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题名: 几个微生物次生代谢后修饰基因功能探索
作者: 耿召良
学位类别: 博士
答辩日期: 2009-02-16
授予单位: 中国科学院昆明植物研究所
授予地点: 昆明植物研究所
导师: 郝小江
关键词: 植物内生菌 ; 宏基因组文库 ; 异源表达 ; 美登木素生物合成 ; 酰基转移酶 ; FADH2依植物内生菌 ; FADH2依赖性卤代酶
学位专业: 植物学
中文摘要: 本论文主要对从本实验室构建的滑桃树(Mallotus nudiflorus)茎皮内生菌宏基因组文库中筛选到的两个FADH2依赖性卤代酶进行了克隆、异源表达和体外酶活性分析;另外,利用接合转移、异源表达、体外酶活性检测等手段对安丝菌素生物合成中的两个重要后修饰基因asm19(酰基转移酶基因)和asm12(卤代酶基因)进行了初步研究。 以滑桃树茎皮内生菌宏基因组文库为基础,应用FADH2依赖性卤代酶基因的简并引物,通过逐级PCR技术对其中约75000个单克隆进行了筛选,获得了13个FADH2依赖性卤代酶阳性克隆。对获得的阳性克隆进一步通过亚克隆和步移测序技术,以获得卤代酶基因的全长。但由于文库Fosmid插入片段的GC含量高、有高级结构等原因,仅获得了63D9Hal II和64E9Hal两个基因全长(序列提交GenBank,注册号FJ493227和FJ493228)。 用特异引物分别对63D9Hal II和64E9Hal进行了克隆,将两个卤代酶基因分别和三个表达载体连接构建了重组表达载体,分别命名为pKIB001(pRSET B/64E9Hal),pKIB002(pRSET B/63D9Hal II),pKIB003(pET28a(+)/64E9Hal),pKIB004(pET28a(+)/63D9Hal II),pKIB007(pET32a(+)/64E9Hal)和pKIB008(pET32a(+)/63D9Hal II)。将六个重组载体在BL21(DE3)pLysE中进行了表达,结果表达产物大部分以包涵体的形式出现。在对表达的温度、IPTG诱导浓度、培养基成分和表达菌株等进行因素改进后,表达效果仍然没有明显改善。 以Tryptophan、MCD、Proansamitocin和Tryptamine为底物进行了体外酶活性检测。LC-MS检测结果表明,除Tryptophan检测到了微弱的可能产物峰261外,其余三种底物均未能检测到相应的产物峰。另外,将63D9Hal II和64E9Hal在链霉菌表达宿主Streptomyces lividans T7 中进行表达,但没有发现明显的表达蛋白和体外活性。推测无明显体外活性主要是因为卤代酶基因是从宏基因组文库中获得的,对其表达中诸如基因来源背景、最适合表达菌株和天然底物等一无所知,从而造成了表达和活性检测的困难。 Asm19和Asm12在安丝菌素生物合成途径中分别执行酰基转移酶和卤代酶的功能,二者的功能和安丝菌素的生物活性有极大关系。通过接合转移成功将含有asm19的pHGF9071质粒转入其突变株Actinosynnema pretiosum ssp. auranticum ATCC31565/△asm19中,通过菌块抑酵母菌Filobasidium uniguttulatum实验和转化子发酵产物的LC-MS检测,结果证实所挑取的转化子和其野生型菌株ATCC31565类似,具有较强的抑酵母菌Filobasidium uniguttulatum活性,并在其发酵产物中检测到了AP1-4系列产物。转化子的抑菌活性不一致,可能是回补后基因插入突变体染色体的位置不一致所导致的。 对asm19进行了异源表达,并对影响表达的温度、时间和IPTG浓度等因素进行了优化。研究发现异源表达的Asm19对酰基CoA具有宽泛的适应性且可以表现相当高的活性,但对一些人工合成的脂肪酰基底物不表现活性;同时对该酶低温下的稳定性、影响粗酶反应的时间、温度因素等特性进行了初步研究。利用亲和层析技术对重组蛋白进行了纯化,对Wash buffer和Elution buffer进行了优化,经SDS-PAGE检测和Lab Works 4.6软件分析,发现纯化后Asm19表达蛋白的含量为45-48%。 另外,利用丙氨酸来源的人工合成底物HGsy14对野生型ATCC31565和asm19加强型ATCC31565-AP-2菌株进行饲喂实验。根据LC-MS分析,在不同浓度饲喂产物提取物中几乎均发现了预期主产物H5(Maytansinol-HGsy14),但该产物峰值很低,需要将饲喂后的产物进一步分离鉴定以证实实验结果。该饲喂实验的尝试对Asm19功能的拓展和对植物中美登木素类化合物起源问题的理解有重要参考价值。 同时,利用pET32a(+)对属于FADH2依赖性卤代酶基因的后修饰酶基因asm12进行了克隆和异源表达,发现该基因在37 ℃时的表达主要是以包涵体形式出现的,16 ℃诱导的可溶性效果优于37 ℃。利用亲和层析技术对Asm12表达重组蛋白进行了纯化,纯化后目的蛋白纯度已经达90%以上,为下一步利用化学动力学对该酶系统研究打下了基础。对Asm12重组蛋白进行了以Tryptophan和proansamitocin(天然底物)为底物的体外活性检测,结果没有发现Tryptophan预期的产物峰261,而在粗酶产物中发现了预期的proansamitocin产物峰,但其产物峰值较低,不能确定该峰是否是目的产物,说明表达的Asm12在体外的卤代催化活性不明显。 附论一主要通过尝试利用不同的固定剂,对滑桃树茎皮内生菌进行富集并提取DNA,利用基于16S rDNA 技术的微生物多样性检测等方法对所提取DNA的微生物多样性进行评价。该部分工作对于探索富集植物组织内生微生物的方法学有重要意义。附论二对采自云南西双版纳的楝科植物老虎楝(Trichilia connaroides)茎叶的化学成分进行了分离鉴定,共分离鉴定了10个化合物,包括三个新的四降三萜TrichilinA、Trichilin B和Trichilin C。
英文摘要: Two FADH2 dependent halogenases were cloned, heterologously expressed and characterized in vitro, screened out from the metagenomic library of microbiota enriched from the stem barks of Mallotus nudiflorus. Acyltransferase gene asm19 and halogenase gene asm12 proved to be involved in the post-PKS modifications of ansamitocin, were studied through conjugal transfer, heterologous expression and in vitro enzymatic characterization. A stepwise PCR screening approach was used on 75000 clones of the metagenomic library and thus 13 positive clones were isolated. As high GC content or complex spatial structures of fomsid in metagenomic library, through subcloning technique and walking sequencing, only 2 halogenase genes named 63D9Hal II and 64E9Hal were obtained (GenBank accession number: FJ493227 and FJ493228). With specific primers,63D9Hal II and 64E9Hal were cloned into 3 expression vectors respectively. Thus, six recombinant expression vectors were constructed, named pKIB001 (pRSET B/64E9Hal), pKIB002 (pRSET B/63D9Hal II), pKIB003 (pET28a(+)/64E9Hal), pKIB004 (pET28a(+)/63D9Hal II), pKIB007 (pET32a(+)/64E9Hal), pKIB008 (pET32a(+)/63D9Hal II). These recombinant vectors were transformed into BL21(DE3)pLysE and expressed, but the majority of expressed product were inclusion bodies. Though we try to optimized the temperature, IPTG concentration, medium and engineered strains, the result was not improved obviously. L-tryptophan、MCD、proansamitocin and tryptamine were used as substrates to detect the biological activity in vitro, but no obvious activity was detected except for a low peak of possible 7-chloro-L-tryptophan through LC-MS. In addition, 63D9Hal II and 64E9Hal were also expressed in Streptomyces lividans T7 strain, but no obvious expressing protein and biological activity were detected either. As we know nothing about the origin, the fittest expressing strain and natural substrates of the two new haolgenase genes, it is disadvantage to their heterologous expression and biological activity detection in vitro. Asm19 and Asm12 act as acyltransferase and halogenase in the post-PKS modifications of ansamitocin, which were proved to be very important to its biological activity. Through conjugal transfer, pHGF9071 containing asm19 was transformed into Actinosynnema pretiosum ssp. auranticum ATCC31565/△asm19. Analysis of antagonistic action to Filobasidium uniguttulatum and LC-MS detection of transformants approved their antibacterial activity and AP1-4 in their fermentation production, similar to wild type strain ATCC31565. Probably, the different inserted position of asm19 resulted in the difference of their antibacterial activity. asm19 was heterologously expressed,and similarly,the induction temperature, time and IPTG concentration were optimized. As a result, Asm19 has extensive adaptability to acetyl-CoA except some artificial synthesized fatty acyl enzyme substrate. The stability and influencing factors to crude enzymatic reactions were also investigated preliminarily. With affinity chromatography, the recombinant protein was purified, and the Wash buffer and Elution buffer were optimized. According to SDS-PAGE and analysis of Lab Works 4.6 software, the content of Asm19 in purified recombinant protein was about 45-48%. Besides, an artificial synthesized substrate named HGsy14 from alanine was fed to ATCC31565 and ATCC31565 AP-2. According to LC-MS analysis of product fed with different HGsy14 concentrations, anticipated main product H5 (Maytansinol-HGsy14) was observed, but the peak was very low. The final submission is to be confirmed by means of the product structure determination next. This is valuable for understanding of Asm19’s function and the origin of maytansinoids in plants. With pET32a(+) vector,asm12 (belonging to FADH2 dependent halogenase genes) was cloned and heterologously expressed. At the induction temperature of 37 ℃, Asm12 were expressed in the form of inclusion bodies, but more soluble protein was produced at 16 ℃. The recombinant protein was purified with affinity chromatography, and over 90% soluble pure protein was obtained. Analyzed through LC-MS, crude protein of Asm12 from E. coli BL21(DE3)pLysE possibly can convert proansamitocin to 19-chloroproansamitocin (low peak of 500 in LC-MS map) but with no activity on L-tryptophan (no peak of 261 in LC-MS map). As a result, Asm12 has no obvious biological activity to proansamitocin and L-tryptophan. Besides, with different fixation solution, endophytes in Mallotus nudiflorus were enriched and their DNA was extracted. Then, the 16S rDNA analysis was used to check whether this method could produce more endophytes pellet. It is meaningful to explore the method of enriching endophytes from plant tissues. In addition, 10 compounds were isolated from twigs and leaves of Trichilia connaroides, which were collected from Xishuangbanna ,Yunnan, China. Among them, TrichilinA、Trichilin B and Trichilin C are new compounds. Among these, Trichilin B is a limonoid having an unprecedented highly rearranged ring system.
语种: 中文
内容类型: 学位论文
URI标识: http://ir.kib.ac.cn/handle/151853/258
Appears in Collections:昆明植物所硕博研究生毕业学位论文_学位论文

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几个微生物次生代谢后修饰基因功能探索.耿召良[d].中国科学院昆明植物研究所,2009.20-25
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