Construction Of Lactate Dehydrogenase-deficient Mutant Of Klebsiella Pneumoniae And Its Application

1,3-Propanediol(PDO) is an important organic chemical material, it is widely used in the fields of polymers, cosmetics, foods, lubricants, and medicines, etc., and the most important characteristics of 1,3-Propanediol is its usage in the polyester industry, as it is the monomer for making the excellent new polyester fiber Polytrimethlene Terephthalate(PTT). 1,3-propanediol production by fermentation has recently become a research hot spot for its amiability of the environment. In practical fed-batch culture with fermentation of glycerol by Klebsiella pneumonia for PDO, a number of byproducts, such as lactate and acetate etc. will be produced simultaneously. Cell growth inhibition of lactate will impair the 1,3-propanediol production and make purification a troublesome task, while this can be effectively solved by blocking the pathways leading to lactate.Target gene ldh (780bp) encoding an essential enzyme of lactate pathway was amplified by PCR using K. pneumoniae AC02 genomic DNA as template. The primers for ldh referred to the sequence of K. pneumoniae MGH 78578 published on NCBI. A suicide plasmid applied to K. pneumoniae was constructed from pGP704-Ap after the Ap-resistant gene of which was replaced by Cm from pMCm. Finally, a recombinant suicide plasmid pGP704-Cm-ldh was constructed, and transferred into K. pneumoniae through the procedure of electronic transformation and double parents conjugation. The partial plasmid integrated into K. pneumoniae due to homologous recombination. Thus ldh gene was disrupted and a mutant with ldh gene knocked out was obtained.One lactate dehydrogenase-deficient mutant was obtained by knocking out the ldh gene of lactate dehydrogenase (LDH) of K. pneumonia AC02. The LDH activity of the mutant was only account for 7% of that of the parental strain. The fed-batch fermentation of 1,3-PDO by mutant was studied. When glycerol fermented to generate for PDO under microaerobic conditions, the concentration of 1,3-PDO and 2,3-BD, and conversion rate were 15%, 88% and 32% higher than that of wild strain K. pneumonia AC02, respectively. And lactate was 98% lower than that of K. pneumonia AC02. Besides, it helped to simplify the process of purification for 1,3-PDO, and reduce 21% consumption of base.