Recombination And Metabolites Analysis Of Klebsiella Pneumoniae

Diacetyl is a valuable flavor naturally existing in laurel oil, strawberry, butter, cherry, honey, beer, etc.. The low diacetyl productivity is the choke point on the development of microorganism fermentation technology. It is a potential method for enhancing diacetyl production by using molecular biological technology.2,3-butanediol oxidoreductase (BDH) is the only enzyme that acts on the catabolism of diacetyl.α-Acetolactate synthase (ALS) is the first enzyme involved in the anabolism of diacetyl, and also the critical rate-limiting enzyme involved in the catabolism of pyruvate. The two enzymes are very important to diacetyl production. In this study, we took the budC gene and als gene of Klebsiella pneumoniae as a target in order to enhance the production of diactyl by genetic engineering means.The homologous recombination linear fragment up-5'budC-Tcr-3':budC-down was constructed by fusion PCR technology using K. pneumoniae CICC10011 as host cell. By inserting tetracycline resistance gene (Tcr) into the coding region of budC+153～+604 and transforming the DNA fragment up-5'budC-Tcr-3'budC-down into K. pneumoniae CICC10011, the mutant strain budC- was sceened, which was named as K. pneumoniae CICC10011-0.The wild type K. pneumoniae CICC 10011 did not produce diacetyl, while the yield of the recombinant K. pneumoniae CICC 10011-0 was 0.14g/l. Compared with the wild type K. pneumoniae CICC10011, the glucose use ratio decreased by 81.25%, acetoin,2,3-butanediol and ethanol concentration decreased by 100%, and acetate concentration increased by 209.33%, up to 5,67g/l.The als gene from Klebsiella pneumoniae CICC10011, encoding a-acetolactate synthase, was cloned and linked into pBR322. The recombinant plasmid pBR322-als, obtained by subclonig, was transformed into Klebsiella pneumoniae CICC 10011 by electro-pulse. The recombinant strains, screened by ampicillin resistance and als PCR, were named as Klebsiella pneumoniae CICC 10011-1.The enzyme activity reached 0.6367U/mg crude protein when K. pneumoniae CICC10011-1 was fermented 24 hours in shaking flask, which was three times of that of K. pneumoniae CICC 10011. Compared with the parent strain of K. pneumoniae CICC 10011, the yields of acetoin and 2,3-butanediol increased a little, and the yields of ethanol and acetate decreased a little. There was not diacetyl in the fermented fluids of K. pneumoniae CICC 10011 and K. pneumoniae CICC 10011-1. As we known above, diacetyl acted feedback suppression on a-acetolactate synthase andα-acetolactate decarboxylase probably; a-Acetolactate synthase was not the critical control point of diacetyl, acetoin and 2,3-butanediol.