Mechanism Of2,3-Butanediol Stereoisomer Formation In Klebsiella Pneumoniae

Klebsiella pneumoniae is the most promising industrial strain for the production of2,3-butanediol(2,3-BD).The stereoisomers of the2,3-BD produced by this strain were analyzed, and it was found to contain all three stereoisomers. The mechanism of2,3-BD synthesis was investigated by the construction of mutant strains which lost the genes involved in the2,3-BD synthesis pathway, and the characteristics of enzyme reactions in vitro. The method of producing R-acetoin was developed, which has a higher economic prospect. And a tool for homologous recombination in K. pneumoniae was developed, which has a higher efficiency for homologous recombination.The ratio of2R,3R-BD to all isomers obtained using glycerol as the carbon source was higher than that obtained using glucose as the carbon source. Therefore, enzymes involved in glycerol metabolism are likely related to2R,3R-BD formation. dhaD gene encoded glycerol dehydrogenase. K. pneumoniae-△dhaD lost the ability to synthesize2R,3R-BD. budA gene encoded α-acetolactate decarboxylase. K. pneumoniae AbudA reduced the yield of meso-2,3-BD and2R,3R-BD. budC gene encoded butanediol dehydrogenase genes. K. pneumoniae AbudC obviously decreased the ability to synthesize meso-2,3-BD, and increased the ability to2R,3R-BD. K. pneumoniae AbudC produced a high level of R-acetoin using glucose as the carbon source, and2R,3R-butanediol using glycerol as the carbon source.. The heterologous protein expression vectors of E. coli BL21/dhaD and E. coli BL21/budC were constructed. In vitro reactions show that glycerol dehydrogenase catalyzes the stereospecific conversion of R-acetoin to2R,3R-butanediol and S-acetoin to meso-2,3-butanediol. Butanediol dehydrogenase exhibits high (S)-enantioselectivity in ketone reduction. These results demonstrated the mechanism of the2,3-butanediol stereoisomer synthesis pathway. Glycerol dehydrogenase, exhibited2R,3R-butanediol dehydrogenase activity and was responsible for2R,3R-butanediol synthesis from R-acetoin. This enzyme also contributed to meso-2,3-butanediol synthesis from S-acetoin. Butanediol dehydrogenase, was the only enzyme that catalyzed the conversion of diacetyl to S-acetoin and further to2S,3S-butanediol.The study about the synthesis of R-acetoin discovered that K. pneumoniae AbudC produced a high level of R-acetoin with58.99g/L after48hours of fermentation. Aco gene encoding acetoin dehydrogenase enzyme system and dhaD gene were disrupted in K. pneumoniae AbudC. However, the result showed that R-acetoin production were increased a little in the double gene mutant and treble gene mutant. The method for red homologous recombinant using in the construction of mutants, in K. pneumoniae, which efficiency is less than in E.coli, and need longer length of homologous arm. It is speculated that the Gam subunit has litter inhibiting effect to exonuclease V. A plasmid that expressing recombinase without gam gene was constructed, and a recB mutant strain was constructed. The efficiency of homologous recombinant without Gam was obviously decreased, and the recB mutant strain showed a high efficiency in recombination. The result showed that the activity of RecBCD was not inhibited by Gam subunit completely. The study provided a menthod for red homologous in K. Pneumoniae with higher efficiency.