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题名	产3-羟基丙酸皱褶假丝酵母丙酰辅酶A脱氢酶的研究及蛛丝蛋白异源表达改造
作者	周凤丽
学位类别	博士
答辩日期	2012-05
授予单位	中国科学院研究生院
授予地点	北京
导师	咸漠研究员
关键词	3-羟基丙酸 丙酰辅酶A脱氢酶 克隆 表达 蛛丝蛋白 静电纺丝 机械力学性能
学位专业	生物化学与分子生物学
中文摘要	3-羟基丙酸（3-HP）与乳酸互为同分异构体，是近年来兴起的一种重要平台化合物。与化学法合成3-HP相比，微生物发酵具有操作简单、条件温和、副产物少、生产成本低及绿色环保等优点，显示了较好的应用前景。目前，在微生物体内主要有7条代谢葡萄糖生成3-羟基丙酸的途径被提出，其中，新型β-氧化途径，即glucose → propionic acid → propionyl-CoA → acrylyl-CoA → β-hydropropionyl-CoA → 3-HP被认为是最有前景的一条。前期研究过程中，通过菌种筛选，并结合相关诱变手段，我们获得了一株可直接发酵葡萄糖产3-羟基丙酸的皱褶假丝酵母（Candida. rugosa）突变菌株；发酵初期添加1%（w/v）的丙酸，菌体干重受影响不大，发酵液中3-羟基丙酸可大量积累（达到18 g/L左右），且丙酸积累有所下降；而对照实验发酵后期3-羟基丙酸仅积累5 g/L左右。我们推测这可能是发酵前期丙酸添加强化了3-羟基丙酸合成途径的代谢通量的结果。结合目前已被揭示的皱褶假丝酵母胞内的丙酸β-氧化途径，我们继续推测3-羟基丙酸代谢通量的强化可能与丙酸调控丙酰辅酶A转化生成丙烯酰辅酶A的酶（丙酰辅酶A脱氢酶）的活力有关。通过cDNA RACE方法首次克隆了皱褶假丝酵母丙酰辅酶A脱氢酶（PACD）基因全长。首先根据GeneBank已报道的源自其它微生物（如假丝酵母，羧菌等）的酰基辅酶A脱氢酶基因序列高同源区设计简并引物，以皱褶假丝酵母的基因组DNA为模板进行PCR扩增，我们获得了一条大小为514 bp的片段，测序分析和GenBank比对结果证实该片段为皱褶假丝酵母丙酰辅酶A脱氢酶基因中间段序列；根据已扩增的丙酰辅酶A脱氢酶基因中间序列设计特异性引物，采用RACE试剂盒扩增目标基因5’端和3’端，通过测序，我们分别获得了614 bp和820 bp的5’端和3’端；通过拼接中间段、5’和3’端序列，获得了1408 bp的全长cNDA序列；根据cDNA全长序列设计特异性引物进行头尾PCR扩增出PACD的开放阅读框1329 bp，编码442个氨基酸。通过测序以及软件预测，序列包含有两个保守性的活性位点“KHYAEGAGY”和“IHHCMRMIGV”，两个糖基化位点“NISC”和“NHSL”。采用Invitrogen公司的pPIC9K载体以及相应的Pichia pastoris GS115表达宿主菌株，将目标基因进行异源表达。SDS-PAGE结果显示，重组菌胞内总蛋白在49 kDa处有条特异性的条带出现，而对照没有。通过Ni柱纯化，重组菌P. pastoris（PACD-pPIC9K-6His）在49 kDa处有一条单一的条带，而对照没有，这和软件预测的结果是一致的。Western blotting结果进一步表明，该49 kDa的特异性条带就是重组目的蛋白6His-PACD。我们对PACD进行动力学参数及酶学性质表征。根据双倒数法作图，通过计算可得出该酶的特征常数：Vmax＝1/1.4414=0.693±0.1 U·mg-1·min-1，Km=58.902/1.4414=40.86 ± 5μM，k cat=0.566 (s-1)；在其它条件一定下，酶反应速率随温度变化的结果如下：以30°C测得的相对酶活力为100%，在25°C~30°C，活性逐渐增加，30°C达到最大，大于30°C酶活逐渐下降，大于60°C时，酶活几乎降为0；以pH7.0测得的相对酶活力为100%，在pH 6.5~7.0之间，酶活逐渐增加，pH大于7.5时酶活逐渐降低；我们考察了常见金属离子对该重组酶的作用，结果显示镁离子对酶有一定的激活作用，钙离子，锰离子等对酶有较弱的抑制作用；该酶在30°C，40°C，50°C下处理2 h后相对残余酶活为76.32%，30.11% 和 4.32%。通过斑点杂交检测了皱褶假丝酵母胞内丙酰辅酶A脱氢酶在丙酸钠/未添加丙酸钠诱导条件下，不同阶段（12 h，24 h，36 h，48 h，60 h）的mRNA表达状况。首先提取皱褶假丝酵母不同发酵阶段总RNA（同时添加和不添加丙酸钠为对照）并反转录为cDNA，以RACE PCR获得的PACD ORF为探针进行地高辛标记，和各阶段cDNA进行膜杂交。初步获得的结果是：皱褶假丝酵母各发酵阶段总RNA随着发酵时间的增加与PACD探针杂交信号逐渐增强，前期36 h由于处于生长阶段，杂交信号较弱，但48 h之后杂交信号逐渐增强直至稳定；同时，添加丙酸钠各阶段杂交信号明显高于未添加丙酸钠。该结果验证了前期丙酸添加强化代谢通量的推测。本研究有助于阐明皱褶假丝酵母3-羟基丙酸代谢途径。在葡萄糖产3-羟基丙酸的新型β-氧化代谢途径中，PACD催化关键步骤丙酰辅酶A生成丙烯酰辅酶A，PACD编码基因的克隆及其重组酶酶学性质的测定将有助于进一步了解和优化生物质糖产3-羟基丙酸的代谢过程。本论文的另外一部分实验针对重组蛛丝蛋白进行了研究。蜘蛛丝是一种天然蛋白质纤维，具有高强度、高弹性、高断裂能等机械性能以及显著的可降解性、组织相容性等生物学特性，在生物医学、材料、纺织和军事装备等领域均有重大潜在应用价值。通过对蛛丝蛋白原始序列11R26的定点突变（部分序列突变为半胱氨酸、异亮氨酸和赖氨酸），利用基因工程的手段，大肠杆菌过量表达和AKTA纯化系统Ni柱纯化带有6His标签的重组蛛丝蛋白，获得了突变的重组蛛丝蛋白ZF4/ZF5/ZF6。同时我们通过分子生物学的手段将其基因序列加倍，获得了其高倍体6倍体的大肠杆菌表达产物。在5 L发酵罐水平进行发酵优化，使蛋白产量达到1 g/L左右。将Ni柱纯化的突变体蛋白进行去离子水透析，收集，冰冻干燥机冻干，-80oC保存。将冻干的蛛丝蛋白样品溶于强挥发性有机溶剂六氟异丙醇HFIP，浓度10% (100 mg/ml），注入1 ml注射器，静电纺丝，扫描电镜观察，结果显示蛛丝蛋白成丝的直径在1~2 µm左右。原子力显微镜(HARMONIX模式）检测未突变11R26和突变后的ZF4/ZF5/ZF6纳米纤维机械力学性能，结果显示，突变体ZF4 (ser-cys)蛋白丝的硬度比未突变前的蛋白丝硬度提高两倍左右；突变体ZF5/ZF6相对突变前硬度基本没有变化。通过比较蛋白丝纤维的弹性模量变化图，11R26弹性模量变化值大部分分布在小于0.6 GPa区域，最大弹性模量值约为1.385 GPa，平均弹性模量值为0.421左右；ZF4弹性模量值主要分布在0.8 以上区域，最大弹性模量值约为2.312 GPa，平均弹性模量值为1.347左右；ZF5弹性模量值主要分布在0.6 以上区域，最大弹性模量值约为1.305 GPa，平均弹性模量值为0.913左右；ZF6弹性模量值主要分布在0.8 以下区域，最大弹性模量值1.481 GPa，平均弹性模量值为0.585 GPa左右；上述结果说明突变后的ZF4/ZF5/ZF6比突变前蛋白丝的硬度和强度都有了一定提高。通过测定，突变体ZF4的二硫键数目比突变前增加了3.5倍左右，进一步验证了二硫键数目变化引起的力学性能的变化。
英文摘要	3-Hydroxypropionic acid, the isomer of lactic acid, is a promising platform chemical for the production of industrially important polymer materials. The biosynthetic pathway for 3-HP by microbial fermentation has shown good prospect for its mild operation conditions, few by-products and reduced environmental load as compared with the chemical process. Mainly seven metabolic pathways from glucose to 3-HP have been proposed in microorganism, among which, the modified β-oxidation pathway (namely propionic acid (PA) pathway, glucose → propionic acid → propionyl-CoA → acrylyl-CoA → β-hydropropionyl-CoA → 3-HP) is proposed as the most promising one. We obtained a mutant of C. rugosa which could directly produce 3-HP from glucose with PA as a selective inducer. The productivity of 3-HP was up to 18 g/L when 1% (w/v) PA was added in the fermentation media at the initial stage, while the control strain only produced 5 g/L of 3-HP. Combined with the modified β-oxidation pathway in C. rugosa, we continue to speculate that the enhancement of metabolic flux of 3-HP could be associated with the propionate regulation in the step from propionyl-CoA to acrylyl-CoA catalyzed by propionyl-CoA dehydrogenase (PACD).The propionyl-CoA dehydrogenase (PACD) gene was firstly cloned from C. rugosa by the cDNA RACE technique. Firstly, with C.rugosa genome as template, a 514 bp internal fragment was abtained by degenerate PCR with primers designed according to the high homologous sequences from yeast and bacterial; then 5’ and 3’ cDNA (614 bp and 820 bp) was abtained by designing specific primers according to the internal fragment; the full length cDNA (1408 bp) of PACD was contiged with internal, 5’cDNA and 3’cDNA fragments; finally, end to end PCR was processed with specific primers designed according to the full length cDNA to amplify the open reading frame (ORF). The ORF of the PACD gene has a length of 1329 bp, coding for 442 amino acids with the active sites of “KHYAEGAGY” and “IHHCMRMIGV” and two glycosylation sites “NISC” and “NHSL”.The cDNA of PACD was cloned into the expression plasmid pPIC9K and transformed into Pichia pastoris GS115. The recombinant intracellular total protein had a specific band at 49 kDa while no band was detected in the control by SDS-PAGE. The recombinant protein was purified by Ni-NTA affinity chromatography, and its size was observed to be approximately 49 kDa as estimated by SDS-PAGE. Anti-His antibodies were used to characterise the recombinant PACD by western-blot analysis. The recombinant protein retained the activity of catalysing propionyl-CoA to acryloyl-CoA. The kinetics of PACD was detected in detail. The K m, k cat and V max values of the purified PACD were calculated to be 40.86 μM, 0.566 (s-1) and 0.693 U·mg-1·min-1. The optimal temperature and pH of the purified PACD were 30°C and 7.0, respectively. Mg2+ had an activating effect on the enzyme, while Mn2+, Ca2+, Zn2+ and Cu2+ had slight inhibition effects. The recombinant PACD maintained 76.3%, 30.1% and 4.3% of its original activity after 2 h incubation in standard buffer at 30°C, 40°C and 50°C, respectively. We detected the transcription levels of the PACD at different stages (12 h, 24 h, 36 h, 48 h, 60 h) of the fermentation experiments (with and without propionate induction) in C. rugosa. Total RNA at different growth stages was extracted and reverse transcripted to cDNA. The PACD ORF was probed with Digoxin and hybridized with cDNA at different stages. The results of dot-blotting hybridisation using a PACD cDNA probe indicated that the PACD mRNA level was modified at different stages: mRNA levels were low for the first 36 h, then increased through 48 h and eventually reached a stable level; the hybridization signal was greater in propionate induction group than the signal in control group without propionate treatment at different stages. These results indicate that propionate induction could significantly activate PACD mRNA expression.Information from this study will be helpful in elucidating the metabolic pathway for 3-hydroxypropionic acid production in C. rugosa. Since PACD catalyzed the key step (from propionyl-CoA to acrylyl-CoA) in the modified β-oxidation pathway from glucose to 3-Hydroxypropionic acid (3-HP), the integration of recombinant PACD could benefit effective production of 3-HP from the most abundant biomass – sugars.The other objective of theis thesis is to research the recombinant spider silk protein and its physical properties. Spider silk is a natural protein fiber with high strength, high elasticity and significant degradation, tissue compatibility and other biological characteristics, thus has promising value in the biomedical, materials, textiles and other areas of military equipment. ZF4/ZF5/ZF6 were obtained by site-directed mutagenesis from the original sequence of spider silk protein 11R26. ZF4/ZF5/ZF6 and 11R26 were both expressed in E.coli and purified by Ni column, also the diploid form of the recombinant ZF4/ZF5/ZF6 and 11R26 were obtained by doubling their original ORFs in the expression plasmids. The recombinant protein production reached 1 g/L by 5 L fermentation optimization.The purified protein was dialyzed with ddH2O, freeze drying and dissolved in HFIP with the concentration of 100 mg/ml and then injected it in the 1 ml syringe and manufactured by electrospunning. Scanning electron microscopy showed that the diameter was about 1~2 μm. The mechanical properties were detected, which showed that the hardness of ZF4 was two times the increase compared with 11R26, while ZF5/ZF6 changed slightly in physical properties compared with 11R26. By comparing the changes in the elastic modulus map, the elastic modulus values of ZF4 mainly focused in the area above 0.8 GPa, the maximum elastic modulus was about 2.312 GPa and the average was 1.347 GPa, while the 11R26 mainly distributed in the area below 0.6 GPa, the maximum elastic modulus was about 1.385 GPa and the average was 0.421 GPa; the elastic modulus values of ZF5 mainly focused in the area above 0.6 GPa, the maximum was 1.307 GPa and the average was 0.913 GPa; the elastic modulus value of ZF6 was below 0.8 GPa, the maximum was 1.481 GPa and the average was 0.585 GPa. These results indicated that the hardness and strength of the mutants were higher than non-mutant 11R26. The number of disulfide of mutant ZF4 had a increase of about 3.5 times compared with non-mutant, which conformed the mechanical improvement caused by the change of disulfide number.
语种	中文
学科主题	生物基化学品
公开日期	2012-11-13
内容类型	学位论文
源URL	[http://ir.qibebt.ac.cn:8080/handle/337004/1351]
专题	青岛生物能源与过程研究所_材料生物技术研究中心
推荐引用方式 GB/T 7714	周凤丽. 产3-羟基丙酸皱褶假丝酵母丙酰辅酶A脱氢酶的研究及蛛丝蛋白异源表达改造[D]. 北京. 中国科学院研究生院. 2012.

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