Knockout Of Acetyltransferase Gene In Xanthomonas Campestris And Studies On Mutants Genetic Characteristics

Xanthan gum is an extracellular polysaccharide produced by the fermentation of Xanthomonas campestris. Because of its special physical and chemical features, xanthan gum is widely used in various industries. The biosynthesis of xanthan gum is a meticulous and complex pathway which should require the participation of all kinds of enzymes and protein factors, such as acetyltransferase, uridine diphosphate glucose dehydrogenase(UDPGD), ketal pyruvate transferase, five specific transferases and polymerase.The acetyltransferase is a kind of very important enzyme in the production of xanthan gum, because it will affect the content of xanthan gum in the acetyl groups. While the stability and partial physical and chemical properties of xanthan gum are determined by its degree of acetylation. Therefore, the acetyltransferase gene of Xanthomonas campestris was selected as the object of this study.Firstly, the genome DNA was extracted when Xanthomonas campestris CICC 10258 acted as the test strain. According to the gene sequence, specific primers for PCR amplification of the acetyltransferase gene fragments were designed. The amplification of gene fragments were connected with pMD19-T vector and got the recombinant plasmids,and successfully cloned the acetyltransferase gene. Then, the recombinant plasmids were digested with EcoRI to obtain the acetyltransferase gene fragments and so did suicide plasmids of pK18 mobSac B, and they were linked together to construct a recombinant suicide plasmid vector. After confirmation, these recombinant suicide plasmid vectors were transformed into the competent cells of Xanthomonas campestris CICC 10258. Finally,mutants of the acetyltransferase gene were successfully obtained. Taking Xanthomonas campestris CICC 10258 as a control, at last, the genetic characteristics of the mutant were analyzed and detected.The results showed that the experiments knocked out the acetyltransferase gene and got mutants. The growth of mutants was restrained in the nutrient broth medium. At the same time, the production of xanthan gum reduced and three kinds of extracellular enzymes activity also was suppressed for the mutant. It was proved that the acetyltransferase gene had a relationship with the synthesis of xanthan gum and three kinds of extracellular enzymes activity, taking Xanthomonas campestris CICC 10258 as a control.Not only does this research yield foundation about the construction of genetically engineered bacteria, but it can provide some experimental dates and theoretical basis for the research of Xanthomonas campestris produced xanthan gum.