Breeding Of L-Isoleucine High Producing Strain Via Global Transcription Machinery Engineering

L-isoleucine is one of the three branched chain amino acids.Due to its special structure and function,L-isoleucine plays an important role in the metabolism of mammalian.In order to get stable mutants with higher L-isoleucine production,directed mutation was exerted on an L-isoleucine producing strain of BF3512 based on global transcriptional machinery engineering strategy.The main contents and results are as follows:1.Gene sigA encoding the main sigma factor was cloned from brevibacterium flavum BF420,recombinant cloning vector pMD18-T-sigA was constructed and transformed to Ecoli JM109,and the sigA gene of BF420 was sequenced.Two rounds of error-prone PCR was performed using sigA gene as the template,an approximately 106 mutant library was constructed by ligating the error-prone PCR products with pZ8-1 plasmid and transformed to Ecoli JM109.Plenty recombinant plasmids DNA harboring mutant sigA were extracted and transformed into brevibacterium flavum BF3512,which contributed to a transcript mutant library of approximately 104 mutanted clones.2.The mutant library was screened by growth circle method:The mutant library with approximately 104 strains harboring the mutated sigA gene was screened by growth circle method using an L-isoleucine auxotrophic strain Corynebacterium crenatum as indicator bacteria,and 47 strains showing larger growth ring than the starting strain were selected.Of which,19 excellent mutants with L-isoleucine production over 4.5g/L were screened out via growth circle method and paper chromatography.The genetic stability of mutant strain BF3-45 was studied,the strain BF3-45 showed genetic stability after 5 times genetation passage with L-isoleucine production at about 4.87g/L,which was increased about 60%compared to its parental strain BF3512.3.The flask-shaking fermention conditions of the strain BF3-45 were optimized.The optium seed medium was determined by testing biomass and the L-isoleucine yeild.The composition of the fermentation medium and fermentation condition were decided via single factor method,the optimal component ratio were optimized by response surface methodology.After 96h culturing under the optium fermentation condition,the L-isoleucine yield of BF3-45 reached to 9.17 g/L,which was increased 72%compared to that of the initial fermentation conditions.