Improvement Of 4-hydroxyisoleucine Biosysnthesis In Recombinant Corynebacterium Glutamicum Through Ribosome Binding Sites

4-Hydroxyisoleucine?4-HIL?is a promising drug for treating diabetes.Currently,the easiest way for producing 4-HIL is catalyzed by L-isoleucine dioxygenase?IDO?,using L-isoleucine?Ile?,?-ketoglutarate??-KG?,and oxygen?O₂?as substrates.In our previous work,the ido gene was cloned and expressed in Corynebacterium glutamicum ssp.lactofermentum SN01,an Ile producer,and 4-HIL was de novo-synthesized from glucose.In this study,RBS was used to optimize the expression of ido and fine-tune the expression of several related genes,and some branch pathway of pyruvate node was blocked to improve the synthesis of 4-HIL in SN01.The main contents and results are as follows.1.In order to optimize the expression of ido,ido-expressing strains under different RBS sequences were constructed,resulting in 18 strains of nI.Their fermentation showed that 4-HIL production of 5 strains were higher than that of SN02 which expressed ido under R0,but 4-HIL production of 13 other strains decreased more or less.Among them,the 4-HIL production of 8I?104.22±0.53 mM?was the highest,9.9%more than that of SN02?94.83±3.22 mM?.Moreover,the IDO activity of 8I was also increased by 9.4%as compared to that of SN02.2.Oxaloacetate?OAA?is the precursor of both Ile and?-KG.To increase the supply of OAA,ppc guided by 7 RBS sequences with different strength were expressed in 8I,resulting in 7 strains of8I-nP.Their fermentation showed that 4-HIL production of only 8I-0P?115.24±4.10 mM?exceeded that of original strain 8I,while the 4-HIL production of other ppc-expressing strains decreased by50.5%-64.2%as compared to 8I,and a large amount of Ile was accumulated in these strains.3.Unphosphorylated Odh I protein can inhibit the activity of?-ketoglutarate dehydrogenase complex.To increase the supply of?-KG,odhI guided by 6 RBS sequences with different strength were expressed in 8I,resulting in 6 strains of 8I-nO.Their fermentation showed that the 4-HIL production of all the 6 strains were lower than that of original strain 8I.The 4-HIL production of8I-10O was 81.3%of 8I,while that of 8I-15O and 8I-11O was extremely low.Meanwhile,the glutamate?Glu?production of others 3 strains increased largely,indicating that the excess?-KG was used to synthesize Glu.4.Vitreoscilla hemoglobin?VHB?can increase the oxygen uptake rate of cells.In order to increase the supply of O₂ in 8I-nO,vgb was further expressed in 8I-15O,8I-10O and 8I-11O,resulting in the 3 strains of 8I-nO-V.Their fermentation showed that the 4-HIL production of these 3strains changed sharply as compared to their original strains.The 4-HIL production of 8I-15O-V and8I-11O-V increased to 119.27±5.03 mM and 86.46±1.07 mM,respectively,whereas that of 8I-10O was extremely low.5.In order to reduce the content of alanine?Ala?,a by-product of 4-HIL biosynthesis,an amino acid transaminase encoding gene avtA was knocked out in SN01,and two strains?avtA-8I-0P and?avtA-8I-15O-V were constructed.Their fermentation showed that the Ala production of both strains decreased by about 80%,and the 4-HIL production increased to 123.91±3.97 mM and 122.16±5.18mM,respectively.6.In order to reduce the pyruvate-related by-products,the gene cluster ldhA-pyk2 which encodes lactate dehydrogenase and pyruvate kinase was knocked out in SN01,and two strains?ldhA?pyk2-8I-0P and?ldhA?pyk2-8I-15O-V were constructed.Their fermentation showed that the lactic acid,acetic acid and Ala production of both strains decreased,and the 4-HIL production increased to 118.33±5.67 mM and 139.82±1.56 mM,respectively.