【作者】 朱汇源；

【导师】 汪天虹；

【作者基本信息】 山东大学 ， 微生物遗传与分子生物学， 2010， 博士

【摘要】 植物细胞壁主要由纤维素、半纤维素及木质素等物质组成。木聚糖是植物半纤维素的重要组分,是自然界中除纤维素外最为丰富的多糖,也是自然界中主要的可再生资源。随着人口增长和资源消耗,对于可再生资源的开发与利用成为学术界共同关注的问题。丝状真菌黑曲霉作为具有重大经济价值的植物物质水解酶工业生产菌株已有多年历史,其全基因组测序已经完成。黑曲霉生产的半纤维素酶和降解复杂多糖的其它水解酶类已被广泛应用于食品、纺织和制浆造纸工业等行业。黑曲霉能够产生高活力的木聚糖酶。丝状真菌木聚糖酶基因已经大量克隆并进行了表达,研究的水平已深入到分子水平,而且对基因表达调控也进行了一定的研究,但是研究主要针对单基因或几个基因,从基因组水平和蛋白组水平上进行全面地、系统性地研究的很少,无法反映出丝状真菌木聚糖分解代谢的全貌,而且至今对木聚糖酶的分子诱导机制了解还比较少。因此,从基因组水平上研究丝状真菌半纤维素分解在基础理论与应用开发研究中都显得很有必要。其中一些重要新基因功能的阐明有助于明晰黑曲霉木聚糖酶的诱导机理、代谢网络调控或重要的信号转导机制,对优化黑曲霉的发酵、产酶过程及菌株的定向遗传改造将具有积极的推动作用。本文以黑曲霉为研究对象,采用抑制性消减杂交技术对在木聚糖诱导下及未诱导下进行基因差异表达的研究。目前对丝状真菌的宿主表达系统主要集中在Trichoderma reesei和A. niger,关于产黄青霉的研究较少,主要是对其抗生素的合成调控进行了细致深入的研究。产黄青霉是许多酶制剂和抗生素等的工业生产菌,能分泌合成蛋白酶、木聚糖酶和葡萄糖氧化酶。它具有极好的合成蛋白和分泌蛋白的能力,当在含有1%木聚糖的培养基中培养时,发酵液中木聚糖酶的含量高达40%,这表明产黄青霉的木聚糖酶启动子属于强启动子,能够向胞外分泌大量的蛋白,因此产黄青霉是优良的真核生物基因表达系统,是一种潜在的“细胞工厂”。另一方面,产黄青霉纤维素酶活性极低。这些特点对于表达用于改善纸浆质量和生物漂白的碱性木聚糖酶基因是非常有利的,本文究选用产黄青霉作为表达的宿主系统高效对来自嗜碱杆菌的木聚糖酶基因进行表达。本文的主要研究内容如下：1.高产木聚糖酶黑曲霉菌株的诱变及培养条件的优化采用UV和硫酸二乙酯（DES）对黑曲霉进行诱变,筛选到一株酶活达6235U/ml的突变菌株,编号为An-19。对其菌落形态进行了扫描电镜观察,发现与出发菌株相比,突变株的分生孢子呈现棕黄色,体积偏大,菌丝直径增粗。生长速度快,3 d就能覆盖整个平板。对突变株和出发株的木聚糖酶基因进行了克隆,测序发现两者基因的相似度为100%,说明诱变没有改变木聚糖酶基因的一级结构。对突变株的遗传稳定性进行了研究,发现经10代后突变株的木聚糖酶活性保持不变,说明突变株是一株产木聚糖酶稳定的菌株。采用中心组合试验和Design-Expert 7.0软件对突变株的培养条件进行响应面优化,结果得到最佳培养基为麸皮20g/L, NH4NO3 10g/L和碳酸钙20g/L,优化条件下酶活高达8516U/mL,比出发菌株提高56.3l%。2.黑曲霉抑制性消减杂交文库的构建及差异序列的分析为了分离和鉴定黑曲霉木聚糖代谢相关基因,以经过1.0%木聚糖诱导的菌丝体为实验组,未诱导的为驱动组,用抑制消减杂交法（SSH）构建了黑曲霉木聚糖诱导正向消减文库。菌落PCR显示插入片段在250～1000 bp之间,逆向斑点杂交验证黑曲霉消减文库质量良好。从黑曲霉木聚糖诱导的消减文库中,随机选取了119个不同插入片段的阳性克隆进行测序。测序结果经BLASTx分析,表明这些序列大部分与丝状真菌木聚糖代谢的相关酶基因具有较高的相似性。总共筛选出41个有效的差异表达基因cDNA片段,其中有7个cDNA片段编码转运蛋白,占测序克隆的5.9%,包括糖转运蛋白编码基因4个,分别为木二糖转运蛋白、木糖转运蛋白、阿拉伯糖转运蛋白和己糖转运蛋白基因。共有22个cDNA片段编码水解酶,占测序克隆的18.4%；其中,编码糖苷水解酶的基因有12个,包括内切木聚糖酶A和B、β-木糖苷酶、阿拉伯糖苷酶、α-半乳糖苷酶、β-葡萄糖苷酶、D-木酮糖激酶,以及木糖醇脱氢酶、醛糖差向异构酶、内切葡聚糖酶A、α-葡萄糖醛酸酶和阿魏酸酯酶基因。搜索黑曲霉的基因组序列,获得特异表达序列的全长序列。对完整基因序列分析发现,特异表达的糖苷水解酶基因除了阿拉伯糖苷酶和β-葡萄糖苷酶基因外,其余基因的启动子区域中均含有特异序列5’-GGCTAA,而该序列可以和XlnR发生特异性结合,从而增强木聚糖酶系基因的表达。此外,发现纤维素酶系中的内切葡聚糖酶Ⅲ和β-葡萄糖苷酶基因启动子区也含有5’-GGCTAA序列,表明受到XlnR的调控,这说明XlnR除了作为木聚糖酶转录激活因子外,还参与调控纤维素降解酶部分基因的转录调控。对筛选出来的6个木聚糖水解酶基因进行了表达分析,结果在诱导组RT-PCR中均扩增获得明亮的PCR条带,试验结果表明这6个基因是在木聚糖诱导下能够进行特异表达,而在以葡萄糖为唯一碳源的培养基中培养时则基本不表达。特异基因RT-PCR结果进一步验证了所构建黑曲霉消减文库的质量。黑曲霉消减文库中分离得到的木聚糖特异表达基因的分离为深入研究木聚糖诱导与代谢调控奠定了基础。3.产黄青霉胞外蛋白酶的分离纯化及蛋白酶缺陷型菌株的构建采用硫铵分级沉淀、DEAE离子交换层析和分子筛层析对产黄青霉FS010的胞外蛋白酶进行初步的分离。SDS-PAGE电泳分析显示产黄青霉FS010菌株所产的胞外蛋白酶种类较少,在分子量约43kDa处有1条明显的蛋白带,对分离纯化的蛋白酶的酶学性质进行研究,研究表明该蛋白酶的最适作用温度为35℃,最适作用pH为9.0。蛋白酶抑制剂的研究显示该酶属于丝氨酸蛋白酶。以黑曲霉、微紫青霉的天冬氨酸蛋白酶PepA作为检索内容,对Penicillium chrysogenum的pepA同源基因进行搜索。结构搜索到1个基因编号为8313187,蛋白编号为XP₀02567415.1的基因,将其命名为pepA,该基因初步认为编码天冬氨酸蛋白酶A,属于酸性蛋白酶。将搜索到的基因片段的上游非编码区和下游区也进行整理,最终获得pepA的全长序列,其中上游非编码区长度为1099bp,下游非编码区为563bp。体外成功构建了产黄青霉天冬氨酸蛋白酶基因pepA敲除载体pApepA,并将其转化产黄青霉原生质体,成功筛选获得3株转化子,经过PCR验证和Southern杂交证实这3株转化子的pepA基因已经被敲除。对3株缺转化子的发酵上清中总蛋白及蛋白酶活性进行了测定,证实转化子ΔPEP14的胞外蛋白酶活性降低最显著,相比出发菌株降低了80.9%；而另外两株转化子的胞外蛋白酶活性比出发菌株分别降低了61.2%和40.2%。酶活测定结果表明产黄青霉天冬氨酸蛋白酶A的缺失能降低发酵液胞外蛋白酶活性,但发酵液总蛋白含量保持相对恒定。对pepA缺失转化子ΔPEP14的遗传稳定性进行了研究,结果显示ΔPEP14转化子具有较好的遗传稳定性,传10代后仍能保持较低胞外蛋白酶活性。4.碱性木聚糖酶基因在产黄青霉蛋白酶缺陷菌株中的分泌表达根据产黄青霉中密码子的偏爱性,利用Codon-Adaptation tool对来自于嗜碱杆菌碱性木聚糖酶基因序列xyl进行密码子优化。优化后的xyll序列的CAI值为0.99,GC含量为60.94%,比出发序列提高了61.2%。Blast分析发现优化后的序列和原始序列核酸相似性为74%。以质粒pUCl9为载体骨架,选用产黄青霉酸性木聚糖酶基因（xyn）的强启动子Pxyn及终止子Txyn序列构建细菌碱性木聚糖酶基因表达盒,成功得到重组表达载体pxyl-1。将pxyl-1转化产黄青霉原生质体,筛选获得4株转化子。Southern杂交及RT-PCR分析证实这4株转化子的碱性木聚糖酶基因xyll能够进行特异表达。对产黄青霉pxyl-1转化子XY35所产的木聚糖酶的酶学性质进行了初步研究。结果证实重组碱性木聚糖酶在产黄青霉中可以分泌到胞外,用产黄青霉pxyl-1转化子XY35发酵上清液进行了SDS-PAGE分析和碱性木聚糖酶活性测定。试验结果显示,与出发菌株相比较,转化子XY35胞外蛋白样品在约4lkDa处出现了1条蛋白带。酶活测定结果显示,出发菌株木聚糖酶活为254.5 U/mg,上清液总蛋白含量为0.42 mg/mL,而转化子为1217.9 U/mg,发酵上清液总蛋白含量为0.48 mg/mL。凝胶扫描分析表明木聚糖酶占胞外总蛋白的15.9%。对转化子XY35的木聚糖酶粗酶液与出发菌株所产酸性木聚糖酶的性质进行了比较分析,结果显示重组碱性木聚糖酶最适作用pH值为8.0,而出发菌株所产的酶为6.0。重组酶稳定pH范围在6.0-11.0之间,在3-9.5范围内可保留80%以上酶活力,碱稳定性较强,该菌株所产碱性木聚糖酶可望用于纸浆的预漂白。重组木聚糖酶和出发菌株的酸性酶的最适作用温度均为40℃C,温度超过50℃后,重组酶的活力丧失大部分活性,到55℃,仅保留原有活性的42.3%,其耐热稳定性需要进一步提高。本文利用产黄青霉作为宿主过表达外源基因的研究为产黄青霉的工业育种提供了试验范例和进行了有益的探讨。高产碱性木聚糖酶菌株的构建使其在环境保护、生物制浆和其它生物技术过程中具有潜在的应用前景。更多还原

【Abstract】 Plant cell walls are mainly composed of cellulose, hemicellulose and lignin and other substances. In addition to cellulose, xylan, a principal component of plant hemicellulose, is the second most abundant polysaccharide in nature. It is also the main renewable natural resources. With the increase of population and resource consumption, the exploitation and utilization of renewable resources had become academic issues of common concern.The filamentous ascomycete Aspergillus niger is a well-known over-producer of enzymes, in particular plant cell-wall hydrolyzing enzymes, such as cellulases and xylanases. The full-genome sequencing of A. niger had been completed. Hemicellulase and other hydrolases to degrade complex polysaccharides produced by the strain had been widely used in food, textile and pulp and paper industry and other industries. A. niger has a considerable strong xylanase activity. Filamentous fungal xylanase gene has been extensively cloned and expressed in several host systems. Studies on xylanase have been refined to the molecular level, and some research on xylanase gene regulation had been performed. However, the study mainly focuses on a single gene or several genes. Comprehensive and systematic research on xylanase regulation at the genomic and proteomic level is not very detailed, therefore it can not reflect the general view of the xylan metabolism in filamentous fungi and only a few researches on the molecular mechanism of xylanase induction had been carried out. Consequently, research on hemicellulose catabolism of filamentous fungi at the genomic level is very necessary in fundamental research and practical application. The function clarification of some novel genes will contribute to understand xylanase induction mechanism and regulation of metabolic networks or important signal transduction mechanism in A. niger. In this paper, suppression subtractive hybridization （SSH） protocol was used to study the differential gene expression under xylan induction and non-induction with A. niger as materials.Aspergillus niger and Trichoderma reesei have been extensively used as model organisms for diverse transformation and expression systems, only a few research on Penicillium chrysogenum was performed. The detailed research mainly focused on the regulation ofβ-lactam antibiotics. The filamentous fungus P. chrysogenum is well known by its ability to synthesizeβ-lactam antibiotics like benzylpenicillin and isopenicillin N as well as other secondary metabolites, and it could secrete various kinds of protease, xylanase and glucose oxidase. The strain has outstanding ability to synthesize and secrete different proteins. The content of xylanase was 40% when the strain was cultured in the medium containing of 1% xylan, indicating that the promoter of xylanase gene from P. chysogenum belonged to strong promoter and it could secrete large amounts of protein into the fermentation broth. Therefore, P. chysogenum is an excellent eukaryotic gene expression system and a potent cell factory for white biotechnology. In addition, cellulase activity of the strain is very low. These features are beneficial to the expression of alkaline xylanase gene to improve the quality of pulp and bio-bleaching. In this paper, P. chrysogenum as a host system is used to high-efficiently express the xylanase gene from alkalophilic Bacillus.The main research works in this thesis are as follows:1、Mutagenesis and optimization for the production of xylanase in A. nigerIn order to screening the mutants with higher xylanase activities, the mutation breeding of the strain NA1003 was carried out by ultraviolet and diethyl sulfate （DES）mutagenesis. A mutant strain whose xylanase activity is 6235U/ml was selected and numbered as An-19. The colonial morphology was observed by scanning electron microscope. The result showed that the conidia of the mutant strain are brown and the conidia volume and hyphal diameter become larger compared with original strain. The mutant strain grows fast and can cover the plate in 3 days. The xylanase genes from the original and mutant strain were cloned and sequenced. The sequencing results has showed that the identity of the gene is 100%, indicating that the mutation does not change the primary structure of xylanase gene. The genetic stability of mutant stain An-19 was performed and the results showed that the strain was a stable strain to produce a large number xylanase. The culture conditions of mutant strain were optimized by response surface analysis using Center Composite Design （CCD） and Design-Expert 7.0 software. The culture medium were optimized as follows:wheat bran 20 g/L, NH4NO3 10 g/L and CaCO3 20 g/L. The xylanase activity was up to 8516U/mL under the optimum conditions.2、Cloning and sequence analysis of differentially expressed genes from A. niger induced by xylanTo identify xylan-related genes in A. niger, suppression subtractive hybridization （SSH） was performed to generate a subtracted cDNA library between induced （Tester） and non-induced A. niger （Driver） using the PCR-SelectTM cDNA Subtraction Kit according to the manufacturer’s protocol. PCR analysis showed that the length of inserted fragments in white bacteria clones were in the range of 250-1 000 bp. The quality of SSH library was identified to be favorable by reverse dot blotting.119 positive clones with different inserts was randomly selected and sequenced. The sequencing results were analyzed by BlastX software, indicating that the different expressional cDNA fragments are mostly relate to the xylanase gene. A series of ESTs of 41 kinds of proteins including sugar transporter, xylan hydrolymes, transcriptional factor and other functional proteins as well as unknown proteins, were isolated from the subtractive libarary.7 cDNAs encoding 4 kinds of sugar transporter including xylobiose transporter, xylose transport, arabinose transport and hexose transporter accounted for 5% of the total sequences.22 cDNAs encoding 12 kinds of xylanases including endo-xylanase A and B,β-xylosidase, arabinosidase,α-galactosidase,β-glucosidase, Xylulokinase, as well as xylitol dehydrogenase, aldose epimerase, endoglucanase C, a-glucuronidase, and ferulic acid esterase accounted for 18.4% of the total sequences. The full lengths of specific genes were cloned by searching the genomic data of A. niger. The sequence analysis of full-length genes showed that the promoter regions of enzyme-coding genes except arabinosidase bandβ-glucosidase had a specific sequence of 5’-GGCTAA, which could be specificially binded by XlnR and consequently enhance the expression of xylanase system. In addtion, XlnR not only could be used as the transricptional factor of xylanase, but also involved in the transcripton regulation of part of celluase. Six specific genes were analyzed by RT-PCR and the results revealed that six genes was expressed under the induction of xlan. RT-PCR analysis further confirm that the qulity of constructed SSH libarary was good. The construction of SSH library and cloning of differential genes have established a solid foundation for cloning xylanase-relevant genes and further studying xylanase induction mechanism from filamentous fungi.3、Purification of extracellular protease from P. chrysogenum and construction of protease-deficient strainAn extracellular alkaline serine protease from P. chrysogenum FS010 has been purified. The purification procedure involved:ammonium sulfate precipitation, DEAE ion-exchange chromatography and sephadex G-100 gel chromatography. SDS-PAGE of the purified enzyme indicated a molecular weight of about 43,000Da. The protease has a maximum activity at pH 9.0 and 35℃. PMSF and DFP are its specific inhibitors, indicating the enzyme blongs to serine protease.A TBLASTN search using PEPA from A. niger and P.janthinellum, as query identified a single orthologous gene in the genome sequence of P. chrysogenum. The gene （gene id:8313187, protein id:XP₀02567415.1） was named pepA. The promoter and terminator of pepA from P. chrysogenum were also cloned. The promoter region of pep A was 1099bp, and the terminator region of pep A was 563bp. To knock-out the pep A from P. chrysogenum, a deletion vector pApepA was constructed in which the complete coding region of pep A was replaced by a phleomycin resistance expression cassette. The deletion vector pApepA was amplified by PCR and transformed into the P. chrysogenum strain FS010. Three transformants were screened and shown to be deleted in the pepA gene by the PCR amplification and Southern blotting. Protease activities of supernatant from three transformants were measured and the results showed that the protease activtity of transformantΔPEP14 was the lowest, which was 80.9% lower than of control strain and the other two transformants were 61.2%和40.2% lower than of control strain, respectively. The measurement of protease activity showed that the absence of aspartic proteinase of P. chrysogenum could decrease the major extracellular protease activity of fermentation broth, but the content of total protein in fermentation broth remained stable. The genetic stability of transformantΔPEP14 was studied and the result showed that transformantΔPEP14 was a genetic stable strain with low protease activity.4、Secretion expression of an alkaline xylanase gene in protease-deficient Penicillium chrysogenumAccording to the codon usage bias in P. chrysogenum, Codon-Adaptation tool was performed to adapt the alkaline xylanase gene xyl from alkalophilic Bacillus. The adapted xyl1 sequence of CAI value was 0.99 and GC content was 60.94%, which was 61.2% higher than that of starting sequence. Blast analysis showed that the sequence similarity of the adapted xyl1 with the original xyl was only 74%.The plasmid pUC19 was used as the carbon backbone of the expreesion vector. The strong xylanase promoter （Pxyn） and terminator sequences （Txyn） from P. chrysogenum were used to express bacterial alkaline xylanase gene, resulting the expression vector pxyl-1. The expression vector pxyl-1 was amplified and transformed into the P. chrysogenum strainΔPEP14. Four transformants were screened and confirmed by PCR amplification and Southern blotting. Southern blot and RT-PCR analysis further confirmed that four transformants contained the alkaline xylanase gene xyl1 and the gene could be specifically transcribed in transformants.The enzymatic properties of recombinant xylanase produced by P. chiysogenum transformants XY35 were preliminary studied. The results confirmed that recombinant xylanase could be secreted into the culture. SDS-PAGE of the extracellular proteins indicated a molecular weight of about 41,000Da. The xylanase activity of control strain was 67.4 U/mg, whereas enzyme activity of transformant XY35 was 572.4U/mg. The content of total protein of fermented broth is 3.45mg/mL. Gel scanning analysis showed that the extracellular xylanase accounted for 15.9% of total protein.The properties of crude enzyme from Transformant XY35 and original strain were preliminary studied and the results showed that the optimal temperature and pH for recombinant xylanase was 40℃and 8.0, respectively. The optimal temperature and pH of acidic xylanase from original strainΔPEP 14 was 40℃and 6.0, respectively. Comparison of characteristics of xylanase from transformant XY35 and original strain suggested that the alkaline xylanase gene had been successfully expressed. Recombinant alkaline xylanase showed nearly 80% of its maximal activity in the pH range 3.0-9.5, indicating that the recombinant xylanase could be used for pulp pre-bleaching. The recombinant enzyme lost most of the activity when the temperature exceeds 50℃, and retaining only 40% of the maximum activity at 55℃, indicating that thermal stability of the xylanase should be further improved. The research of gene expression and regulation in P. chrysogenum could provide the necessary theoretical basis for the industrial breeding of Penicillium. Alkaline xylanase has potential applications in environmental protection, bio-pulping process and other biotechnology.更多还原