| Deficiency of feed intake and increased metabolic demand will usually lead to negative energy balance in periparturient dairy cows. Selenomonas ruminantium is one of dominant bacteria in the rumen. Carbohydrates from feed can be utilized by the bacteria to generate propionic acid, acetic acid and other metabolites in order to provide enegy for ruminant. To study the cell metabolic network of dominant microbial in rumen and finally improve negative energy balance in periparturient dairy cows, we knocked out acetate kinase gene which codes the key enzyme in generation process of acetic acid and constructed the Selenomonas ruminantium ack deleted mutant K6Δ(ack). Finally, the fermentation characteristics of the K6Δ(ack) was analyzed.Firstly, we analyzed the evolutional position of Selenomonas ruminantium K6 and screened out 6 strains from all the bacterium who had near genetic relationship with K6. Amino acid sequence of ack from 6 strains were submitted to BlockMaker program to make BLOCKS from MOTIF. We found 9 relatively conservative BLOCKS and for every BLOCK design degenerate primer for acetate kinase of Selenomonas ruminantium using CODEHOP program. Chose one pair of degenerate primers named ACKSe and used the propionic acid producing bacteria Selenomonas ruminantium K6 genome DNA as template to make degenerate PCR. 749bp PCR product was obtained. Similarity alignment showed that the products of the cloned DNA were similar to those of acetate kinase gene.Acid-resistant lactobacilli was successfully isolated from rumen fluid of healthy dairy cow by the anaerobic isolation culture method. The bacteria was preliminary identificated to Lacbobacillus casel by Gram staining method and biochemistry identification morphology. After that, the 16S rDNA phylogenetic analysis were used to identify the bacteria. The 16S ribosomal RNA primer was designed on the basis of the sequence of GenBank,and 16S ribosomal RNA gene was cloned.The result showed that the obtained sequence was 99% identical with those of Lacbobacillus casel in GenBank. Based on the above mentioned results,it was confirmed as Lacbobacillus casel. The 16S rDNA sequence and ffh gene full-length sequence of Lactobacillus casei were submitted to Genbank, and their accession numbers were FJ171331 and FJ227541 respectively. The ack fragment (749bp) cloned from Selenomonas ruminantium K6 was linked with vector pUC18 to construct the pUC18-ack plasmid. The acid-resistant gene ffh cloned from Lactobacillus casei was inserted into plasmid vector pUC18-ack, thus the suicide plasmid vector pUC18-ack:: ffh was obtained. Then ack gene of K6 genome was inactivated due to site-directed inserting of the pUC18-ack::ffh by homologous recombination, so the acetic acid can not be generated. Finally three ack-deleted mutants were successfully screened from the numerous transconjugants with selective culture medium containing fluoroacetic acid. There numbers were 081110-1, 081110-2 and 081110-3 respectively. the three mutants were identified by 16S rDNA phylogenetic analysis, the result showed that the obtained sequence was 100% identical with Selenomonas ruminantium K6. Enzyme activity assays showed that the AK activity in the three mutants decreased significantly in compared with the wild type K6. Based on the above mentioned results,it was confirmed that ack gene deleted mutant K6Δ(ack) of Selenomonas ruminantium was obtained.Acid-resistant experiments showed that both strains were inhibition for growth in CDC plate which pH is 5.5, while no bacterial colony can be observed in either CDC plate when pH is 5.0. This experiments showed that K6Δ(ack) has no better acid resistance than the wild strain K6. Possible reason is that ffh gene is a major acid-resistant genes, may not be the only acid-resistant gene. Compared to the wild type, the mutant grew more slowly at pH 6.0 and 37℃, with a lower specific growth rate, likely due to the partially impaired pta-ack pathway.Primary substrates (lactic acid and pyruvate) were cocultured with K6 and K6Δ(ack) for 48h, respectively, in order to observe the fermentation of pure culture medium in vitro. Culture fluid was sampled at 0,2,4,6,8,10,12,24,36,48h for analysis of pH and VFA. The results were as follows: the lactate and pyroracemic acid in the culture fluid were utilized by the strains of K6 and K6Δ(ack). Compared to strains K6, propionic acid production of K6Δ(ack) did not significantly enhanced, lactic acid generation was significantly inhibited, only few acetic acid was detected. The ratio of acetate to propionate were obviously reduced in the culture fluid of the strain K6Δ(ack). The effect demonstrating that the transponed engineering bacteria K6Δ(ack) belonged to a deleted strain of acetic acid-producing gene.In vitro fermentation of ruminal fluid, primary substrates (lactic acid and pyruvate) were cultured with K6 and ack deleted mutant K6Δ(ack) for 48h, respectively. Culture fluid was sampled at 0,2,4,6,8,10,12,24,36,48h for analysis of pH and VFA. The results were as follows: substrate in the culture fluid was utilized by the strains of K6 and K6Δ(ack). Compared to strains K6, propionic acid production and acetic acid production of K6Δ(ack) were both decreased. In addition, there were no differences in production of butyric acid, pH, concentration of lactic acid, the ratio of acetate to propionate between K6 and K6Δ(ack). This result may be relationship with slow growth rate of K6Δ(ack). The next step will focus on improving the growth and vitality of the engineering strain K6Δ(ack).
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