| 题名 | 基于代谢物组学和发酵优化及其整合策略的热纤梭菌乙醇耐受性研究 |
| 作者 | 朱新术 |
| 学位类别 | 博士 |
| 答辩日期 | 2012-11 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 崔球 研究员 |
| 关键词 | 热纤梭菌 乙醇耐受 代谢物组学 培养基优化 整合策略 |
| 学位专业 | 生物化学与分子生物学 |
| 中文摘要 | 热纤梭菌(Clostridium thermocellum)是利用联合生物加工技术（Consolidated BioProcessing，CBP）生产纤维素乙醇的候选菌株之一，然而生长和发酵过程中的乙醇耐受性问题严重制约了其商业化应用。本研究分别用代谢物组学和发酵培养基优化以及两者相结合的策略研究该问题。1、利用NMR和GC-MS以及多元统计学方法进行了热纤梭菌野生株(WT)和乙醇耐受株(ET)的胞内代谢物组学研究，结果发现与野生株相比，乙醇耐受株的代谢路径发生了广泛的变化，主要涉及糖酵解/TCA循环、GS/GOGAT循环、PPP路径和嘧啶与嘌呤代谢路径以及脂肪酸代谢路径。经过分析我们提出热纤梭菌乙醇耐受性获得的机制的“纤维二糖-纤维糊精”假说，即由于胞内碳水化合物代谢发生改变，纤维二糖被菌体合成为纤维糊精以抵抗乙醇的胁迫。2、选取氨基酸、糖类、有机酸和部分重要阳离子作为热纤梭菌胞外靶向代谢物组的分析目标，并用离子色谱(ion chromatography, IC)获取这些分析目标的定性定量结果，数据进一步通过多元统计学方法进行深入分析。结果发现ET与WT的代谢足迹存在很显著的差异，在所选取的四类靶向分析代谢物中均有所表现。这些结果支持了前述的“纤维二糖-纤维糊精”假说。并且发现热纤梭菌可以直接摄取培养基中的部分有机酸类代谢物，减少了从头合成的麻烦，同时发现ET的TCA循环存在“溢出”现象，以致向培养基中排出苹果酸。3、针对ET存在的一些突出问题，诸如提高乙醇耐受性的常规方法相当繁琐，菌株生长相对缓慢，乙醇产量相对较低以及副产物所占比例过高等，我们提出了利用发酵培养基优化来提高热纤梭菌乙醇耐受性（通过相应的生物量大小评估）并改善其他发酵表型的研究思路。首先利用Plackett–Burman Design(PBD)筛选到了培养基中影响热纤梭菌乙醇耐受性的三个关键培养基成分，即MgCl2·6H2O、CaCl2·2H2O和半胱氨酸盐酸盐。再通过均匀设计(Uniform Design,UD) 对这三个成分的浓度进行优化，目的是为后续的偏最小二乘回归（Partial Least Squares Regression, PLSR）建模以及与代谢物组学整合研究提供典型而又精简的研究样本。然后分别建立了三种培养基关键成分与生物量、乙醇产量、副产物乙酸和乳酸浓度等优化目标之间的PLSR模型，最后用遗传算法（Genetic Algorithm,GA）获得了单个优化目标的最优解与最优值，以及优化目标两两配对的Pareto解。结果发现发酵培养基优化方法可以分别实现提高热纤梭菌乙醇耐受性和乙醇产量，以及降低副产物乙酸与乳酸的含量等四个优化目标。但是在现行优化体系下，无法同时实现这些优化目标，然而另一方面，利用多目标优化方法却可以探究出优化目标之间的内在联系，为相应的遗传改造工作提供有价值的目标。4、依据代谢物组学是系统生物学水平上研究生物学问题的有力武器，以及发酵培养基优化可以提高热纤梭菌乙醇耐受性而内部机制不明朗这两个事实，本文提出了一种整合这两种研究方法的新型研究策略。利用前述建立的离子色谱法获取热纤梭菌乙醇耐受株UD培养基优化实验样品的胞内胞外代谢物组数据，并用主成分分析（principal component analysis，PCA）和偏最小二乘（partial least squares，PLS）对这些数据进行深入分析。结果发现不仅UD实验三种关键成分与其相应的胞内胞外代谢物组所建立的PLS模型的潜变量之间的相关性很强，并且UD实验胞内胞外代谢物组和相应的优化目标(表型)所建立的PLS模型的潜变量之间也存在较强的相关性。这样利用代谢物组学这一“媒介”，初步探讨了发酵培养基优化这一“黑箱模型”的内在机制，并首次在系统生物学层面阐明了传统发酵培养基优化策略的内在合理性。同时，还具体分析了UD三种关键成分是如何影响相应的胞内胞外代谢物组，并最终影响设定的优化目标。此外，本文通过考察所建立的宏观发酵培养基成分和胞外/内代谢物组，以及胞外/内代谢物组和最终的表型之间PLS模型相关系数的大小，提出了一种判断代谢物组分析是否完备的一种方法。最后，基于以上研究，本文还提出一种传统发酵优化技术和代谢足迹技术相结合的新型发酵优化策略。
总之，本文研究不仅加深了对其耐受机制的研究，有助于纤维素乙醇的研发，并且首次在系统生物学水平上初步揭示了传统发酵培养基优化方法黑箱模型的内部机制和潜在合理性，还为代谢物组学的发展提供了新思路。 |
| 英文摘要 | Clostridium thermocellum is one of candidate microorganisms for cellulosic ethanol production with Consolidated BioProcessing(CBP), but its low ethanol tolerance is a big obstacle for the commercial application. In this study, we employed metabolomics, medium optimization and their integrated strategy to solve the problem. Firstly, the intracellular metabolome of wild type strain (WT) and ethanol tolerant strain (ET) of C. thermocellum were analyzed via NMR and GC-MS as well as multivariate statistical methods. Compared with WT, ET showed various differences in many metabolic pathways, involving glycolysis/TCA cycle, GS/GOGAT cycle, PPP pathway as well as pyrimidine and purine metabolisms. These results indicated that the mechanism of obtaining the ethanol tolerance in C. thermocellum might be due to the change of the metabolism of intracellular carbohydrate, i.e., cellobiose was transformed into cellodextrin in order to resist the ethanol stress, namely, a “cellobiose-cellodextrin” hypothesis was presented.Secondly, four types metabolites, i.e. amino acids, carbohydrates, organic acids and some key cations were selected as analytical goals for studying the extracellular metabolome of C. thermocellum, and the qualitative and quantitative data of these analytical goals were got by ion chromatography (IC), subsequently, data were further analyzed by multivariate statistical methods. Noticeable differences between WT and ET existing in all the four types of selected metabolites were discovered. The results supported the “cellobiose-cellodextrin” hypothesis. In addition, we found C. thermocellum could directly assimilate some organic acids from the medium, in order to reduce de novo synthesis. Furthermore, malic acid secreting into the medium in ET was also observed, which maybe meaned that a metabolic overflow appearing in TCA cycle in ET.Thirdly，aimed at some prominent problems associated with ethanol tolerant strain of C. thermocellum, such as conventional method for improving the ethanol tolerance being quite troublesome, the relatively poor growth, the lower ethanol production and the relatively higher proportion of byproducts, we performed medium optimization in this study for improving ethanol tolerance (assessed by corresponding biomass) and fermenting pattern of C. thermocellum. MgCl2·6H2O, CaCl2·2H2O and cysteine hydrochloride were firstly screened by Plackett–Burman Design(PBD) as the crucial components in the medium regarding to ethanol tolerance, and their concentration were further optimized according to Uniform Design (UD) as well as subsequent partial least squares regression(PLSR) modeling regarding with biomass, ethanol production, and byproduct (i.e., acetic acid and lactic acid) production, respectively, the UD also might be beneficial for offering representative and terse samples for the followed integrated research with metabolomics. And then the optimal solution and corresponding optimal value for every optimized target as well as Pareto solution for each pair optimized target were calculated by Genetic Algorithm (GA). The results showed that medium optimization could be used to improve the ethanol tolerance and ethanol production, and reduce the byproduct production, respectively. However, It might be difficult to optimize multi-targets simultaneously under the present optimizing conditions, while the internal relationships among these optimized targets could be explored by this strategy,which could provide potential target for genetic engineering.Fourthly, according to the facts that metabolomics is a powerful weapon in solving the biological problem at the level of system biology and the ethanol tolerance of C. thermocellum could improve assuredly by medium optimization yet with ambiguous internal mechanism, a new research strategy that integrating the metabolomics and media optimization was proposeed in this study. At first, the intracellular and extracellular metabolome of the UD samples (ET cultivated in UD optimized medium) were obtained qualitatively and quantitatively by IC, and the data were further analyzed by principal component analysis (PCA) and PLS. The results indicated that strong correlation, not only between the latent variables of the PLS model of the three key media components in the UD on corresponding intracellular and extracellular metabolome, but also between the latent variables of the PLS model of intracellular and extracellular metabolome on corresponding optimized targets (phenotypes). So making the metabolomics as a “media”, we explored the internal mechanism of “black box” of medium optimization, and demonstrated firstly the intrinsic rationality of traditional medium optimization at the level of system biology. Meanwhile, we also analyzed how the three key media components in UD influence concretely corresponding intracellular and extracellular metabolome and then the final optimized targets. Furthermore, according to the big or small of related coefficient of PLS models between the medium components on corresponding intracellular and extracellular metabolome, as well as intracellular and extracellular metabolome on corresponding final phenotypes, a new method was presented that for judging the metabolome analyzing whether complete or not. Finally, according to these study, a new strategy of medium optimization coupling with traditional medium optimization methods and metabolic footprinting was proposed. In summary, this work improved the recognition on ethanol tolerance of C. thermocellum and was helpful for the production of cellulosic ethanol. Furthermore, this study preliminarily explored the internal mechanism and intrinsic rationality of traditional medium optimization at the level of system biology, which provided noval ways for medium optimization and metabolomics research. |
| 语种 | 中文 |
| 学科主题 | 代谢物组学 |
| 公开日期 | 2012-12-12 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.qibebt.ac.cn:8080/handle/337004/1433]  |
| 专题 | 青岛生物能源与过程研究所_代谢物组学团队 |
| 推荐引用方式 GB/T 7714 | 朱新术. 基于代谢物组学和发酵优化及其整合策略的热纤梭菌乙醇耐受性研究[D]. 北京. 中国科学院研究生院. 2012. |
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