Construction of Klebsiella Oxytoca HP1 Mutants by Homologous Recombination to Enhance Hydrogen Evolution
|Keywords||Klebsiella oxytoca HP1 adhE gene LDHa gene Biohydrogen Gene insertion inactivation|
Hydrogen bacterial hydrogen production capacity is not high biological hydrogen production technology is to limit the development of important factors . By means of the use of molecular biology to transform bacteria , is to improve the efficiency of the important biological hydrogen production method . Ethanol and lactic acid are Klebsiella Klebsiella oxytoca HP1 main anaerobic fermentation by-product H2 , cut useless byproducts increased H2 production rate and substrate conversion . In this study, K. oxytoca HP1 as a receptor , homologous arms was constructed integration vector pTA-Str and pLDH-Tr. Platform using endogenous double exchange principle , carried on the plasmid pMHE6 glucosaminidase adenosine transferase gene (Aminoglycoside-3'-adenyltransferase, aadA) exogenous expression cassette sequence ( containing a T7 promoter , aadA coding frame , T4 terminator, ) inserted into the recipient strain designated chromosome alcohol dehydrogenase ( containing acetaldehyde dehydrogenase and alcohol dehydrogenase ) gene adhE 868 ~ 869 zones (adhE start coding frame of 1 ) loci . And on this basis plasmid pTA-Str K. oxytoca HP1 adhE mediated insertion inactivation obtained with adhE- streptomycin resistant mutant . Using a similar method to carry on the pBR322 tetracycline resistance gene (tetracycline resistance protein, tet) exogenous expression cassette sequences ( including P1, P2 promoter , tet coding frame ) inserted into the recipient strain designated chromosome lactate dehydrogenase gene LDHa 518 ~ 519 zones (LDHa start coding frame of 1 ) loci . Based on this plasmid pLDH-Tr -mediated insertion of K. oxytoca HP1 LDHa inactivation obtained with tetracycline LDHa- resistant mutants . Experimental results show that the fermentation of glucose , the same fermentation conditions , adhE- mutant strain than in the wild hydrogen production increased 16.07% , ethanol production fell by 70.47%; LDHa- mutant strain than in the wild hydrogen production increased 20.35% . This study constructed Klebsiella oxytoca HP1 mutant adhE- and LDHa-, further research will lay the foundation for efficient hydrogen production system .