Study On The Design And Application Of Rare Codons For L-lysine Of Corynebacterium Glutamicum

L-lysine is the only basic amino acid in the aspartic acid family with a primary amino side chain on the?-carbon atom that can be used for protein synthesis.It is widely used in feed,food and pharmaceutical industries mainly in the form of L-lysine hydrochloride and L-lysine sulfate.In this paper,we analyzed the Corynebacterium glutamicum 23604 genome by whole-genome sequencing,replaced the L-lysine transporter RNA promoter by homologous double exchange method to make AAA an L-lysine rare codon,and used the L-lysine rare codon to establish a link between the fluorescence intensity of cellular expression and intracellular L-lysine content.And the strains with enhanced L-lysine production were successfully screened after breeding by ARTP mutagenesis.The main results are as follows.(1)The C.glutamicum 23604 genome was sequenced by Illumina Mi Seq and Pac Bio RSII to obtain the whole genome sequence of C.glutamicum 23604.the GC content of the C.glutamicum 23604 genome was 54%,with a total of 2962 protein-coding genes.After functional annotation of the genes,2872 protein sequences,1990 GOs and 1196KEGG annotations were obtained.After advanced annotation of the genome,2872functional annotations of virulence factors,88 annotations related to carbohydrate-active enzymes,63 annotations of genes and their corresponding pathogen-host interactions,187annotations of signal peptides,2872 annotations of lipoproteins,and subcellular localization information of proteins were obtained.By analyzing the distribution of amino acid ratios and the frequency of use of each amino acid codon in the genome,it was determined that C.glutamicum 23604 does not have a natural L-lysine rare codon.(2)The gene sequences of green fluorescent protein egfp,ebfp and m Cherry were obtained by NCBI search,and the codon AAG encoding L-lysine was replaced with the L-lysine rare codon AAA to obtain the mutant genes egfp ~M,ebfp ~M and m Cherry ~M,respectively,and were ligated into the pEC-XK99E expression vector to obtain pEC-XK99E-egfp ~M,pEC-XK99E-ebfp ~M and pEC-XK99E-m Cherry ~M.pEC-XK99E-egfp ~M was transferred into C.glutamicum by electrotransformation and detected and screened after10 h of expression induction by 0.6?M IPTG.Compared with the control strain,C.glutamicum transfected with pEC-XK99E-egfp ~M had good fluorescence signal,green fluorescent protein containing L-lysine rare codon AAA could be expressed normally in C.glutamicum,and single cells with strong fluorescence intensity could be sorted using flow cytometry,so the mutant gene egfp ~M was identified as the screening marker.(3)The gene sequence of the leaky expression promoter Pgit1 was obtained by NCBI search,and the L-lysine transporter RNA promoter of C.glutamicum with anticodon TTT was replaced by using the knockout vector p K19mobsac B-Pgit1 containing kanamycin resistance gene and sucrase gene to reduce the expression of tRNA with anticodon TTT level.The obtained recombinant C.glutamicum Pgit1 was transformed into pEC-XK99E-egfp ~M and subjected to ARTP mutagenesis,and the mutagenized strains were cultured and screened by flow cytometry for a total of 31 single cells with high fluorescence intensity.The L-lysine content of ARTP mutagenized bacteria 21 increased by 12.5%,compared with the control bacteria,indicating that the strains with improved L-lysine production were successfully screened by using L-lysine rare codons.