Construction Of ACK Gene Deletion Engineering Bacteria Of Veillonella Parvula And Its Characterization

Ketosis and fatty liver (pregnancy toxemia or fat cow syndrome), are important common dairy cow disease, their primary pathology characteristics are negative energy balance. Energe negative balance is caused by propionic acid deficiency and/or increase of demand accounts for the disorders in energy metabolism. Under normal circumstances, carbohydrates in the rumen of ruminant animals are fermented to volatile fatty acids (VFA), such as acetic acid, propionic acid and butyric acid, by a variety of bacteria that contain the corresponding enzymes. VFA are precursors for the synthesis of fat and glucose. Approximately 90% of glucose required by ruminant animals is derived from gluconeogenesis. The major glycotropic precursor is propionic acid. Ketosis is one of the most harmful cow diseases, and is caused by decreased dry matter intake and insufficient propionic acid intake The traditional preventative and therapeutic measures involve supplying cows with precursors of glucose, such as propionate. Successful production of propionic acid via ruminal propionic acid-generating bacteria has not yet been reported. The aim of this study was to investigate the effect of propionic acid-generating bacteria on ruminal propionic acid metabolism in vitro and, thus, to demonstrate the possibility of producing propionic acid in vivo via ruminal microbial fermentation.In this study, we extracted the genome of veillonella parvula, and obtained the two wings of the ACK gene by anchoring PCR. By BLAST, the results showed the sequence obtained was ACK gene.On the basis of the sequence of GenBank that the ACK protein amino acid sequence, we found the active center of veillonella parvula (about 900 bp), introduced a acid-resistant gene between them, and finally it was linked with pUC18 vector building suicide plasmid using homologous recombination successfully. Two engineering bacteria were screened from the plenty transconjugants with selectivity culture medium through donor and receptor bonding test. PCR identification and ACK analysis of enzyme activity revealed that they belong to ACK gene deletion strains resistant fluoroacetic.Primary substrates were cultured with wild-type strains and genetic engineering bacteria for 48 h respectively in vitro fermentation of ruminal fluid. The results were shown as follows: the lactate in the culture fluid was used by wild-type strain and genetic engineering bacteria, VFA produced is mainly propionic acid, the type of their fermentation was prone to propionic acid-type. Compared to wild-type strains, the concentration of lactate and the ratio of acetate to propionate were obviously decreased in the culture fluid with genetic engineering bacteria.