阴沟肠杆菌WL1318代谢工程改造及发酵棉秆水解糖液产氢过程调控提高生物氢产率

Hydrogen production from dark fermentation using lignocellulose as substrate has become a promising method of biohydrogen production, however, there is less study on biohydrogen production from dark fermentation by single strain, and also the biohydrogen production rate obtained by wild strain is relatively low through traditional metabolic pathways, so the wild strain needs reconstruction via metabolic engineering to improve biohydrogen production. Our group has previously isolated an Enterobacter cloacae, which can produce biohydrgen from fermentation of cotton stalk hydrolysate. In this project, an engineering strain of Enterobacter cloacae, which will be engineered toward key enzyme genes cloned in hydrogen production pathway, and constructed with Lactate dehydrogenase ldhA gene, H2-uptake hydrogenase hybC subunit gene deleted, and [Fe] hydrogenase hydA gene, formate hydrogen lyase activator fhlA gene overexpressed, will be used as inoculum for hydrogen production from fermentation of cotton stalk hydrolysate. In the fermentation process, the fermentative condition will be optimized, the fermentative dynamic model will be constructed, and metabolic flux analysis (MFA) will be introduced to analyze the mass flow and energy flow, then the key step and regulation of pathway will be understood and used as guidance for hydrogen production from fermentation of cotton stalk hydrolysate. In addition, the biohydrogen production will be contrasted between the wild strain and engineering strain, and the mechanism that metabolic engineering improving biohydrogen productin capability of Enterobacter cloacae will be illustrated. This project will provide theoretical reference and technical support for efficient hydrogen production from fermentation of cotton stalk hydrolysate.

木质纤维素暗发酵产氢近年来已成为一种有效的生物制氢方式，然而其以单菌株发酵产氢的研究相对较少，并且野生菌株通过传统代谢途径获得的产氢率也较低，因此野生菌株往往通过代谢工程手段进行改造以期实现生物氢产量的提高。课题组前期研究工作中已筛选获得一株能发酵棉秆水解糖液产氢的阴沟肠杆菌，本项目拟针对该菌株已克隆的产氢代谢关键酶基因，构建乳酸脱氢酶ldhA基因和吸氢酶hybC亚基基因沉默、铁氢酶hydA基因及甲酸-氢裂解酶fhlA基因强化表达的工程菌，用于发酵棉秆水解糖液产氢，实现发酵产氢条件优化，建立产氢发酵动力学模型，对产氢过程中的物质流和能量流进行代谢通量（MFA）分析，弄清工程菌产氢的关键步骤和调控路径，用以指导棉秆水解糖液发酵产氢过程，对比工程菌和野生菌株发酵棉秆水解糖液的生物氢产量，阐明代谢工程改造对阴沟肠杆菌产氢能力提高的影响机理，为实现棉秆水解糖液高效发酵产氢提供理论依据和技术支持。

木质纤维素暗发酵产氢近年来已成为一种有效的生物制氢方式，然而单一野生菌株通过传统代谢途径获得的产氢率相对较低，因此野生菌株往往通过代谢工程手段进行改造以期实现生物氢产量的提高。本项目针对课题组前期研究工作中筛选获得的一株能发酵棉秆水解糖液产氢的阴沟肠杆菌的产氢代谢关键酶基因，构建乳酸脱氢酶ldhA基因和吸氢酶hybC亚基基因沉默、铁氢酶hydA基因及甲酸-氢裂解酶fhlA基因强化表达的工程菌。研究首先通过同源重组方式进行菌株ldhA基因和hybC亚基基因敲除，从一系列突变株中筛选获得2株生长良好且具较强产氢潜力的基因敲除菌株。在这2株基因敲除菌株中超量表达hydA基因和fhlA基因，进一步筛选获得6株铁氢酶和甲酸氢裂解酶活性较高的阳性菌株，用其发酵棉秆水解糖液产氢，发现6个阳性菌株的生物氢产量较之野生菌株WT均有提高，产氢量相较野生菌株最高提高约2.5倍。其中2株工程菌ΔldhΔhybfhlAhydA-1和ΔldhΔhybfhlAhydA-2具有较强的产氢潜力，其通过Gompertz模型拟合累积产氢量的最大产氢速率Rm可达42.613和43.852 mL L-1h-1，其产氢潜力P可达3032.125和3053.067 mL L-1。对工程菌发酵棉秆水解糖液产氢过程进行了代谢通量分析，结果表明超表达hydA和fhlA基因增强了生物氢合成支路的代谢流分布，有效提高了生物氢合成量和合成速率。本研究结果为获得高生产力的产氢菌株，实现棉秆水解糖液高效发酵产氢提供了理论依据和技术支持。