光合细菌产氢关键基因的增强表达对产氢速率的影响机理研究

Low hydrogen production rate is one of the main bottlenecks in photo-fermentation using purple nonsulfur bacteria. In order to make some breakthrough on this issue, Rhodobacter sphaeroides HY01 with relative high hydrogen production rate will be employed as the model strain. On the basis of increasing the ratio of electron flux to nitrogenase by knocking out the calvin cycle, hydrogen uptake process and polyhydroxylbutyrate synthesis process that compete with hydrogen production, the rate of electron flux to nitrogenase is expected to be increased by overexpression the three key gene clusters of rnfABCDGEH,fdxN and nifHKD in the electron transport chain of nitrogenase hydrogen production, and consequently increase the hydrogen production rate remarkably.By constructing the promoter-lacZ fusion plasmids for testing and selecting the promoters with proper expression intensity in R.sphaeroides HY01, the promoters of the key gene clusters will be replaced by these selected promoters for optimizing the expression level of these genes,and some gene engineering strains will be constructed. The transcription level and expression level of these genes will be tested with RT-qPCR and Western-blot, respectively, and the matching mechanisms of the key gene clusters and their effect mechanism on hydrogen production rate will be revealed; the effects and mechnism of expression level of these genes under hydrogen production conditions on enhancing the tricarboxylic acid cycle, photosynthetic phosphorylation process and hydrogen production rate will be studied by testing the hydrogen production rate, reducing power and energy density of these strains,and the gene engineering strains with optimized genotype will be constructed based on the above studies. The results of this project will offer theoretical and experimental basis for improving photo-fermentative hydrogen production rate.

产氢速率低是光合细菌产氢的主要瓶颈之一。针对该问题,本课题拟以R.sphaeroides HY01为模型菌，在敲除开尔文循环，氢气吸收及聚羟基丁酸合成等与产氢竞争的途径，提高电子流向固氮酶的比例的基础上，通过优化表达固氮酶产氢的关键基因簇rnfABCDGEH，fdxN和nifHKD，提高电子流向固氮酶的速率，提高光合产氢速率。构建lacZ融合表达质粒测试并筛选该菌株中表达强度合适的增强子,并用合适的增强子置换上述三个基因簇的增强子，优化基因表达量，构建基因工程菌株；利用RT-qPCR及Western-blot研究关键基因簇的转录水平及表达量，研究其表达量匹配机制及对产氢速率的影响机理；通过测试细菌的产氢速率，还原力及能量水平，研究关键基因簇的表达量对三羧酸循环，光合磷酸化和产氢速率的相互促进规律与机制，获得优化基因表达的工程菌株，为进一步提高光合发酵产氢速率提供理论基础与实验依据。

产氢速率低是光合细菌产氢的主要瓶颈之一。针对该问题,本课题拟以R.sphaeroides HY01 为模型菌，在敲除开尔文循环，氢气吸收以及聚羟基丁酸合成等与产氢竞争的途径，提高电子流向固氮酶的比例的基础上，通过优化表达固氮酶产氢的关键基因簇rnfABCDGEH，fdxN 和nifHKD，提高电子流向固氮酶的速率，提高光合产氢速率。. 对HY01中的多个基因的增强子进行了表达强度测试，并以此为基础对rnfABCDGEH，fdxN，nifHKD基因进行了增强表达；研究了HY01中hupSL及spbA基因缺失对其固氮酶活性和产氢性能的影响机理；研究了ATP合成酶结构基因f0及f1操纵子过表达对细菌ATP浓度、固氮酶活性和产氢性能的关系；研究了Mo-固氮酶和Fe-固氮酶共表达对光合产氢性能的影响。研究结果表明，通过用nifH和pucBA基因增强子修饰rnfABCDGEH基因后，所得突变子的光合产氢速率分别提高16%及20%；对fdxN进行过表达后，突变子具有比野生菌更高的产氢活性和稳定性；敲除hupSL及spbA后，突变子WH04(hupSL-)及WSH10(spbA-)的最大产氢速率分别较野生菌提高29.9%及55%；其产氢量也分别提高了19.4%和21.8%，且WSH10具有一定的抗铵抑制性能；过表达ATP合成酶结构基因f0及f1后，其产氢速率分别提高了10.5%和20.6%；通过敲除mopAB基因，实现了Mo-固氮酶及Fe-固氮酶的共表达，显著提高了固氮酶活性(提高42%)及最大产氢速率(提高了24%)。. 研究结果表明，为提高光合产氢效率，一方面可通过敲除与固氮酶产氢相竞争的途径，提高电子和能量流向固氮酶产氢的比例；另一方面可以增强固氮酶相关基因的表达量，拓宽电子和能量流向固氮酶产氢的通道，这为后续进一步提高光合发酵产氢效率提供了数据支持和理论依据。