Combining Metabolic Engineering And Adaptive Evolution To Construct High-production Strains Of L-ornithine In C.glutamicum

L-ornithine is an intermediate of polyamine anabolism and an importantconstituent of the urea cycle. It is effective in the treatments of liver diseases andrelease deposit of amine. L-ornithine has been found an increasingly wide utilizationin medicine, healthy and food fields. Corynebacterium glutamicum is the industrialproduction strain of several important amino acids such as L-lysine, L-arginine andL-glutamate，containing L-ornithine. Adaptive laboratory evolution（ALE） is culturingthe microorganism in a certain environment pressure, and long-term evolution forgaining an improved strain. ALE is applied widely for selecting resistance, extendingsubstrate, producing special product and improving production.This study applied gene over-expression, gene knockout and adaptive evolutionto construct the high-productivity strains of L-ornithine based on C.glutamicum（ΔargFΔproB）（ΔAP）. The results of the paper were as following.This experiment constructed over-expression vectors containing pgi, pfkA, gap,pyk, pyc, gltA, gdh, argB and argJ, then transfered the resulting plasmids into△AP,respectively. The results showed over-expression of these genes could improve theL-ornithine production with the expection of pyc and gltA. The over-expression of argB and argJ could enhance L-ornithine production significantly in△AP. The genesof gap and gdh enhance L-ornithine production and couple with the regenerating ofreducing power. Therefore, we over-expressed the gene of NADP-dependentglyceraldehyde-3-phosphate dehydrogenase （gapC） and that of NADH-dependentglutamate dehydrogenase（rocG）. The results showed the over-expressions of gapCand rocG could improve L-ornithine concentration effectively. Further weco-overexpressed above several genes in△AP. The co-expression of pyk-pfkA couldenhance L-orntihine production, that of gapC-rocG couldn’t. According to the results,we infered that enhancing the supply of glutamate can improve L-ornithineproduction, and the reactions catalyzed by acetylglutamate kinase and bifunctionalornithine acetyltransferase/N-acetylglutamate synthase are the rate-limiting nodes forL-ornithine synthesis in△AP.we deleted the speE gene to block the passway of ornithine to polyamine in△AP. The ornithine production of the resulting strains, C.glutamicum（ΔargFΔproBΔspeE）（ΔAPE）, increased10%compared with parent strain. Theeffect of CaCO₃and glucose on ornithine production in ΔAPE also was investigated.The investigation indicated that CaCO₃and glucose affected significantly ornithineproduction, and the fermention containing8%glucose and3%CaCO₃could maintainneutral pH value and facilitate ornithine producing in flask. The deletion of argR wasperformed in ΔAP and ΔAPE, respectively. The resulting strains, C. glutamicum（ΔargFΔproBΔargR）（ΔAPR） and C. glutamicum（ΔargFΔproBΔspeEΔargR）（ΔAPER）, produced more ornithine than the parent ΔAP, increasing15%and22%,respectively. Knockout of argR decreased the growth markedly.The tac-M is a strong promoter in corynebacteria. This paper ligated it to gapCand rocG, then inserted the resulting fragments into expression vector pEC-XK99Eand integration vector pK-JL. The resulting over-expression plasmids and integarationplasmids were electro-transfered into ΔAPER, respectively, to construct a series ofstrains△APER::Ptac-M-gapC,△APRE::Ptac-M-rocG,△APE::rocG-R::gapC and△APER-Ptac-M-gapC,△APER-Ptac-M-rocG,△APER-Ptac-M-gapC-rocG. Theintracellular NADPH concentration of the strains containing gapC or rocG was positively related to L-ornithine, and both was higher than that of ΔAPER. The outputof qPCR showed the mRNA levels of the genes, relative to generating NADPH （gndand icd） and NADH（gap and odhA） in these strains were downregulated comparedwith that in ΔAPER. It implied that the expression of gapC and rocG did improveNADPH content and L-ornithine production. However, the required fermentioncondition and orntihtine accumulation rate were different in these engineered strains.In this paper, we used adaptive evolution strategy to get a high-yield ornithinestrain, APE6937, originated in APE. The strain harbors improved properties ingrowth and adaptive ability to tween80and temperature. No base mutants were foundin genes of pgi, fkA,gap,pyk,pyc,gltA,gdh,argB and argJ in the evolved strain. But themRNA levels of above genes were more than2-fold high with respect of the parent’s.The mRNA levels of gdh and argJ were highest, and that of argB was relative low,among them. So we over-expressed argB in APE6937, and found it improve9%L-ornithine production.To take into account argB is regulated by ArgR, we infered that the relative lowmRNA levels of argB, are caused by the repressor of ArgR in APE6937. Therefore,we deleted the argR from APE6937, to get APE6937R42. The L-ornithineproduction increased27%compared with APE6937. The mRNA levels of genes inthe pathway of glutamate were upgrade more than2-fold, and argB and argJ wereupgrade over9-fold compared with APE. It confirmed that ArgR is the rate-limitingfactor for L-ornithine producing in APE6937, and increasing glutamate availabilitycan facilitate L-ornithine production in APE6937R42.APER was not as good as APE6937R42in growth and L-ornithinesynthesis. The later had more than3-fold NADPH content, and2-fold mRNA levelsof zwf, gnd and icd, and1.8-fold mRNA levels of ppnK, compared with that of theformer. It implied that improved intracellular NADPH content led to the increase ofL-ornithine production in APE6937R42. L-ornithine production of APE6937R42reached24.1g/L in5-L fermentor.