【作者】 刘国栋；

【导师】 曲音波；

【作者基本信息】 山东大学 ， 微生物学， 2012， 博士

【摘要】 利用木质纤维素类生物质生产液体燃料和化合物是保障人类社会可持续发展的重要替代途径。在被广泛接受的技术路线中,来自真菌的木质纤维素降解酶系通常被用来将木质纤维素材料水解为可发酵的糖。然而,目前木质纤维素降解酶系的生产水平和水解活性都有待提高。丝状真菌斜卧青霉(Penicillium decumbens)已被用于工业水平的纤维素酶生产多年,经诱变选育的突变株的纤维素酶容积生产率与广泛使用的纤维素酶生产菌里氏木霉相当,且酶系组成更为合理。因此,对该真菌进行深入研究对降低木质纤维素降解酶系的生产成本、认识真菌中木质纤维素降解酶的合成调控机制具有重要意义。本论文的主要研究内容及结果如下：1.斜卧青霉野生株114-2的基因组测序与分析对斜卧青霉野生株114-2的基因组序列进行了测定,对木质纤维素降解酶及其它多个重要生理过程相关蛋白进行了人工汁释。比较基因组学分析显示,斜卧青霉尽管在系统进化上与产黄青霉较为接近,却含有较少的细胞代谢、调控相关蛋白,其功能蛋白数目与里氏木霉更为接近。相对于广泛使用的产纤维素酶真菌里氏木霉,斜卧青霉含有种类、数量更为丰富的木质纤维素降解酶编码基因,尤其是参与半纤维素降解的蛋白和含纤维素结合域蛋白的编码基因。在木质纤维素降解酶合成的调控机制方面,斜卧青霉与里氏木霉存在一些共性,但基因组角度的分析与功能实验也显示二者之间存在若干相关调控机制的差异。此外发现,斜卧青霉基因组上存在植物细胞壁降解酶编码基因密集分布的区域,部分木质纤维素降解酶编码基因可能来自进化中的水平转移、扩张事件。2.斜卧青霉野生株与木质纤维素降解酶高产株的比较基因组学与转录组学研究通过对斜卧青霉野生菌株114-2和木质纤维素降解酶高产突变株JU-A10-S、JU-A10-T进行比较基因组学分析,发现野生株114-2与两株突变株在基因组水平存在大量差异,而两株突变株之间仅存在少量的序列差异。推测最早分离的菌株114可能为异核体,且在后续的菌株分离、诱变选育中得到了纯化。对野生株与突变株之间含有氨基酸序列变异的蛋白的功能分析显示了转录因的富集,为进一步研究突变株的木质纤维素降解酶高产机理提供了参考。通过序列比对,还找到了突变株孢子色素合成能力缺失的可能原因。通过对野生株与突变株之间比较转录组学数据的分析,确认了分泌组学的研究结果,发现了菌株间若干胞内生物学过程表达水平的差异,为产木质纤维素降解酶真菌的进一步改造指出了方向。此外,确认了野生株、突变株以及另一株斜卧青霉Peni-1的交配型属性,为在斜卧青霉中探索有性杂交提供了可能。3.斜卧青霉内切-β-1,4-葡聚糖酶Cel45A与Cel5C的异源表达与重组酶的性质研究实现了来自斜卧青霉的内切β-1,4-葡聚糖酶Cel45A和Cel5C的活性重组表达,发现这两种酶水解纤维素的产物均主要为纤维寡糖。与已报道的多数真菌来源内切β-1,4-葡聚糖酶相比,rCel45A与rCel5C对CMC-Na的水解活性较低,可能与它们与底物的结合机制有关。与其它GH45家族蛋白类似,rCel45A具有较高的最适作用温度和较强的热稳定性,有望用于纤维素酶系的改良。此外,通过结构域缺失方法,发现第447位至655位的氨基酸在Cel5C水解活性的发挥中具有重要作用,其作用机制可能与货白稳定性的保持有关。4.斜卧青霉丝氨酸蛋白酶PrtB生物学功能的初步研究通过同源蛋白搜索,发现构巢曲霉中参与菌体自溶的向丁质酶ChiB、中性金属蛋白酶PepJ和内切-β-1,3-葡聚糖酶EngA在斜卧青霉中均存在直系同源蛋白,而碱性丝氨酸蛋白酶PrtA在斜卧青霉中不存在直系同源蛋白。基因敲除研究发现ChiB、PepJ和EngA在斜卧青霉对碳源饥饿的响应及纤维素酶诱导表达中可能不具有重要作用。与PrtA序列一致度最高的斜卧青霉丝氨酸蛋白酶PrtB的编码基因被敲除后,导致碳源饥饿条件下孢子发生受阻,胞外酸性蛋白酶活力上升。在纤维素酶诱导条件下,PrtB编码基因的敲除导致胞外多种蛋白产生水平的下降,但酸性蛋白酶活力与出发株相比未发现明显差异。更多还原

【Abstract】 The use of lignocellulosic biomass for the production of liquid fuels and chemicals has long been considered to be an important alternative for sustaining the human economy and society. In one of the most widely accepted biorefinery schemes, lignocellulosic materials are first hydrolyzed to fermentable sugars by fungal enzymes and then converted to various products. Currently, both the production level and the performance of lignocellulolytic enzymes still need to be improved to make the process economically competitive. The filamentous fungus Penicillium decumbens has been used for industrial-scale cellulase production in China for16years. Cellulase productivities of P. decumbens mutants are comparable with those produced by the widely used cellulase producer Trichoderma reesei. Furthermore, the enzyme system shows a more balanced protein composition than that from T. reseei. Thus, deep investigation of P. decumbens would be important for reducing the cost of lignocellulolytic enzymes production, as well as the understanding of regulation mechanisms for lignocellulolytic enzymes production in fungi.The main results of the research are as follows.1. Genome sequencing and analysis of P. decumbens wild-type strain114-2Lignocellulolytic enzymes and proteins involved in several other biological processes were annotated based on the genome sequence of P. decumbens wild-type strain114-2. Although more related to Penicillium chrysogenum phylogenetically, P. decumbens contains less proteins involved in cellular metabolism and regulation, while the numbers are similar with those of T. reesei. Compared with T. reesei, P. decumbens has genes encoding a set of plant cell wall-degrading enzymes with more diverse components, particularly for cellulose binding domain-containing proteins and several key enzymes to decompose hemicellulose. P. decumbens shares some regulation mechanisms for lignocellulolytic enzymes synthesis with T. reesei, but some different mechanisms between the two species were also indicated by genomic analysis. In addition, genomic regions rich in plant cell wall-degrading enzyme genes were found in P. decumbens, and some genetic events including horizontal gene transfer and gene expansion were observed for lignocellulolytic enzyme-encoding genes.2. Comparative genomic and transcriptome analysis of wild strain and lignocellulolytic enzyme hyper-producing mutants of P. decumbensNumerous genetic variations were found between the wild strain114-2and two lignocellulolytic enzyme hyper-producing mutants JU-A10-S and JU-A10-T through comparative genomic analysis. On the other hand, much less genetic variations were detected between the two mutants. The result indicated that the parent strain114was likely a heterokaryon, and current strains have been purified by subsequent isolation processes. Proteins with sequence differences between strains114-2and JU-A10-T were significantly enriched for those involved in transcriptional regulation, highlighting the important roles of these proteins in the phenotypic differences between the strains. A frame-shift mutation possibly accounting for the abolishment of conidial pigment synthesis in mutants were also found. The result of previous secretome analysis was confirmed, and differential expression of several intracellular biological processes was found, by comparative transcriptome analysis between strains. In addition, the mating-types of wild strain, mutants and another P. decumbens strain Peni-1were established, which providing the possibility for sexual cross in P. decumbens.3. Heterologous expression and characteriztion of endo-p-1,4-glucanases Ce145A and Cel5C from P. decumbensTwo endo-P-1,4-glucanases Ce145A and Ce15C from P. decumbens were recombinantly expressed and characterized for enzymatic properties. Both enzymes produced cellodextrins from cellulose. Compared with reported endo-p-1,4-glucanases from fungi, rCe145A and rCe15C showed relatively lower activities on CMC-Na, which might be related to the mechanisms of enzyme-substrate interactions. Similar to other GH family45proteins, rCe145A had higher temperature optimum and thermal stability, thus indicating the potential for improvement of cellulolytic enzyme systems. In addition, amino acids from position447to655were found to play an important role in the hydrolytic activity of Ce15C, and a protein stability-related mechanism was proposed.4. Preliminary study of the biological function of serine protease PrtB in P. decumbensOrthologs of three hydrolytic enzymes involved in autolysis in Aspergillus nidulans, including chitinase ChiB, metal protease PepJ and endo-β-1,3-glucanase EngA, were found in P. decumbens. However, another autolysis-involved protease PrtA of A. nidulans had no ortholog in P. decumbens. Characterization of gene deletion strains of ChiB, PepJ or EngA revealed that they might not play important roles in carbon starvation response and cellulase production in P. decumbens. Serine protease PrtB of P. decumbens showed the highest sequence identity with A. nidulans PrtA. When prtB was deleted, conidiation was blocked and extracellular acid protease activity was elevated under carbon starvation condition. In addition, deletion of prtB resulted in reduced extracellular protein yields under cellulase inducing condition, while extracellular acid protease activity was similar with that of the parent strain.更多还原