Breeding Of L-isoleucine Producing Strain And Optimization Of The Fermentation Conditions On Escherichia Coli

Original strain Escherichia coli N12 with lifting the repression of L-isoleucine was modified by metabolic control of fermentation,then a high yield strain with valuable of industrial production were obtained.The main contents and results are as follows:1.The amino acid concentration was determined using reversed-phase high-pressure liquid chroatography with 2,4 Dinitrochlorobenzene and established the quantitative determination of L-isoleucine,conditions and a calculation method:50%acetonitrile:NaAc= 3:2,column temperature 30?,flow rate is 1mL/min,the detection wavelength is 360nm.Original strain Escherichia coli N12 with lifting the repression of L-isoleucine was mutated mutagenesis by diethylsulfate(DES),then L-isoleucine producing strain named E.coli NML were obtained.At 48 h,L-isoleucine production in the fermentation medium of NML(Met-+ Leu')was 2.77g/L and increased 23.3%compared to N12.E.coli NML was mutated mutagenesis by diethylsulfate(DES),then L-isoleucine producing strain named E.coli NMLL(Met-+ Leu-+ Lys-)were obtained.At 48h,L-isoleucine production in the fermentation medium of NMLL was 3.29g/L and increased 18.8%compared to NML.The amino acid concentration was determined using reversed-phase high-pressure liquid chroatography with 2,4-Dinitrochlorobenzene and established the quantitative determination of L-isoleucine,conditions and a calculation method:50%acetonitrile:NaAc= 3:2,column temperature 30 ?,flow rate is ImL/min,the detection wavelength is 360nm.2.The fermentation medium of a L-isoleucine producing strain Escherichia coli NMLL was optimized with univariate and the software Design Expert,a response surface model was established using the Box-Behnken design(BBD).The results showed that the optimum conditions for NMLL to produce L-isoleucine were as follows:glucose 116.40g/L,corn steep liquor 12.03mL/L,(NH4)2SO440.00g/L,KH2PO4 0.05g/L,MgSO4 · 7H20 2.5g/L,FeSO4 · 7H20.01g/L,CaCO3 2.0g/L.At 48h,L-isoleucine production of NMLL was 4.62g/L and increased 40.4%under optimal conditions.3.Amplified the aspartate kinase gene encoding lysC of Pseudomonas fluorescens F113 by PCR and connected it with the expression vector pSTV28,recombinant plasmid pSTV28-lysC was obtained and transferred it into E.coli NMLL.At 48h,NMLL's production was 0.83g/L,and NMLL-lysC's production was 1.35g/L,increased 62.65%compared with NMLL by inducing by isopropy1-?-D-thiogalactoside(IPTG).Indicates that the aspartate kinase gene encoding lysC cloned from Pseudomonas fluorescens F113 genome was excessive expressed in E.coli,can play its original function to promote the accumulation of L-isoleucine.Previous research found both of mutation breeding and metabolic engineering can improve L-isoleucine production,but acid production is lower than mutation breeding because of engineerin g bacteria fermentation conditions were not studied,engineering bacteria fermentation conditions should be further studied.