Studies On Genetic Recombination To Enhance Hydrogen Production Of Enterobacter Aerogenes

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.