Construction Of Engineered Strains For L-ornithine Production Based On Metabolic Engineering And Fermentation

L-Ornithine is an important chemical ingredient with applications in the food and pharmaceutical industries as a dietary supplement,as it is kown to be efficacious in the treatment of liver diseases and wound healing.We performed metabolic pathway engineering of a hyper arginine producing Corynebacterium crenatum?subspecies of C.glutamicum?for improved L-ornithine production.We initially attempted to enhance L-ornithine accumulation by deleting the side pathway and alleviating the ornithine inhibition of OATase but instead recorded unexpectedly low enzyme activity.Thus,to enhance L-ornithine biosynthesis,in our next attempt,we successfully co-expressed the E.coli argA and S.marcescens argE genes in C.crenatum in order to mimic a linear transacetylation pathway.Using E.coli as orginal strain,deleting the side pathway and tandam expression gene to improve L-ornithine production.Based on the mutated strain,argF was deleted to block the conversion of L-ornithine to L-citrulline and L-arginine by pK18 plasmid.The resulting strain,Cc-QF-1,became an L-arginine and L-proline auxotroph,the 4.2 g/L titer of L-ornithine obtained from the Cc-QF-1strain was thought to be due to the stronger feedback inhibition of CcOATase by ornithine in C.crenatum.Homologous alignment and analysis of the structure model indicated that the side chain of the catalytic residues T150 and M176 were potential L-ornithine binding sites.Thus,site-saturation mutagenesis was applied to these two residues.The specific activity of the wild-type CcOATase is 112.6 U/mg,and its I^R_0.5.5 is 5.2 mM.These results suggested that different sizes and shapes of the side chains of residues 150 and 176 had a significant impact on the substrate affinity and catalytic efficiency of CcOATase and indicated that these two residues could be not only the product binding site but also the substrate binding sites and thus not effective for alleviating L-ornithine inhibition with regard to site-directed mutagenesis.All enzymes for ornithine synthesis were obtained through BLAST searches of the genomic sequences.the recombinant argE?encoding NAOD?from S.marcescens showed higher activity than the NAOD genes from E.coli BL21?DE3?,K.pneumoniae and B.subtilis.The specific activities of the recombinant EcNAGS and SmNAOD at 25°C were 50.4 U/mg and 580.5 U/mg,respectively.The optimal temperature of the recombinant EcNAGS was 37°C.The optimal pH of the recombinant EcNAGS was 9.0,slightly different from NAGS from other organisms,which exhibited an optimal pH of 8.0.Additionally,the EcNAGS exhibited good stability at pH values ranging from 6.0 to 9.0.The argA and argE co-expression system was used to mimic the linear route of the L-ornithine biosynthesis pathway.In shaker-flask experiments,the recombinant strain Cc-QF-4 allowed the production of 12.6 g/L of L-ornithine,with an overall productivity of 0.673 g/L/h at 72 h after inoculation.The production performance of the strains Cc-QF-1,Cc-QF-2,and Cc-QF-4 was investigated in a 5-L bioreactor fermentation.the concentrations of cells,glucose,and L-ornithine,and the L-ornithine production rate was detected.The recombinant strains showed a slight advantage in L-ornithine production between 24 and 36 h.At the end of fermentation,L-ornithine yield increased markedly,to 40.4 g/L,with an overall productivity of 0.673 g/L/h in the recombinant strain Cc-QF-4,which was approximately 28.2 and 41.8%higher than those of Cc-QF-2 and Cc-QF-1,respectively.The results obtained in this work demonstrate transacetylation increased was efficient to improve L-ornithine production.Metabolic engineering to construct recombinant E.coli for higher L-ornithine production,the results indicated knock out triple genes argI,speF,and speC was accelerated the accumulation of L-ornithine.After 36 h fermentation,the recombinant strain EC-O-0,produced0.35 g/L L-ornithine.Moreover,Tandem expression genes for the L-ornithine synthesis pathway in the expression vector pET-28a by Gibson assembly.The recombinant plasmid was transformed to EC-O-0,obtained strains EC-O-5/EC-O-6.In shaker-flask experiments,the recombinant strain EC-O-5/EC-O-6 allowed the production of 4.28,4.71 g/L of L-ornithine respectively at 36 h after inoculation.This study indicates that overexpression of key enzymes in metabolic pathways has a significant effect on L-ornithine production,further demonstrating that the use of metabolic engineering to make amino acid-producing bacteria is one of the best ways.