Preliminary Analysis Genome Of Corynebacterium Glutamicum ATCC14067 And Construction Of Its Recombinant Producing L-Arginine

L-arginine is a semi-essential amino acid. It is also an important metabolite of the body, with a wide range of application in food, medicine, feed and other industries. Brevibacterium flavum is a kind of gram-positive and facultative anaerobic conynebacte ria, which is used to produce amino acids. In this research, we preliminary analysis the genome sequencing results of ATCC14067. In order to improve the production of arginine, we try several methods to constructing knockout system in ATCC14067. In additio n, the arg B, arg C, arg D, arg E, arg J and car AB and arginine transporter gene lys E and arginine transporter were over-expressed. The specific contents of this study are as follows:We preliminary analysis ATCC14067 genome sequencing results which using Sanger sequencing and Solexa sequencing techniques work together to complete. Meanwhile, by the use of relevant software, we obtain the basic information of genome, analysis the tandem repeat sequences, insertion sequence, genomic island and phage sequence.Try several methods to knock out, including Cre/loxp knockout system and suicide vector pk18 mobsac B. Cre/loxp knockout system use overlap extension PC R to get the cre/loxp knockout box, it directly transferred into ATCC14067 in the form of fragment or with the help of a vector. Suicide vector p K18 mobsac B-Δarg B was constructed by crossover PCR and then it was transferred into ATCC14067 by electroporation.By PCR amplification we get gene lys E and arginine transporter, plasmids p EC-XK99E-csp-lys E and p EC-XK99E-csp-arg transporter was transformed to ATCC14067 to get recombinant strain 14067-p EC-XK99E-csp-lys E and 14067-p EC-XK99E-csp-arg transporter. Then, recombinant plasmids p EC-T18-arg C, p EC-T18-arg C-arg B, p EC-T18-arg C-arg JBD, p EC-T18-arg C-arg E, p EC-T18-arg E, p EC-T18-arg E-arg B, p EC-T18-arg E-arg JBD, p EC-T18-car AB, p EC-T18-car AB-arg B, p EC-T18-car AB-arg E and p EC-T18-car AB-arg JBD were transformed to above- mentioned strains respectively. The best expression of L-arginine strain increasing by 91% relative to ATCC14067, in the other hand, the fluorescence quantitative PCR analysis indicated that the expressions of gene arg B, arg C, arg D, arg E and arg J were 2.1 times respectively of that in strain ATCC14067 on m RNA level. Gene lys E has the highest expression in 14067-T18-AB-JBD-lys E on m RNA level, its expression was 2.6 times respectively of that in strain ATCC14067 on m RNA level. Gene arg transporter has the highest expression in 14067-T18-C-JBD-at on m RNA level, its expression was 2.52 times respectively of that in strain ATCC14067.