耐酸型高效产氢细菌成团泛菌BH18产氢关键基因及代谢途径研究

Fermentative hydrogen production is important for providing clean energy and is considered to have good application prospect. However, hydrogen yield by fermentative bacteria is very low until now. Especially, the accumulation of organic acids in the process of hydrogen production causes an inhibition of hydrogen yield. Isolation of acid tolerant bacteria that can decrease the limit of pH is one of the most important techniques for high hydrogen production. This project will take hydrogen-producing bacterium Pantoea agglomerans BH18 isolated from mangrove sludge as the study material. Acid tolerant bacteria with high efficiency of hydrogen production are obtained by random mutation. Identification of key genes and metabolic pathway required for hydrogen production will be studied. First, based on the generation of Pantoea agglomerans BH18 library by the previous work in our lab, mutant screens under acid stress condition are conducted for isolation of mutant strains with different phenotypes. These mutants are used to analyze the transposon insertion sites and reveal which genes are essential for hydrogen production. Furthermore, the regulatory expression of these correlative genes during the process of hydrogen production will be determined to analyze the roles of the key genes required for hydrogen production. Finally, according to the results of roles of key genes required for hydrogen production and comparison of fermentation types and metabolic rates, transformation of metabolic pathway by acid tolerant hydrogen-producing bacterium Pantoea agglomerans BH18 will be analyzed. The results in this project will make contributions to the mechanisms of hydrogen production by aciduric fermentative hydrogen-producing bacteria and aid the construction of engineering strain with high efficiency of hydrogen production. Also, this research may improve the study process on the mechanisms of hydrogen production by anaerobic fermentative bacteria.

厌氧发酵细菌产氢对提供清洁能源具有重要意义，被认为有较好的应用前景。目前，厌氧发酵细菌产氢水平较低，尤其是产氢过程中的有机酸反馈抑制作用明显降低了产氢效率。耐酸型产氢菌株对于提高厌氧发酵细菌产氢能力具有重要意义。本申请项目拟以红树林污泥中分离成团泛菌BH18为材料，采用随机突变方法获得耐酸型产氢菌株，开展耐酸型厌氧发酵细菌产氢关键基因及代谢途径研究。首先，在已构建菌株BH18转座子突变文库基础上，利用反向PCR技术鉴定被阻断的靶位基因，确定产氢代谢途径关键基因；然后，调控表达产氢关键基因，检测基因表达水平，分析产氢关键基因在代谢途径中功能；最后，结合分析产氢关键基因功能、发酵代谢产物类型和代谢物之间的代谢流比率，解析成团泛菌BH18产氢代谢途径的改造。研究结果将揭示耐酸型厌氧发酵细菌高效产氢分子机制。可为构建高效产氢工程菌株提供理论基础，也有助于推动有关厌氧发酵细菌产氢机理的研究进程。

目前，厌氧发酵细菌的产氢能力不高，限制了该技术规模化应用。在基因水平上（主要是基于氢酶基因和产氢代谢途径相关基因）对野生型产氢菌株进行改造，被认为是一种有效提高菌株产氢能力的途径。项目在成团泛菌BH18转座子突变文库基础上，筛选产氢正突变菌株和产氢负突变菌株。基于野生菌株全基因组序列，筛选产氢相关基因，确定了产氢代谢途径关键基因。在此基础上，根据正突变菌株、负突变菌株以及野生菌株转录组数据，分析产氢关键基因的差异表达情况，解析成团泛菌BH18产氢代谢途径。研究结果不仅为构建高效产氢工程菌株提供理论基础，也有助于推动有关厌氧发酵细菌产氢机理的研究进程。.1、在转座子Tn7构建了成团泛菌BH18突变文库基础上，同时结合物理诱变法以及抗生素筛选，获得产氢正突变菌株TB26和产氢负突变菌株TB30，为构建产氢工程菌株和研究产氢相关基因提供了实验支持。.2、在获得产氢菌株全基因组序列数据基础上，通过比对已公开产氢基因序列，确定了肠杆菌产氢相关基因序列。.3、在获取产氢相关基因数据基础上，通过对比产氢正突变菌株和产氢负突变菌株的差异表达基因。确定了肠杆菌产氢关键基因，并解析了氢化酶、丙铜酸甲酸裂解酶以及铁氧还蛋白酶的产氢代谢通路。`