【作者】 刘可可；

【导师】 夏金兰；

【作者基本信息】 中南大学 ， 生物工程， 2011， 硕士

【摘要】 聚-β-羟基丁酸酯（poly-β-hydroxybutyrate,以下简称为PHB）是细胞内的一类生物聚酯,是制备“生物可降解塑料”的理想原料。隐藏嗜酸菌株DX1-1（CCTCC M208056）筛自有机质严重缺乏的酸性矿坑水中,是一种兼养性铁还原和硫氧化菌,具备累积体内PHB的能力。为了阐明培养条件对菌株DX1-1生长和PHB累积相关代谢途径的影响机制,开发菌株DX1-1在生长和PHB合成相关的有用基因资源,本文开展了如下研究工作：（1）比较研究了菌株DX1-1在自养（元素硫和/或硫酸铁）、异养（葡萄糖）、微异养（限制性葡萄糖+硫和/或硫酸铁）的混合养条件下的9K基础培养基中的生长和PHB累积情况；（2）克隆和分析了菌株DX1-1生长和PHB累积相关代谢系统功能基因；（3）采用Realtime PCR比较分析了菌株DX1-1在不同能源底物条件下生长和PHB累积相关功能基因的差异表达。取得如下研究结果。在异养条件下,菌株生长良好,能累积胞内PHB,在自养条件下,菌株不累积PHB,且生长较慢,其中在仅含铁（Ⅲ）为能源底物时不生长；在加入有限碳源,如0.1%的葡萄糖的混合养条件下,菌株无论在单质硫和/或硫酸铁的无机能源基质中,都能生长良好以及累积胞内PHB,且比相同限制性浓度的葡萄糖下的异养生长更快,PHB累积更多,在96 h时菌株DX1-1的细胞量为9.89×1 08个/mL,PHB产量为2.07g/L。在这些培养条件下,菌株DX1-1生长和生产PHB递减顺序为：GSF>GS>GF>G>>S,SF>>F（培养基名称参见表2-2）。克隆了菌株DX1-1的1个内标基因和26个生长与PHB累积相关代谢途径基因,分别为：6个PHB积累基因相关基因（包括一个表达相对稳定的基因）；10个二氧化碳固定相关基因；7个硫代谢相关基因；3个铁代谢相关基因。相关代谢基因Arcy₃030（β-羟基丁酸聚合酶）,Arcy₂759（聚β-羟基丁酸解聚酶）,Arcy₀824（核酮糖二磷酸羧化酶）,Arcy₀626（乙酰辅酶A合成酶）, Arcy₁318（硫酸腺苷转移酶,亚基2）Arcy 2800（磷酸腺苷磷酸硫酸酯）,Arcy₂799（亚硫酸盐还原酶p亚基）,Arcy₀256（ABC型周质铁转运蛋白复合体）在限制性葡萄糖异养的混合养条件下较自养和异养条件均有明显上调。表明在0.1%的有限葡萄糖浓度的混合养情况下,细胞的PHB代谢相关基因更为活跃且利用了空气中的CO2进行了PHB的累积,意味着少量葡萄糖可以促进菌株DX1-1以自养方式积累PHB。更多还原

【Abstract】 Poly-β-hydroxybutyrate （PHB） is a kind of polyester that stored in microbial cells. It is a ideal materiel for producing "bio-degradation plastic". Acdiphilium cryptum DX1-1 （CCTCC M208056） was isolated from acidic mine drainage, a site strictly lacking in onganic nutrients. It is a faculty strain capable of accumulation intracellular PHB with ferric iron reduction and sulfur oxidation as well as heterotrophic metabolism.In order to explore the genes related PHB synthesis of it and clarify the molecular mechanism of the effect of culture conditions on PHB accumulation of it, the present work focusing on A. cryptum DX1-1 accumulating PHB are as follows:the growth and accumulation of PHB in heterotrophy, autotrophy and mixotrophy that were provided glucose, sulfur and/or ferric iron, and glucose+sulfur and/or ferric iron as the energy substrates, respectively; cloning, sequencing and homology comparison analysis of related genes of A. cryptum DX1-1 metabolic system; related genes were investigated by RT-qPCR analysis of the differential expressions of genes encoding the key enzymes of PHB, carbon, sulfur, and ferrum metabolisms in heterotrophy, autotrophy and mixotrophy as different energy substrates. And the primary results are as follows:A. cryptum DX1-1 grows well and accumulates great amount of PHB in heterotrophic condition but grows slow and doesn’t accumulate PHB in autotrophy, and it can not grow in ferric iron as its only energy source. After adding limited organic carbon source like 0.1% glucose, DX 1-1 also grows better and accumulates more PHB in sulfur and/or ferric iron as the energy substrates than with the some concertration glucose in heterotrophy. The cell amount is up to 9.89×108mL, and the accumulation of PHB is 2.07 g/L in mixtrophic condition as sulfur and ferric iron as the energy substrates. In all conditions, In these culture conditions, the growth and PHB production of the strain decreased in the order in terms of energy substrates:GSF>GS>GF>G>>S, SF>>F （abbreviations see Table 2-2）. Referring to genome of A. cryptum JF-5 （JGI, http://genome.orn 1.gov/cgi-bin/JGI_microbial/kegg categories.cgi）, twenty-six useful homologous genes as well as the 16S rDNA gene to A. cryptum DX1-1 were successfully cloned, including 6 genes of PHB metabolism,10 gemes of carbon metabolism,7 genes of sulfur metabolism and 3 genes of ferrum metabolism. The sequences of the PCR products of these genes are basically matched to that of strain JF-5.After RT-PCR analysis of the differential expressions under different energy source, the result showed that in the mixotrophic cultures all the key enzyme-encoding genes of PHB, carbon, sulfur and ferrum metabolisms are basically up-regulated, especially PHB polymerase （Arcy₃030）, isocitrate dehydrogenase （Acry₀565）, Rubiscase （Acry₀565）, ribose 5-phosphate isomerase （Acry₁272）, ribulose 5-phosphate 3-epimerase （Acry₀827）, phosphoadenosine phosphosulfate reductase （Arcy₂800）, and sulfite reductase （Arcy₂799） encoding genes were significantly up-graduated.更多还原