Screening And Metabolic Optimization Of Corynebacterium Glutamicum For L-isoleucine Production

C.glutamicum LD320 is an L-isoleucine producing strain obtained by multiple of random mutagenesis, therefore,it is very difficult to further improve the yield of L-isoleucine in this strain by random mutagenesis. In this work, metabonomics technology and proteomic analysis were employed to reveal the L-isoleucine production mechanism in C.glutamicum LD320, and metabolic engineering strategies were employed to further increase its L-isoleucine production level. The main results are listed below:(1) Through overexpressing the wild type and the mutant type of key enzymes from L-isoleucine synthetic ways in the C.glutamicum LD320 found that the mutant threonine dehydratase(TD1) and acetohydroxy acid synthase(AHAS1) could increase more yield of L-isoleucine.(2) The TD1 not only showed completely resistance to L-isoleucine inhibition, but also showed enhanced activity. The AHAS1 showed more resistance to L-isoleucine inhibition than the wild type. Co-expression of the TD1 and AHAS1 in C.glutamicum LD320 led to increase of L-isoleucine production. In shaking flask tests, LD320/pXMJ19-ilvBN1-ilvA1 produced L-isoleucine 4.55 g/L, which was increased by 29% than the control strain.(3) Overexpressing the mutant type brnFE operon in C.glutamicum LD320 could improve L-isoleucine secretion and increase L-isoleucine productivity obviously. In shaking flask tests, LD320/pXMJ19-brnFE1 produced L-isoleucine 4.44 g/L, which was increased 21% than the control strain.(4) To further increase the L-isoleucine production level, the mutant typy ilvA1 、ilvBN1 and brnFE1 were Co-expressed in C.glutamicum LD320. In shaking flask tests, LD320/pXMJ19-ilvBN1-ilvA1-brnFE1 produced L-isoleucine 5.17 g/L, which was increased by 41.3% than the control strain.(5) Optimized the medium of LD320/pXMJ19-ilvBN1-ilvA1-brnFE1 for L-isoleucine fermentation, based on the level of shake flask. The optimal concentration of biotin was 0.5 mg/L. The optimal precursor was threonine and its optimal concentrations was 1 g/L. The surfactant of Tween-80 played a key role in cell growth and L-isoleucine production.The suitable concertration of Tween-80 was 0.5 g/L with adding it at 16 h.(6) LD320/pXMJ19-ilvBN1-ilvA1-brnFE1 was investigated in a 7 L fermentor with an combination of the above optimization conditions. The concentration of L-isoleucine increased from 18.53 g/L to 25.45 g/L, which was increased by 37% than the control strain.