Dynamic Regulation Of Corynebacterium Glutamicum For Producing 4-hydroxyisoleucine By Isoleucine Biosensor

4-Hydroxyisoleucine?4-HIL?is a promising drug for treating type II diabetes.In the previous study of our group,the ido gene encoding L-isoleucine dioxygenase?IDO?was introduced into an isoleucine?Ile?producing strain Corynebacterium glutamicum ssp.lactofermentum SN01,and 4-HIL was synthesized from its own generated Ile.In this study,the P_brnFE promoter of Ile-induced Lrp-P_brnFE biosensor?Leucine-responsive regulator protein,Lrp?was modified and several strong promoters were selected out,subsequently,to increase4-HIL production,these different P_brnFE promoters were utilized to dynamically regulate the the expression of ido and dynamically increase the supply of?-ketoglutarate and oxygen.The main research contents and results are as follows:?1?The sensitivity and regulation range of Lrp-P_brnFE biosensor to isoleucine was characterized.The P_brnFE promoter of Lrp-P_brnFE biosensor responsed to the Ile in the range of1 m M to 10 m M,with the activation ranging from 1.40-to 2.30-fold.However,the strength of P_brnFE promoter after activation was still 52.14—70.70%lower than that of P_{tac M} promoter.?2?Based on the Lrp-P_brnFE biosensor,the mutation library of P_brnFE promoter was constructed,and tetracycline efflux protein?Tet A?screening marker were used for genetic dual selection.Five mutant PbrnFE promoters,i.e.PbrnFE1,PbrnFE5,PbrnFE7,PbrnFE9 and PbrnFE13were selected.After induction with 1 m M Ile,the activation fold of these five promoters reached 3.60 times,4.00 times,5.61 times,2.39 times and 2.36 times respectively.?3?The natural and five mutant P_brnFE promoters were used to construct the six different plasmids plrp-P_brnFEN-ido,which dynamically regulate the expression of ido.And they were transformed into the strain SN01 to obtain the strains ST01—ST06.Compared with the natural promoter-controlled strain ST01,the five mutant promoters-regulated strains exhibited the increased 4-HIL production.The strian ST04 which harbored the plasmid plrp-P_brnFE7-ido owned the highest 4-HIL production,74.40 m M,which was 201.70%higher than that of ST01.?4?Based on the strain ST04,?-ketoglutarate was then dynamically supplied through expressing oxoglutarate dehydrogenase inhibitor?Odh I?dynamically by P_brnFE,P_brnFE1 and P_brnFE7 promoters,resulting in three dual-dynamic strains ST07-ST09.The 4-HIL production of these three strains were lower than that of the control strain ST04,which may be due to the insufficient oxygen supply.?5?Based on the strains ST07,ST08 and ST09,the oxygen uptake rate of the strain was increased.Therefore,the P_brnFE,P_brnFE1 and P_brnFE7 promoters were used to dynamically regulate the expression of the vitreoscilla hemoglobin?VHb?and 3×3 tri-dynamic strains ST10-ST17 were constructed.The 4-HIL production of these tri-dynamic strains were higher than that of the corresponding dual-dynamic strains.Among them,the strain SN01/plrp-P_brnFE7-ido-P_brnFE7-odh I-P_brnFE7-vgb,i.e.ST17,owned the highest 4-HIL production,135.34 m M.The yield increased by 81.91%as compared to the best single dynamic strain ST04.These results indicated that the dynamic supply of?-ketoglutarate and oxygen could greatly increase 4-HIL production.