【作者】 赵锦芳；

【导师】 张传溪；

【作者基本信息】 浙江大学 ， 环境生物学， 2009， 博士

【摘要】 随着环保要求的迫切和化石能源的日益短缺,氢能作为清洁高效的可再生能源受到人们的普遍重视。目前通过基因工程和代谢调控手段改造产氢生物,是生物制氢领域的研究热点。本文以高效产氢细菌产气肠杆菌Enterobacter aerogenesATCC13408为主要研究对象,对其产氢代谢途径中的相关基因进行了以下研究:克隆阴沟肠杆菌E.cloacaeⅡT-BT 08的铁氢酶hydA基因,并在大肠杆菌E.coli BL21、产气肠杆菌E.aerogenes ATCC13408和阴沟肠杆菌E.cloacaeCICC10017中实现异源表达,重组后细菌的产氢量均有不同程度的提高:使原本不产氢的E.coli BL21氢气产量达到0.5mol H₂/mol葡萄糖,使产气肠杆菌的氢气产量从原菌的1.19 mol H₂/mol葡萄糖提高到2.31 mol H₂/mol葡萄糖,使阴沟肠杆菌的氢气产量从原菌的1.286 H₂/mol葡萄糖提高到2.545 mol H₂/mol葡萄糖。fhlA基因是产气肠杆菌甲酸-氢裂解酶系统FHL的转录激活蛋白,本文通过设计简并性引物和genomic walking技术,成功克隆了fhlA基因,完整的ORF全长2073 bp,编码690个氨基酸。FhlA成功地在产气肠杆菌中实现过量表达,重组后的细菌的产氢能力由原菌的1.23 mol H₂/mol葡萄糖提高到了1.48 molH₂/mol葡萄糖,提高了20.36%。产气肠杆菌氢酶属于典型的[NiFe]氢酶,由多亚基共同组成。本文扩增出E.aerogenes ATCC13408的hyc操纵子全长,共有9个亚基的ORF,其中HycE、HycG分别编码氢酶的大、小亚基,hycE和hycG基因的过量表达使产气肠杆菌的底物产氢潜力由1.14 mol H₂/mol葡萄糖提高到了2.05 mol H₂/mol葡萄糖和2.08 mol H₂/mol葡萄糖,分别提高了80.1%和82.65%。蓝藻Synechocystis sp.PCC 6803的hoxEFUYH基因全长6493 bp,由E、F、U、Y、H共5个亚基组成一个NAD-还原型氢酶操纵子。本文克隆了蓝藻Synechocystis sp.PCC 6803的hoxEFUYH氢酶基因,并在产气肠杆菌中表达,结果表明:蓝藻HoxEFUYH蛋白的异源表达提高了细菌的产氢能力,最大产氢速率和累积产氢量均比原菌有较大幅度提高,其中,表达F和U亚基的重组菌株E.aerogenes/HoxFU的产氢量最高,从未重组前的1.14 mol H₂/mol葡萄糖提高达到2.29 mol H₂/mol葡萄糖,最大产氢速率达到113.4 mL·L^-1·h^-1,表达U、Y和H亚基的重组菌株E.aerogenes/HoxUYH的产氢量最少,从未重组前的1.14mol H₂/mol葡萄糖提高到1.82 mol H₂/mol葡萄糖,最大产氢速率为41.6mL·L^-1·h^-1。但是目前蓝藻氢酶重组后的作用机制还不清楚,有待于进一步的研究。更多还原

【Abstract】 Hydrogen,as a clean energy that can replace conventional fossil fuels,is attracting worldwide attentions.Nowdays,more and more researches focused on producing hydrogen from biological processes modified by genetic techniques and metabolic engineering.In this paper,several genes which are involved in the hydrogen producing metabolic pathway of a facultative anaerobic bacteria, Enterobacter aerogenes ATCC13408 were studied.The results are as following:[Fe]-hydrogenase encoding gene isolated from E.cloacae IIT BT-08 has been overexpressed in fast growing non-hydrogen producing Escherichia coli BL21 using pGEX-4T-2 vector and the yield of hydrogen from glucose using recombinant E.coli BL21 was 0.5 mol H₂/mol glucose.At the same time,the hydA gene was also transformed into two fermentative anaerobic hydrogen-producing bacterial species,E. aerogenes ATCC13408 and E.cloacae CICC10017.The hydrogen yield of the recombinant strain E.aerogenes ATCC13408/hydA was promoted from 1.19 mol H₂/mol glucose to 2.31 mol H₂/mol glucose.And the hydrogen yield of recombinant strain E.cloacae CICC10017/hydA was promoted from 1.286 mol H₂/mol glucose to 2.545 mol H₂/mol glucose.FhlA is the transcriptional activator of the genes coding for the formate hydrogen lyase system in E.aerogenes.By using universal primers and genomic walking,fhlA ORF was successfully cloned and shared high amino acid identities with other bacterial species.Then the gene was transformed into E.aerogenes ATCC13408,and the hydrogen yield of the recombinant strain was promoted from 1.23 mol H₂/mol glucose to 1.48 mol H₂/mol glucose,which increased by 20.36%。E.aerogenes ATCC13408 has a typical[NiFe]-hydrogenase and is composed of 9 subunits,hycE encodes the large subunit,which harboures the active centre,and hycG encodes the small subunit,which containes 2-3 FeS clusters.In this study, HycE and HycG were overexpressed in E.aerogenes ATCC13408,and the hydrogen production of the recombinant strains were promoted form 1.14 mol H₂/mol glucose to 2.05 mol H₂/mol glucose and 2.08 mol H₂/mol glucose,which increased by 80.1% and 82.65%respectively.The hydrogenase gene hoxEFUYH of the Cyanobacteria Synechocystis sp.PCC 6803 was cloned and expressed in E.aerogenes ATCC13408 also.The hydrogen yield of recombinant strain E.aerogenes/HoxFU was promoted from 1.14 mol H₂/mol glucose to 2.29 mol H₂/mol glucose and the maximum hydrogen-producting rate reached 113.4 mL·L^-1·h^-1,which was the best in all the recombinant strains.E. aerogenes/HoxUYH had a lower product,which was 1.82 mol H₂/mol glucose and the maximum hydrogen production rate was 41.6 mL·L^-1·h^-1 only.更多还原