Construction Of Klebsiella Oxytoca HP1 Mutants By Homologous Recombination To Enhance Hydrogen Evolution

The low bacterial H2-production ability would be the choke point on the development of bio-hydrogen technology. It is a potential method for enhancing H2-production by using molecular biological technology. Ethanol and lactic acid are the main byproduct of anaerobic H2-producing fermentation in Klebsiella oxytoca HP1.This can be effectively solved by blocking the pathways leading to the byproducts that are of no benefit to H2-production.In this study,the homologous integration plasmid vector pTA-Str and pLDH-Tr were constructed using K. oxytoca HP1 as host cell. The adhE genes, which encoded for acetaldehyde dehydrogenase and alcohol dehydrogenase, were cloned into plasmid pMD18-T to generate plasmid pTA. By inserting Aminoglycoside-3'-adenyltransferase (aadA) cassette into the coding region of adhE +868～+869 into plasmid pTA, the homologous integration plasmid vector pTA-Str was constructed. The amplified DNA fragment 5'-adhE-aadA-adhE-3'from pTA-Str was transformed into K. oxytoca HP1 and the mutant strain adhE- was sceened.Using the similar strategy, The LDHa genes, which encoded for lactic acid dehydrogenase, were cloned into plasmid pUC19 to generate plasmid pLDH. By inserting tetracycline resistance protein (tet) cassette into the coding region of LDHa +518～+519 into plasmid pLDH, the homologous integration plasmid vector pLDH-Tr was constructed. Mutant strain LDHa- was screened after pLDH-Tr was transformed into K. oxytoca HP1.Compared with the H2-production of wild type K. oxytoca HP1, the hydrogen yield of the mutant strain adhE- increased by 16.07% and ethanol concentration decreased by 77.47%, and the hydrogen yield of the mutant strain LDHa- increased by 20.35%.In this study, we obtained Klebsiella oxytoca HP1 adhE- and LDHa-mutant strains. They offered platform for future research on high bacterial hydrogen production.