Breeding Of L-Isoleucine Producer And Its Optimizing Conditions In Fermentation

In this paper, the fermentation principles of metabolism control were applied into original strainimprovement of Corynebacterium melassecola ATCC17965 for the overproduction of L-Isoleucine, which wasnot utilized into breeding of the L-Isoleucine producer by precursor. Then the compositions of medium and theconditions in fermentation were studied. The metabolic fluxs of the original strain and its mutants weremeasured and their metabolic flux distribution maps were established and compared according the theory ofmetabolic flux analysis. The main research contents and results are as follows:1. Paper chromatography–spectrophotometry was applied into determining the L-Isoleucine in broth wasstudied , the conditions in the course of determining the L-Isoleucine was established, the reclaim of averagepercentage of this method was 99.25%, so this method is accurate to determine the concentration ofL-Isoleucine in broth.2. The L-Isoleucine producer was derived from the original strain Corynebacterium melassecolaATCC17965 by chemical and physical mutation methods, the plate screening with amino acid analogues:sulfaguanidine (SG), L-leumethyl ester HCL (Leu-ME),α-amino-β-hydroxyvalic acid (AHV), ethionine(Eth) and used succinate as sole carbon sourse. The L-Isoleucine producing level of I31-5(SGr+ Leu-MEr+Leu-+AHVr+Sucg+Ethr) was 14~15g/L under un-optimization condition.3. The effects of seed medium compositions and culture conditions on the L-Isoleucine production inflask were considered. The optimum seed medium compositions were determined as: saccharose 30 g/L,CH3COONH4 7.5 g/L, corn steep liquor 40 g/L, KH2PO4 1.0 g/L, MgSO4·7H2O 0.5 g/L, biotin 70μg/L, CaCO310 g/L, pH 7.0;and the better conditions of culture seed was culturing 11 h at 30℃ on a rotary shaker ( 85r/min ) with 40mL culture broth in the flask( 250 mL ) .4. The effects of fermentation medium compositions and conditions in fermentation on the L-Isoleucineproduction in flask were considered. The optimum fermentation medium compositions were determined as:glucose 120 g/L, (NH4)2SO4 50 g/L, corn steep liquor 15 g/L, KH2PO4 2.0 g/L, MgSO4·7H2O 0.5 g/L, biotin5.0μg/L, VB1 600μg/L, Leucine 0.1 g/L, ZnSO4·7H2O 7.5 mg/L, Mn SO4·4H2O 12.5 mg/L, FeSO4·7H2O 7.5mg/L, CaCO3 30 g/L, pH 7.5;and the better conditions of fermentation was fermented 72 h at 30℃ on arotary shaker ( 100 r/min ) with 12.5mL medium in the flask( 250 mL )inoculated by 10 percent . Strain I31-5produced 19.2 g/L of L-Isoleucine on average and 21.3g/L of L-Isoleucine as highest under above conditions,which increased 30%~37% more than before.5. The simplified model of the L-Isoleucine biosynthetic network was constructed based on the theory ofmetabolic flux analysis. The maximal stoichiometric theoretical yields of L-Isoleucine was 0.8 mol/mol, thatwas 0.75 mol/mol while considering assistant factors. The metabolic flux distribution maps of the originalstrain and its mutants were obtained, compared and analyzed. The flux partitioning at the 6-phosphate-glucosenode and phosphoenol-pyruvate node changed greatly. The ratio of HMP and EMP at the6-phosphate-glucose node, which was 3.637∶1 in the original strain, became 1.504∶1 in the strain I31-5. Theflux to the pathways of L-Isoleucine from the phosphoenol-pyruvate node, which was 0 in the original strain,became 33.380 in the strain I31-5, and the flux to the "other acids" and "outgrowth" from thephosphoenol-pyruvate node, which was 100 in the original strain, became 33.240 in the strain I31-5.