【作者】 杨慧林；

【导师】 林影；

【作者基本信息】 华南理工大学 ， 发酵工程， 2012， 博士

【摘要】 本研究以一株鸟苷工业生产菌解淀粉芽孢杆菌XH7为出发菌株，为了提高育种效率、构建高产稳定的鸟苷基因工程菌，探索了将全基因组测序和系统生物学分析方法应用于XH7菌株分子育种的可行性，并获得了如下结果：1.解淀粉芽孢杆菌XH7全基因组测序采用Solexa高通量测序技术，对XH7菌株进行全基因组测序，共获得629Mb大小的原始数据，平均测序深度为160×。使用SOAPdenovo软件对测序产生的reads序列进行拼接和组装得到76个Contigs和23个Scaffolds。将Scaffolds的头尾序列与参考基因组序列进行比对，以确定每个Scaffold的相对位置，然后设计引物PCR扩增内洞和外洞并进行序列鉴定，最终得到XH7菌株的基因组完成图。基因组序列已提交至NCBI数据库，登录号为CP002927。解淀粉芽孢杆菌XH7基因组全长3,939,203bp，GC含量45.82%，共编码4204个蛋白、75个tRNA和7个rRNA操纵子。比较基因组学分析发现，有一段约1.26Mb大小的DNA片段发生了倒置，同时鉴定出多个基因缺失与XH7菌株高产鸟苷相关。2.鸟苷生物合成的系统生物学分析以枯草杆菌基因组尺度代谢网络模型iYO844为基础，以提高鸟苷合成水平为目标，对枯草杆菌鸟苷合成进行了系统生物学分析，包括最大理论产率分析，in silico菌种改造等。使用FDCA和PS-FDCA两套算法预测得到了一些可能提高鸟苷产率的关键基因改造位点，包括基因敲除位点、基因强化位点和基因弱化位点，为后续的分子育种改造工作提供了理论指导。3.解淀粉芽孢杆菌遗传操作体系的建立及分子育种建立了解淀粉芽孢杆菌XH7的电转感受态细胞制备和转化方法，并且将in silico菌种改造预测得到的基因强化位点prs、purF、guaB和透明颤菌血红蛋白基因vgb分别转化到XH7菌株中表达。四个转化子的鸟苷发酵实验结果表明：增强prs和purF基因表达后，鸟苷产量分别提高了10.8%和20.9%；增强guaB基因表达后，鸟苷产量不增反而略有下降，同时转化子的生长速度明显低于野生型；vgb基因的表达没有显著提高鸟苷产量，但是转化子的生长速度明显要比野生型菌株快，这有利于缩短发酵周期和降低能耗。4.解淀粉芽孢杆菌基因敲除体系的建立及分子育种基于温敏复制型质粒pKS1，建立了解淀粉芽孢杆菌XH7的基因敲除体系。根据insilico菌种改造预测得到的基因强化位点（purEKBCLQFMNHD）、弱化位点（ptsGHI）和敲除位点（deoD），分别构建了嘌呤操纵子多拷贝突变株、ptsGHI缺失突变株、deoD缺失突变株。由于嘌呤操纵子全长达12kb，难于直接对其进行分子克隆等操作。通过在嘌呤操纵子终止子末端插入一个氯霉素抗性基因和一段嘌呤操纵子启动子区域约1kb的DNA片段，然后通过不断提高培养基中氯霉素的浓度来诱导整个嘌呤操纵子在基因组上的倍增，qPCR鉴定最多达11个拷贝，并且鸟苷产量提高了25.5%。ptsG和ptsHI缺失突变株的发酵实验结果表明：敲除ptsG基因后，鸟苷产量提高了23.9%；而敲除ptsHI基因后，鸟苷产量降低了81.8%。这可能是因为敲除ptsG基因后，有利于提高葡萄糖进入磷酸戊糖途径的代谢通量，而敲除ptsHI基因后，彻底阻断了菌体对葡萄糖的吸收。deoD缺失突变株的鸟苷产量没有明显变化，表明deoD基因编码的酶基本不降解鸟苷。本研究基于系统生物学分析得到的关键基因改造位点，对解淀粉芽孢杆菌XH7进行了分子育种改造。结果表明，该方法是可靠有效的，为后续的进一步分子育种提供了新的思路和研究方向。本文的研究平台具有良好的普适性，适合于其它各种类别产物的研究。随着系统生物学的发展，该平台也将得到逐步完善并趋于成熟。更多还原

【Abstract】 Aguanosine industrial producer Bacillus amyloliquefaciens XH7was used as starting strain inthis work. In order to improve the efficiency of breeding and create high yielding stablegenetic engineering bacteria of guanosine, it was studied to improve guanosine productionwith whole genome sequencing and systems biology analysis. The main results were achievedand shown as follows:(1) Whole-genome sequencing of Bacillus amyloliquefaciens XH7Whole-genome sequencing of XH7was carried out by Solexa sequencing to produce629Mb filtered sequences, representing a160-fold coverage of the genome. The sequences wereassembled into76contigs and23scaffolds using the SOAPdenovo package. Scaffolds’relative position was identified by blasting with the published Bacillus amyloliquefaciensFZB42genome sequence. Then, special primers were designed to amplify the sequences ofinner gaps and outer gaps of the scaffolds. Finally, the identified PCR products and thescaffold make up of the whole genome sequence. The genome sequence of Bacillusamyloliquefaciens XH7was deposited in GenBank under the accession number CP002927.The complete genomic information of the Bacillus amyloliquefaciens XH7is contained on asingle circular chromosome of3,939,203bp with an average GC content of45.82%. Atotal of4,204protein coding genes,75tRNA genes, and7rRNA operons were identified.Comparative genomics analysis revealed that an approximately1.26Mb DNA fragment wasinverted, and multiple inactive gene realated with guanosine high-yield were identified.(2) Systems biology analysis of guanosine biosynthesisBased on Bacillus subtilis metabolic model iYO844, systems biology methods were usedto analyze the theoretical conversion yields of guanosine and in silico strain modification.Besides, two strategies including the flux distribution comparison analysis (FDCA) methodand product stress-flux distribution comparison analysis (PS-FDCA) method were employedto predict potential gene targets in order to improve the guanosine production. The resultswere reliable and could provide guidance for the follow-up strain modification.(3) Genetic transformation system of Bacillus amyloliquefaciens and molecular breedingA protocol for electroporation-competent cells preparation and transformation of XH7 strain was created. Based on the results of in silico strain improment for guanosine production,four genes (prs, purF, guaB, vgb) were transformed into XH7strain. The results of shakingflask fermentation showed that overexpression of prs and purF genes enhanced the guanosineproduction yield by10.8%and20.9%, while overexpression of guaB genes did not enhancethe guanosine production yield, but caused a slight decrease of guanosine production and asignificant decrease of growth rate. The expression of vgb caused no significant increase ofproduction yield, but vgb expression promoted cell growth, shortened fermentation period andreduced energy consumption.(4) Gene-knockout technology of Bacillus amyloliquefaciens and molecular breedingA gene-knockout technology based on temperature-sensitive plasmid pKS1wasestablished, and pur operon duplication mutants, phosphotransferase system and deoDdeficient mutants were constructed based on the results of in silico strain improment forguanosine production. As the total length of pur operon is12kb, it is very difficult to clone itdirectly. A chloramphenicol resistance gene and a pur operon promoter about1kbhomologous sequence were inserted into3’ end of pur operon terminator. By increasing theconcentration of chloramphenicol in the medium, the pur operon was induced doubling in thegenome. The max copy number of the pur operon was about11by qPCR. Meanwhile, theproduction yield was increased by25.5%. In guanosine fermentation experiments, theguanosine production yields of ptsG genetic defect strains was increased by23.9%, while thatof ptsHI genetic defect strain was decreased by81.8%. These results showed that ptsGdeletion was benefit to increase the carbon flux into the pentose phosphate pathway, whileptsHI deletion completely blocked the glucose use. The deoD genetic defect strain did notincrease the guanosine production yield, which suggested that the protein DeoD encoded bydeoD did not degradate the guanosine.In this study, the molecular breeding of guanosine industrial producer XH7was carriedout based on systems biology. The results show that the method is reliable and effective, andcan provide new ideas and research directions for further follow-up molecular breeding. Theplatform constructed by this work is also suitable for other kinds of products research. Infuture, the platform will get well developed step-by-step with the development of systemsbiology.更多还原