。第三，溶剂耐受这一遗传特点是由基因组水平和转录水平共同控制的。然而随着溶剂耐受能力的增强，基因组改变的比例增加，而转录组改变的比例减小。第四，在整个演化过程中，非同义突变的结构蛋白的比例增加。在演化后期，参与细胞膜代谢的蛋白在突变基因中占较大比例。最后，再解析出上述代谢网络后，我们致力于发展适用于TGPAs的遗传改造系统从而能对这些网络进行重组，构建出满足CBP工业生产需求的工程菌株。所以，我们建立了一种简单、快速以及侵害性较小的，适用于TGPAs的超声波介导的遗传转化方法。利用该转化方法，成功的将含有绿色荧光蛋白的穿梭质粒pHL015和含有热纤梭菌的的β-1，4-内切葡聚糖酶的pIKM2转化到嗜热厌氧产乙醇菌X514中，转化效率为600 转化子/μg DNA。并且，在X514细胞内，功能性的表达外源β-1，4-内切葡聚糖酶使之成为具有CBP特征的的微生物，即在同一细胞内不仅具有产乙醇的能力，还具有纤维水解酶的活性。该方法的创建加速了那些难于遗传操作，但具有工业生产潜能的TGPAs高通量遗传转化体系的发展。总之，本论文中所阐述的嗜热厌氧菌糖代谢模型和乙醇耐受的分子机理，加上建立的超声波遗传转化体系，为合理的改造TGPAs和其它产乙醇菌株以便提高纤维素乙醇产量奠定了坚实的基础。
其他摘要	Lignocellulosic biofuel is a renewable and clean energy source which could help reduce dependence upon fossil energy. CBP, one proposed scheme of lignocellulosic bioethanol, is the most promising, due to lower cost, higher production efficiency and simple processed. However, it has a serious of rigorous requirements for its microorganisms. Thermophilic, Gram-positive, anaerobic bacteria (TGPAs) are very interest in CBP due to extreme-temperature growth and simultaneous pentose and hexose utilization (co-utilization). However, high carbon loading inhibition, sensitive to high concentration ethanol and the lower ethanol production greatly block them in industrial applications. Here, using Thermoanaerobacter sp. X514 as the model, we divised a series of schemes to solve these problems. First of all, we experimentally reconstructed the structure and dynamics of the first genome-wide carbon-utilization network of thermoanaerobes, which is foundation in solving above problems. The network uncovered numerous novel pathways and pinpoint previously unrecognized yet crucial pathway-interactions and the associated key junctions. First, glucose, xylose, fructose and cellobiose catabolism are each featured in distinct functional modules; the transport systems of hexose and pentose are apparently both regulated by the transcriptional antiterminators of the BglG family, which is consistent with pentose and hexose co-utilization. Second, glucose and xylose modules cooperate in that the activity of the former promotes the activity of the latter via activating xylose transport and catabolism, while xylose delays cell lysis by sustaining coenzyme and ion metabolism. Third, the vitamin B12 pathway appears to promote ethanologenesis through ethanolamine and 1, 2-propanediol, while the arginine deiminase pathway probably contributes to cell survival in stationary phase. Moreover, by experimentally validating the distinct yet collaborative nature of glucose and xylose catabolism, we demonstrated that these novel network-derived features can be rationally exploited for product-yield enhancement via optimized timing and balanced loading of the carbon supply in a substrate-specific manner. Thus, this thermoanaerobic glycobiome reveals novel genetic features in carbon catabolism that may have immediate industrial implications and provides novel strategies and targets for fermentation and genome engineering. Next, we presented the first “integrated” view of microbial acquisition of solvent-tolerance traits, using thermoanaerobic ethanologen Thermoanaerobacter sp. X514 as a model. Our work revealed that, first, ethanol stress reshaped carbon flux where Teth5140145 (one of nine adh genes) was overexpressed to mediate the balance-shift from ethanol-deriving to lactate-deriving state, a cellular response shared over the whole process of tolerance-acquisition but distinct from that of E.coli. Second, under ethanol stress, cell-membrane metabolism slowed down and expression of ion/carbon transporters genes inhibited, however vitamin B biosynthetic pathways were activated, a feature not reported in other organisms; moreover, ethanol stress induced expression of efflux transporter systems and general stress response systems to protect cell from the solvent. Third, adaptations at transcript and genomic levels both contributed to the acquisition of tolerance, however along the process, alternations in genome sequences increased while alteration in transcriptome gradually subdue (number of expression-altered genes decreased). Finally, At DNA-level, the proportion of non-synonymous mutated structure proteins increased along trait-development, with the cell membrane metabolism gaining prominence among the mutated structural proteins at later period. Finally, we established first reported for any Gram-positive or thermophiles an ultrasound-based sonoporation as a simple, rapid, and minimally invasive method to genetically transform TGPAs in order to genetic engineering TGPAs, based on above reconstructed networks. Via sonoporation, shuttle vectors pHL015 harboring a jellyfish gfp gene and pIKM2 encoding a Clostridium thermocellum β-1, 4-glucanase gene were delivered into X514 with an efficiency of 6x102 transformants/μg of methylated DNA. Furthermore, the foreign β-1, 4-glucanase gene was functionally expressed in X514, converting the host into a prototypic thermophilic consolidated bioprocessing organism that is not only ethanologenic but cellulolytic. This new DNA-delivery method could significantly improve the throughput in developing genetic systems for TGPAs, many of which are of industrial interest yet remain difficult to manipulate genetically. In summary, together with the established genetic transformation method, the newly discovered networks of thermoanaerobic glycobiome and toleratomes formed a foundation for rational engineering both host-cells in TGPA- and other ethanolgens to improve cellulosic ethanol production.
学科领域	功能基因组
公开日期	2012-06-05
学位类型	博士 ; 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/337004/981
专题	单细胞中心组群
推荐引用方式 GB/T 7714	林璐. 用于纤维素制液体燃料的嗜热厌氧菌产能遗传机制研究[D]. 北京. 中国科学院研究生院,2011.

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