【作者】 周景文；

【导师】 陈坚；

【作者基本信息】 江南大学 ， 发酵工程， 2009， 博士

【摘要】 本文以一株能在胞外大量积累丙酮酸的光滑球拟酵母的（Torulopsis glabrata）四重维生素（硫氨酸、生物素、吡哆醇和烟酸）营养缺陷型菌株CCTCC M202019为研究对象,以阐明能量代谢对酵母生理过程的影响为目标,在初步了解T. glabrata中能量代谢途径的基础上,运用代谢工程和微生物生理学的理论和方法,就ATP代谢途径如何调控酵母胞内微环境,并影响细胞生长与产物积累的机制展开研究。主要研究结果如下:（1）有机整合融合PCR、酵母高效电转化、制霉菌素富集和限制性培养基筛选等技术手段,建立了一种针对酵母的无抗性标记并可重复使用的大片段缺失营养缺陷型菌株构建方法,并成功构建了尿嘧啶缺陷型（Δura3）、精氨酸缺陷型（Δarg8）和尿嘧啶精氨酸双缺陷型（Δura3Δarg8）等三株T. glabrata营养缺陷型菌株。在此基础上,验证了含有2μm片段的酵母载体在T. glabrata中的稳定性,并实现了增强型绿色荧光蛋白的可诱导表达;（2） ATP8、ATP6和ATP9基因分别编码F₀F₁-ATP合成酶的三个重要亚基,均位于线粒体基因组上（mtDNA）。以一个线粒体重新编码的ARG8m基因作为筛选标记,利用同源重组策略敲除ATP8、ATP6和ATP9基因。在这一过程中发现,野生型和转化的mtDNA能同时存在于转化子中,且随着培养条件的改变,两种mtDNA所占的比例会发生规律性变化。作者将这一现象命名为单细胞线粒体基因组多态性（Single cell mitochondrial genome polymorphism,SCMGP）。研究表明,mtDNA不稳定性、线粒体融合/分裂过程和mtDNA的选择性丢失是形成SCMGP现象的主要因素。在理解SCMGP现象形成机制的基础上,建立了利用厌氧培养消除SCMGP现象的策略,获得了三株仅含有目标mtDNA的同质体ATP8、ATP6和ATP9缺失菌株,并分别命名为ATP8、ATP6和ATP9;（3） ATP6缺失可以导致T. glabrata在基本培养基和精氨酸补充培养基中培养24代后的mtDNA丢失率分别达到42%和63%。胞内ATP水平、活性氧（Reactive oxygen species,ROS）浓度、线粒体膜间腔（Mitochondrial intermembrane space,MIMS）中的pH值、跨膜电势（ΔΨm）及乌头酸酶的表达水平与酶活性均表明,MIMS中H+的过量积累是导致ATP6缺失突变株mtDNA不稳定的关键因素。当细胞缺失ATP6基因后,发挥离子通道作用的a亚基丢失,导致MIMS中积累的H+无法通过F₀F₁-ATP合成酶得到释放,导致ROS水平升高,干扰线粒体基质蛋白的定位,系统性的影响mtDNA的稳定性。为了提高ATP6敲除突变菌株mtDNA在不同培养条件下的稳定性,将来源于荚膜组织胞浆菌（Histoplasma capsulatum）的交替氧化酶基因AOX1和来源于乳酸乳球菌（Lactobacillus lactis）的NADH氧化酶基因noxE表达于ATP6缺失突变株中,显著提高了mtDNA稳定性,两株菌分别命名为AOX和NOX;（4）以前面得到的一系列ATP合成酶缺失突变株为对象,研究了胞内ATP水平变化对细胞生理过程的影响。ATP8、ATP6和ATP9三个基因的敲除显著降低了ATP水平,强烈的抑制了细胞的生长,48 h时的菌体干重分别降低了46.9%、44.2%和59.8%。在发酵初期,由于F₀F₁-ATP合成酶活性缺失导致的胞内ATP水平下降,ATP8、ATP6、ATP9和NOX的胞内ATP水平分别为出发菌株的65.5%、62.0%、48.4%和73.0%。ATP水平的降低显著解除了ATP对糖酵解途径关键酶活性的抑制,加速了糖酵解速率。但随着发酵继续进行至28 h后,ATP合成酶缺失菌株的糖酵解速率显著下降。研究表明,ATP合成酶缺失菌株中ATP水平的显著下降和ROS水平的显著提高,导致细胞抵御酸胁迫和渗透压胁迫的能力显著下降。在胞质中表达NADH氧化酶基因noxE可以促进NADH代谢,降低胞内ROS水平,改善胞内微环境,从而促进细胞生长和丙酮酸的积累。代谢网络通量、中心代谢途径关键酶表达水平和酶活性分析表明,F₀F₁-ATP合成酶缺失对胞质中的糖酵解途径具有显著影响,而对位于线粒体基质中的三羧酸循环影响较小,表明线粒体的亚细胞区隔可以有效保护其中进行的物质合成和产能代谢途径;（5）真核微生物细胞依靠一系列ATP酶,利用水解ATP产生的能量,进行H+和其它离子的转运,以维持各亚细胞区隔间的pH梯度。当胞外pH较低时,细胞需要消耗更多的ATP维持更高的pH梯度。为了研究胞内ATP水平在细胞应对胁迫过程中的作用,通过在培养基中添加柠檬酸盐促进ATP供给,研究低pH条件下T. glabrata的细胞生长和丙酮酸生产。结果表明,ATP供给的增强显著促进了依赖于ATP的胞内pH平衡过程,使培养基、胞质和液泡之间的pH梯度得到显著提高。pH梯度与胞内ATP浓度之间的量化关系表明胞内ATP浓度的上升可以显著的促进相关的pH平衡过程。此外,T. glabrata CCTCC M202019的pH平衡能力显著弱于其它常见的酵母菌株,可能也是其可以快速积累丙酮酸的一个重要原因。更多还原

【Abstract】 In this dissertation, a multi-vitamin auxotroph （i. e., thiamine, biotin, pyrodoxin and nicotinic acid） Torulopsis glabrata strain, CCTCC M202019 was used as a model to demonstrate the effect of the energy metabolism on the physiological processes in eukaryotic microorganisms. Based on the well understanding of the energy metabolism, the mechanisms in the role of ATP metabolism in the intracellular micro-environment and related physiological processes were investigated. The main results were described as follows:（1） A reusable method, which systematically integrated the fusion PCR, high-efficiency electroporation transformation, nystatin enrichment and limited media screening, for construction of non-marker large fragment deletion Torulopsis glabrata auxotroph strains was established. By using the method, three T. glabrata auxotroph strains were obtained, i. e., a uracil auxotroph strain （Δura3）, an arginine auxotroph strain （Δarg8） and a uracil arginine double auxtroph strain （Δura3Δarg8）. The stability of 2μm bearing vectors was assayed in the T. glabrataΔura3 strain. The result proved that the 2μm bearing vector was highly stable and could express target green fluorescent protein in T. glabrataΔura3 strain.（2） ATP8, ATP6 and ATP9 are three genes that encode three key subunits of F₀F₁-ATPase, which are essential for the respiration, and locate on the mitochondrial genome （mtDNA）. To knockout of the three genes, a homologous knockout box with a recoded ARG8 gene ARG8m, were transformed into the T. glabrataΔura3Δarg8 strain by biolistic transformation. The mtDNA transformants were primarily screened by the back-mutation of arginine auxotroph. However, it was found out that both the wild-type mtDNA and transformed mtDNA co-existed in the transformant cells and the ratio of the two kinds of mtDNA varied under different culture conditions and growth periods. The phenomenon was designated as Single Cell Mitochondrial Genome Polymorphism （SCMGP）. It was found that the instability of mtDNA, mitochondrial fusion/fission and selective loss of mtDNA played crucial roles in the SCMGP. Three mtDNA homoplasmic cells without ATP8, ATP6 and ATP9 were obtained based on the characterization of the SCMGP and named as ATP8, ATP6 and ATP9, respectively.（3） Deficiency in ATP6 could increase the loss ratio of mtDNA to 42% and 63% on minimum medium and argine supplement medium after 24 generations, respectively. Through the assay of intracellular ATP level, production of reactive oxygen species （ROS）, intracellular ATP level, the pH in mitochonchondrial intermembrane space （MIMS） and the transcriptional and enzyme activity assay of aconitase, it was found out that the H+ accumulation in MIMS was the dominant factor for the mtDNA instability of T. glabrata cells deficient in ATP6. In normal cells, the H+ accumulation in MIMS could be released by the F₀F₁-ATPase accompanied with the ATP synthesis. However, in cells deficient in ATP6, which encodes the ion channel subunit a, the H+ could not be released through the F₀F₁-ATPase. The accumulation of H+ in MIMS further improved theΔΨm and ROS, interrupted the translocation of mitochondrial matrix located proteins, and thus systematically caused the mtDNA instability in T. glabrata cells deficient in ATP6. The expression of two NADH oxidation associated genes, AOX1 from Histoplasma capsulatum and noxE from Lactobacillus lactis, could significantly improve the stability of mtDNA in ATP6. The result further proved that the conclusion. The two strains were names as AOX and NOX, respectively.（4） The effects of ATP metabolism on the central carbon metabolism were systematically investigated using strains ATP8, ATP6, ATP9 and NOX. The deficiency in the F₀F₁-ATPase significantly decreased the intracellular level, inhibited the cell growth and released the inhibitory effect of ATP during the initial 28 h. However, the relased inhibitory effect of ATP was mostly compsensated by decreased ability to deal with the tolerance to acidic stress and osmotic stress. The deficiency of F₀F₁-ATPase resulted in increased intracellular ROS level. The overexpression of noxE could recover the intracellular ROS level caused by the ATP6 deficiency and improved the intracellular micro-environment, thus enhanced the cell growth and pyruvate accumulation. The metabolic networks analysis, transcriptional and enzyme activities analysis of central metabolism key enzymes revealed that the deterioration of intracellular micro-environment caused by the deficiency in F₀F₁-ATPase affected the central carbon metabolism on different levels thus prohibited the further increase of pyruvate production. The phenomenon also suggested the importance of sub-cellular compartmentation in the protection of biomass synthesis and energy metabolism in the mitochondrial matrix.（5） In eukaryotic microorganisms, the pH in different subcellular compartmentations is kept in a suitable range thus the enzymes in these compartmentations could play their normal functions. Eukaryotic microorganisms realized the pH homeostasis in different cell compartmentation through a series of ATPase, which consumed additional ATP besides the normal cell growth. To determine the effect of ATP in the tolerance to lower pH, the intracellular micro-environment under enhanced ATP supply conditions was investigated by the citrate addition. The result showed that the enhanced the ATP supply could facilitate the ATP-dependent H+-transportation processes, thus kept higher pH gradients among extracellular environment, cytoplasm and vacuole. The quantitative relationship between the intracellular ATP content and the pH gradients showed that the increased intracellular ATP level could significantly promoted the pH homeostasis processes. Compared with the pH homeostasis process in Saccharomyces cerevisiae, it was found out that the lower pH homeostasis ability in T. glabrata CCTCC M202019 should be a positive factor for the pyruvate accumulation.更多还原