CRISPR Assisted Engineering Of Corynebacterium Glutamicum SNK118 For L-Ornithine Production

L-Ornithine,an intermediate metabolite of urea cycle,is an alkaline non-essential amino acid with a variety of physiological and biochemical functions.It has wide applications in medicine,food,health products and other fields.Here,Corynebacterium glutamicum SNK118,an L-arginine industrial producer with sequenced genome,was metabolically engineered for L-ornithine production through CRISPR-Cpfl-based genome manipulation and plasmid-based heterologous overexpression.Our results provided a promising 1-ornithine-producing strain and an efficient approach for enhancing cofactor NADPH in C.glutamicum strains.(1)L-Ornithine is a precursor for L-arginine synthesis.Gene argF,encoding ornithine carbamoyltransferase,was deleted to interrupt the degradation of L-ornithine to L-citrulline and L-arginine.Also,argR of arg operon,which is close to argF,was inactivated simultaneously for unnecessary interference.The resulted strain,KBJ01(SNK118?argF?argR)was obtained.The production of L-ornithine by KBJ01 reached 28.24 g·L-1 after 84 h in shaker-flask,which is 46-fold higher than that produced by JML04(SNK118?argR)(0.62 g·L-1).(2)To enhance the supply of coenzyme NADPH for L-ornithine biosynthesis,Clostridizum saccharobutylicum DSM 13864-derived gapC(encoding NADP-dependent glyceraldehyde-3-phosphate dehydrogenase)and Bacillus subtilis HB-1-derived rocG(encoding NADH-dependent glutamate dehydrogenase)were introduced into the strain KBJ01 to construct KBJ05(KBJ01/pXMJ19-CsgapC)and KBJ06(KBJ01/pXMJ19-BsrocG),respectively.The recombinant strain KBJ05 allowed production of 34.75 g·L-1 L-ornithine with a yield of 0.274 g·g-1 glucose after 84-h flask fermentation,which is 28.8%and 15.61%higher than that of the control strain KBJO1/pXMJ19,respectively.In flask fermentation,32.93 g·L-1 L-ornithine with a yield of 0.271 g·g-1 was achieved after 84 h by KBJ06.Compared with the control strain KBJ01/pXMJ19,the L-ornithine production and yield were increased by 22.1%and 14.35%,respectively.(3)Considering the unwanted accumulation of branch chain amino acids,strain KBJ07(SNK118?argR?argF?ncgl2228)was constructed,by deleting ncgl2228(encoding a branched-chain amino acid permease)in KBJ01 to alleviate the competitive branch pathway of pyruvate.After 84 h fermentation in a 500-mL flask,L-ornithine titer of 30.46 g·L-1 with a yield of 0.26 g·g-1 glucose were achieved by KBJ07.(4)Above results demonstrated that ncgl2228 deletion and heterologous expression of CsgapC or BsrocG were favorable to L-ornithine production by C.glutamicum.Hence,strains KBJ09(KBJ07/pXMJ19-CsgapC),KB10(KBJ07/pXMJ19-BsrocG)and KBJ11(KBJ07/pXMJ19-CsgapC-BsrocG)was constructed.After fermentation in shake flasks for 84 h,strain KBJ11 allowed the highest L-ornithine titer of 35.85 g·L-1 with a yield of 0.28 g·g-1 glucose,which was markedly improved compared with those of KBJ08((KBJ07/pXMJ19)(30.34 g·L-1 and 0.249 g·g-1).Notably,the L-ornithine titer produced by strain KBJ11 was 27%higher than that of KBJ01(SNK118?argFAargR).(5)To further investigate the performance of strain KBJ11,fed-batch fermentation in a 5-L bioreactor was carried out.Strain KBJ11 allowed the L-ornithine titer of 60.1 g·L-1 after 85 h of fermentation.By fed-batch fermentation in a 10-L bioreactor,L-Omithine of 88.26 g·L-1 with productivity of 1.23 g·L-1 h-1(over 72 h)and yield of 0.41 g·g-1 glucose was achieved by strain KBJ11 after 72 h of fermentation with optimized culture conditions.This result represents the leading level of L-ornithine production by microbial fermentation.