【作者】 黄蓓蓓；

【导师】 柴逸峰； 陈万生；

【作者基本信息】 第二军医大学 ， 药物分析学， 2009， 博士

【摘要】 萜类化合物广泛分布于植物及微生物初级代谢物和次生代谢物中,其种类繁多,不仅在植物的生命活动中扮演重要的作用,而且还被广泛的应用于工业、医药卫生等方面,如倍半萜中的青蒿素、双萜中的紫杉醇可分别作为抗疟、抗癌的有效药物。但是萜类在生物体内的产量很低,而且分离和纯化操作复杂。由于萜类结构复杂,化学合成需要的步骤烦琐,非常困难。由于酵母自身存在甲羟戊酸(Mevalonate,即MVA)途径,可自我合成萜类前体,为利用代谢工程改造细胞代谢途径为提高萜类产量提供了一个很好的操作平台,然而酵母中萜类前体库不足够提供高产,有必要调节其参与萜类前体生物合成的代谢途径以提高产量。本研究中,我们选择酿酒酵母作为宿主细胞,以酿酒酵母中的萜类生物合成途径为主要研究对象,建立遗传改造的酿酒酵母的分析技术平台,将最先进的分析技术用于测量代谢物及其在一定条件下含量的变化,有助于阐释遗传干预对感兴趣的途径的影响,同时对微生物的应激反应进行整体性无偏的描述,以进一步理解通过这个途径的代谢流的控制分子机制,得到如下结果:(1)首次将基因敲除的分子生物学方法,引入酿酒酵母的次生代谢途径萜类生物合成途径中,以调节其代谢,增加其萜类前体库供给。酿酒酵母中的萜类生物合成途径主要分成六异戊二烯基焦磷酸合成途径以及麦角甾醇生物合成途径两个部分。为了增强通往萜类前体的代谢流,构建敲除盒,将负责FPP转为角鲨烯,竞争FPP的erg9基因,位于萜类前体下游的coq1基因分别敲除,减少旁路消耗。用(2) PCR方法构建敲除盒LoxP-kanMX/URA3–loxP,分别敲除erg9基因和coq1基因,剔除筛选标记ura3后,分别得到新菌型BY4743- erg9和BY4743- coq1。在BY4743- erg9基础上,再利用敲除盒LoxP-URA3–loxP,敲除coq1基因,经PCR验证,并剔除筛选标记ura3,得到新菌型BY4743- erg9- coq1。同时考察其基础时间生长曲线的变化,为下一步的基因-生理机能机制深入研究,提供基础。野生菌株生长最快,缺失株生长较慢,双缺失菌生长最慢,形成的菌落也较小。(3)为了考察上述遗传改造对萜类前体积累的效果,测定酿酒酵母的萜类生物合成途径上重要的指标性成分GGPP (二萜成分的前体),分析它的变化,表征其随erg9, coq1基因缺失的关联性,为阐明酿酒酵母中萜类合成途径的调节分子机制提供现实依据。本研究首次建立LC-MS方法测定遗传改造后酿酒酵母中GGPP含量的变化。应用4.6 mm×25 cm SB-Aq C18 5-μm (Agilent Tech., USA)色谱柱进行液相分离,流动相75%(10mM三丁胺水溶液用15mM醋酸调成pH 4.95):25%甲醇,质谱负离子模式扫描质荷比范围50–600,选择离子检测GGPP([M–H]?),质荷比449.2。标准曲线线性范围0.1–50 ng/ml。在这个范围内的,日内相对标准偏差<10%,日间相对标准偏差<14%,精密度范围96.5-105.4%。结果显示,遗传改造有效,BY4743- erg9- coq1的积累量在72小时后增加非常显著。遗传改造后的菌株与野生型菌株比较,含量有显著差异(P<0.01);随时间的长短,含量显著增高。说明erg9与coq1对GGPP的代谢显著影响。(4)为了研究遗传改造对萜类生物合成途径代谢流的影响,建立GC–MS方法测定萜类生物合成途径上各代谢物及其在酿酒酵母中的应用。本研究首次建立一个精确灵敏的非放射性的方法,同时定量酿酒酵母中萜类合成途径上的GPP,FPP,GGPP,角鲨烯,麦角甾醇和羊毛甾醇,考察代谢流的变化。这个方法建立在GPP,FPP和GGPP去磷酸化成相应的烯萜醇,用GC-SIM-MS分析。本研究考察了三种转化方法:酸解法、碱解法、酶解法。酸解法和碱解法的转化率都低于15%,不能应用于此,酶解法在不同的缓冲体系中的转化率相差很大,50mM Bis-Tris propane/HCl, 1mM MgCl2 , pH 7.0,转化率<1%; 0.1M 2-amino-2-methyl propanol/NaOH, 1mM MgCl2 , pH 10.35,转化率<1%; 50 mM Tris-HCl, 10 mM MgCl2 , pH9.0,转化率<1%; 0.1 M glycine/1 M NaCl/40% MeOH, pH 10.4,转化率<2%, 1 M diethanolamine, 0.5 mM MgCl2 , pH 9.8,转化率可>88%,符合研究的需要。本研究综合比较全发酵液提取法、细胞液离子交换柱提取法、细胞液超声提取法三种提取方法,正己烷、石油醚、乙酸乙酯、氯仿:乙酸乙酯(1:10)四种提取溶剂,0.5min、1.5min、3min这样3个涡旋时间,比较其效率,确定最终的前处理方法为:细胞液超声提取法,正己烷提取,涡旋3min。应用TRACE TR-5MS柱(30m×0.25mm×0.25μm)进行气相分离,单重四极杆质谱选择离子监测GOH,FOH,GGOH,角鲨烯,麦角甾醇和羊毛甾醇,以保留时间和碎片信息进行定性定量分析。结果显示,方法准确有效,标准曲线线性良好,日内和日间精密度良好,加样回收率准确度高。将此方法应用于酿酒酵母中,发现不同基因对于酿酒酵母中萜类合成途径上的各个代谢物的代谢过程的作用是不一样的,双缺失erg9和coq1,使得GPP、FPP、GGPP大量积累,而角鲨烯、麦角甾醇、羊毛甾醇的含量可维持细胞正常生理功能。。(5)通过考察不同遗传改造的酿酒酵母基础时间生长曲线的变化,发现其生理活动发生了变化。为了考察遗传改造对细胞生理活动的影响,建立荧光分析和化学发光方法测定分子操作后,与细胞生理特性和基因功能相关代谢物及其含量的变化,有助于阐明萜类代谢途径的基因敲除后,对细胞功能的影响。利用罗丹明6G,测得BY4743- erg9- coq1离子外排作用低于野生型和单缺失菌,并且酿酒酵母的外排作用是能量依赖型的。利用JC-1,测得erg9与coq1对线粒体膜电位的影响巨大,两者的缺失打破线粒体膜电位分布的平衡,但24h后,不同遗传改造的酿酒酵母建立的新的膜电位平衡无明显差别。利用DCFA-DA,测得erg9与coq1的缺失引发大量内源性活性氧的产生。利用荧光素酶的化学发光,测得BY4743- erg9- coq1ATP的含量低于野生型和单敲除菌。当前的工作力求将代谢工程,化学分析,数据处理等多个领域结合,在细胞水平分析代谢物的变化以详细地认识基因—代谢产物、基因—代谢流之间关联的分子机制,分析生理状态的变化以认识基因功能。本研究涉及多种技术的联用。这些方法包括分子生物学技术,微生物代谢活性瞬时淬灭,化合物的酶法衍生化、相关胞内代谢产物的提取,色谱与质谱的偶联(GC-MS, LC-MS),荧光与化学发光的应用以及统计学上的数据挖掘等等。多种技术的创新和联用必将提供大量的代谢途径信息,有助于反映有机体的生理活动,理解潜在的调节机制,对以酿酒酵母为平台生产多种重要价值的萜类化合物具有重要意义。更多还原

【Abstract】 Isoprenoids with more than 40,000 described compounds are the largest and most structurally diverse group of plant metabolites. Isoprenoids play various biological roles in plants. Depending on the number of isoprene units, isoprenoids can be classified into several groups, such as monoterpenes, sesquiterpenes and diterpenes (respectively 2, 3 and 4 C5 units).Isoprenoids are functionally important in many different parts of cell metabolism such as photosynthesis (carotenoids, chlorophylls, plastoquinone), respiration (ubiquinone), hormonal regulation of metabolism (sterols), regulation of growth and development (gibberellic acid, abscisic acid, brassinosteroids, cytokinins, prenylated proteins), defense against pathogen attack, intracellular signal transduction (Ras proteins), vesicular transport within the cell (Rab proteins) as well as defining membrane structures(sterols, dolichols, carotenoids). Many isoprenoids also have considerable medical and commercial interest as flavors, fragrances (such as limonene, menthol, camphor), food colorants (carotenoids) or pharmaceuticals (such as bisabolol, artemisinin, lycopene, taxol).Isoprenoids are widely present in plant tissues, and extraction from plants has been the traditional option for the large-scale production of these compounds. However, in many cases this method is neither feasible nor economical. Among the drawbacks in using plants as a source for isoprenoid production are influence of geographical location and weather on the composition and concentration of isoprenoids in the plant tissues, low concentration and poor yields for the recovery of isoprenoids from plants, and the high costs associated with extraction and purification. Chemical synthesis of isoprenoids has also been reported, and currently most of the industrially interesting carotenoids are produced via chemical synthesis. However, because of the complex structures of isoprenoids, chemical synthesis, involving many steps, is difficult. Side reactions, unwanted side products, and low yield are other disadvantages. In vitro enzymatic production of isoprenoids through the action of plant isoprenoid synthases is also impractical due to the dependency on the expensive precursors, as well as poor in vitro conversion. There is therefore much interest in using microorganisms as cell factories for the production of isoprenoids. The intracellular pools of isoprenoid precursors in microorganisms appear, to be however, not enough to provide high level production. It may therefore be necessary to deregulate the pathways involved in the biosynthesis of isoprenoid precursors in order to improve production.Yeast therefore has a high inherent capacity for the biosynthesis of isoprenoid precursors that may be directed to the production of heterologous compounds. Besides, tools from other related areas are being incorporated into the metabolic engineer’s repertoire. These developments range from rapid sample collection, instant quenching of microbial metabolic activity, extraction of the relevant intracellular metabolites, quantification of these metabolites using modern high tech hyphenated analytical protocols, mainly chromatographic techniques coupled to mass spectrometry (GC-MS, LC-MS), as well as the mathematical analyses.In this study, we chose S. cerevisiae as a host cell for the accumulation of isoprenoid precursors to extend an understanding of the mechanisms by which the flux through the pathway is controlled. The results are as follows:(1) In order to enhance the flux to isoprenoid precursors, both the erg9 gene which is responsible for conversion of FPP to squalene and the coq1 gene which lies in downstream pathway of GPP, FPP, GGPP were knockout by two disruption cassettes for gene replacement.(2) Upon genetic modification, the change in the concentration of GGPP was measured via LC-MS at batch cultivations time. LC separations were performed on a 4.6 mm×25 cm SB-Aq C18 5-μm column (Agilent Tech., USA) with a mobile phase consisted of 75% eluent A (10mM tributylamine aqueous solution adjusted pH to 4.95 with 15mM acetic acid) and 25% eluent B (methanol), and mass spectra were operated in negative ion mode over a range of m/z 50–600, and selective ion monitors (SIM) were at m/z 449.1-449.2 for GGPP ([M–H]?). The calibration curves were linear over the range of 0.1–50 ng/ml. In this range, relative standard deviations (R.S.D.) were <10% for intra-day precision and <14% for interday precision. The accuracy was within the range of 96.5-105.4%. The disruption of both erg9 and coq1 at 72 h of the fermentation period improved the accumulation of GGPP at most(3) A precise and sensitive nonradioactive method was developed for the simultaneous quantification of the isoprenoid precursors, geranyl diphosphate (GPP), farnesyl diphosphate (FPP), geranylgeranyl diphosphate (GGPP), squalene, ergosterol and lanosterol in recombinant and wild-type S. cerevisiae. The method is based on the dephosphorylation of FPP and GGPP into the respective alcohols and involves their in situ extraction followed by separation and detection using gas chromatography–selective ion-monitoring mass spectrometry (GC-SIM-MS). The analysis of GOH, FOH, GGOH, squalene, ergosterol and lanosterol illustrates robustness and reliability of this method outlined. Quantification of the analytes was performed by external calibration with reference substances and internal standardization. The recovery of the procedure has been evaluated.(4) The application of fluorescence and chemiluminescence analysis to the measurement of metabolites and the changes in metabolite concentrations under the molecular manipulation characterized cellular physiology and gene function, which would help us illuminate the effects of perturbation in pathways of interest, as well as unbiased characterizations of microbial stress responses as a whole. Functional analysis revealed greater energy-dependent efflux activity of membrane transporters, lower intracellular ATP level and mitochondrial membrane potential, more endogenous reactive oxygen species generation in response to gene disruption.The current work is based on the integration of genetic engineering, chemical analysis and data processing, which may provide a convenient format for considerable knowledge of metabolic pathway, as well as a platform for the production of a broad range of high value isoprenoids in yeast. This combination of rigorous analysis and quantitative molecular biology methods has endowed metabolic engineering with an effective synergism that crosses traditional disciplinary bounds. Meantime, the application of metabolic flux analysis contributes to reflect the activities of organisms, understand the underlying regulation mechanisms.更多还原