酪酸梭菌1,3-丙二醇耐受性分子机制研究

1,3-propanediol has a wide range of application in many fields, such as food, chemical, pharmaceutical, and cosmetics. It is a hot ressearch issue that 1,3-propanediol is obtained from the trasformation of crude glycerol through microbial fermentation in these fields. Although Clostridium butyricum is one of the most ideal fermentation strains which can produced 1,3-propanediol throuth biological method, there is still a shortcoming with low tolerance of 1,3-propanediol in this strain. In present project, in order to obtain a mutant strain with high 1,3-propanediol tolerance, genome shuffling technology is used in the directed evolution of C. butyricum which was a original probiotic strain screened from baby feces. On this base, difference genes associated with 1,3-propanediol tolerance will be obtained using reverse metabolic engineering technology through the whole genome sequencing. Subsequently, RNA and protein’s defferences will also be analyzed using the RNA-Seq, 2-DE and iTRAQ technology on transcription and protein levels, respectively. Then, genes and proteins associated with 1,3-propanediol tolerance will be determined by systems analysis. Furthermore, molecular mechanism about 1,3-propanediol tolerance of C. butyricum will be comprehensive analyzed base on the function validation of key genes and proteins. This research will enhance the comprehensive utilization of crude glycerol and the biosynthesis of 1,3-propanediol by C. butyricum on the theoretical and technical levels.

1,3-丙二醇（1,3-PD）在食品、化工、医药等领域具有非常广泛的应用，而通过微生物发酵粗甘油生产1,3-PD是近年来的热点研究问题。酪酸梭菌是生物法生产1,3-PD的理想发酵菌种之一，但该菌仍然存在1,3-PD耐受性低的问题。本课题以自主筛选的益生菌—酪酸梭菌作为出发菌株，通过Genome shuffling技术，定向进化并筛选1,3-PD的高耐受性突变株。在此基础上，采用反向代谢工程技术，通过全基因组测序，获得与1,3-PD耐受性相关的差异基因；然后，利用RNA-Seq、2-DE和iTRAQ技术分别从转录和蛋白质水平，分析样品RNA和蛋白的变化差异情况，综合分析并确定与1,3-PD耐受性相关的基因与蛋白；最后，对其中的关键基因和蛋白进行功能验证，综合解析酪酸梭菌的1,3-PD耐受性机制。该课题的开展可为粗甘油的综合利用及酪酸梭菌的1,3-PD高效生产提供理论和实践上的参考。

1,3-丙二醇（1,3-PD）在食品、化工、医药等领域具有非常广泛的应用，通过微生物发酵甘油生产1,3-PD是近年来的热点研究问题。酪酸梭菌是生物法生产1,3-PD的理想菌种之一，但该菌仍然存在1,3-PD耐受性低而影响产量的问题。课题以自主筛选的益生菌—酪酸梭菌作为出发菌株，建立并优化了酪酸梭菌（C. butyricum）1,3-PD耐受性的平板初筛体系（GPDS）以及高通量复筛体系（GPDH）；以亚硝基胍（NTG）为诱变剂，对1,3-PD高耐受性菌株进行了化学诱变，并对1,3-PD高耐受性菌株进行了Genome shuffling改造，提高了菌株对1,3-PD的耐受性，进而采用ARTP技术进一步提高了菌株1,3-PD的耐受性。针对酪酸梭菌菌株，完成了菌株的全基因组测序，结果显示基因组大小为4.6Mb, 共预测出4220个CDS序列,为后续其他组学分析与关键基因验证提供了基础；随后，完成了耐受性菌株的转录组测定与分析，初步确定了关键差异通路与关键差异基因；进而，完成了蛋白组的测定与分析，并结合基因组和转录组结果，综合分析并确定了与1,3-PD耐受性相关的关键基因和蛋白。最后，通过关键基因的在大肠杆菌的增强表达以及在酪酸梭菌中的增强表达与敲除失活，验证了关键基因和蛋白的功能；在此基础上，解析了酪酸梭菌的1,3-PD耐受性分子机制，综合确定了关键蛋白－ABC转运蛋白、组氨酸激酶和甲基接受化学趋向性蛋白对酪酸梭菌1,3-PD耐受性的增强具有较大影响，过表达这三个蛋白的编码基因均可较大幅度提高菌株对1,3-PD的耐受性；另外，敲除这三个基因均可较大幅度降低菌株对1,3-PD的耐受性。而关键蛋白－异柠檬酸脱氢酶、谷氨酰胺合成酶、铁氧还蛋白和磷酸核糖焦磷酸合成酶对酪酸梭菌1,3-PD的耐受性增强具有一定程度的影响，过表达这四个蛋白编码基因可一定程度提高菌株对1,3-PD的耐受性，但幅度较小；分别敲除这四个基因可一定程度上降低菌株对1,3-PD的耐受性。该项目成果可为甘油的综合利用及1,3-PD的高效生产提供理论和实践上的借鉴。