Influence Of LdhA Knockout On The Microbial Production Of2,3-butanediol By Klebsiella Pneumoniae

Microbial fermentation produces2,3-butanediol (2,3-BD) as a good opportunity to extend the2,3-BD application. To make up for the shortcomings of chemical production of2,3-BD process such as cumbersome, difficult to control, low yield and many other problems, it expands the value of2,3-BD application.2,3-BD can be widely used in the fields of energy sources, food service, chemical industry, and pharmaceutical. As a high-performance fuel additives and its derivatives:diacetyl, methyl ethyl ketone,1,3-butadiene are all high value materials. Kkbsiella as the predominant strain in the production of2,3-BD has a broad substrate spectrum, can completely metabolize substrates to products but with simple by-products, thus is the potential strain for industrial production. However, the accumulation of lactic acid by-product is still an obstacle to2,3-BD conversion. The increase of lactic acid in the broth can inhibit cell growth and reduce product conversion rate. Accumulation of lactic acid isn't conducive to post-processing either, significantly increases the production costs. It has reported that the ldhA gene encoding lactate dehydrogenase is a key enzyme in lactate metabolism branch. Therefore, this paper is going to construct one lactate dehydrogenase deficient recombinant strain of AT. pneumoniae with gene knockout technology.A DNA fragment of approximately990bp marked as ldhA which coding for lactate dehydrogenase was cloned from K. pneumomae CICC10011. Primers were designed according to the Klebsiella pneumomae subsp. pneumoniae MGH78578genome sequence published in Genebank. According to websites of NCBI Blast, the homology of ldhA from K. pneumoniae CICC10011was83%identification with K. pneumoniae MGH78578. Tcr gene (1400bp) coding for tetracycline resistant gene was also cloned from pBR322plasmid. Through splicing operation with endonuclease and ligase in vitro, the Tcr gene inserted into ldhA gene, one homologous recombinant fragment:ldhAl-Tcr-ldhA2was constructed for the gene knockout. After resistance experiments. PCR, activity determination, five recombinants with ldhA gene knocked out obtained by X red recombination system.By shaking flask experiments, the2,3-BD conversion rate was almost the same with the original bacteria, but the acetoin conversion rate significantly reduced, besides other organic acids production increased obviously. To get a high2.3-BD conversion rate, the fermentation conditions need to be optimized.