Molecular Breeding Of Klebsiella Pneumoniae For 1,3-propanediol Production

As an important chemical material, 1,3-propanediol (1,3-PD) has numerous promising properties and applications. Compared with chemical synthesis, microbial fermentation processes to 1,3-propanedio! have many obvious advantages in that they typically use renewable feedstocks and reduce pollution to a great extent. And they have attracted particular attention and become the focuses of research. The inhibition potentials of substrate and products on the growth of microorganism cell will limit the product titer, the yield of 1,3-propanediol on glycerol and the productivity of 1,3-propanediol, and then make the product recovery and purification a troublesome task. This can be effectively solved by blocking the pathways leading to the byproducts that are of no benefit to 1,3-propanedil production.The 1,3-propanediol titers of 36.09 g 1-1 and 45.22 g 1-1 have been obtained by using two-stage fermentation with two substrates and microaerobic fermentation in the fed-batch culture of Klebsiella pneumoniae M5al, respectively, which greatly exceeded the level that can be achieved in China.Two pta- mutants have been selected by using suicide substrate after the mini-Tn5 transposon insertion mutagenesis of Klebsiella pneumoniae M5al. When used in microaerobic fermentation, the amount of acetate produced by the mutants reduced to less than 50% of the parent strain, and the yields improved whereas the 1,3-propanediol titers and productivities decreased.Two suicide vectors of pWW and pVV carrying the 871bp segment ofadhE and 805bp segment of IdhA amplified by PCR respectively were constructed and six mutants ofadhE and four mutants of ldhA- were obtained by homogenous recombination between the partial adhE or IdhA in the vectors and the Klebsiella pneumoniae M5al chromosome. The acetaldehyde dehydrogenase (ACDH) and alcohol dehydrogenase (ADH) of the adhE mutants and parent strain were assayed with a result that the ACDH activities of mutants were less than 10% of the parent strain and the ADH activities of mutants are about 10% of the parent strain. The lactate dehydrogenase (LDH) were also assayed and the LDH activities of IdhA were less than 7% of the parent strain. Whether the mutants are able to improve the 1,3-propanediol titers, yields and productivities will be investigated in the fermentation later.