Multi-Dynamic Regulation Of 4-Hydroxyisoleucine Biosynthesis In Corynebacterium Glutamicum

4-hydroxyisoleucine（4-HIL）has great potential in the treatment of type 2 diabetes.L-isoleucine dioxygenase（IDO）can catalyze the C-4 hydroxylation of L-isoleucine（Ile）to form 4-HIL by consuming O₂ and oxidizingα-ketoglutaric acid（α-KG）.In our previous study,the ido gene was expressed in an Ile producing strain Corynebacterium glutamicum SN01 for de novo biosynthesis of4-HIL.On the basis of SN01 strain,this study used three dynamic control tools with different functions to regulate the expression of ido,coordinate the supply of Ile,α-KG and O₂,and weaken the synthesis of by-product L-lysine（Lys）,so as to increase the yield of 4-HIL and reduce the concentration of Lys.The conversion of Ile to 4-HIL is the last step of the whole 4-HIL synthesis pathway.In order to improve the activity of IDO,the codon of ido was optimized to obtain ido ^U firstly.Then,seven kinds of Ile sensors Lrp-P_{brn FE}N with different strengths were used to dynamically up-regulate the expression of ido ^U and promote the conversion of Ile to 4-HIL.Therefore,seven dynamic expression strains D-_NI were constructed,and the static expression strain of ido ^U controlled by P_{tac M},i.e.SI,was used as the control strain.The fermentation results showed that except for D-₁₃I,the 4-HIL yield of other six strains increased in different degrees.Among them,the 4-HIL yield of the best strain D-₁I reached 111.1±0.8 mmol×L^-1,which was 23.5%higher than that of strain SI(89.9±2.5 mmol×L^-1).α-KG and O₂ are necessary for converting Ile into 4-HIL.In order to improve the supply ofα-KG and O₂,three Lrp-P_{brn FE}N biosensors with low,medium and high strengths were used to dynamically up-regulate the expression of odh I and vgb on the basis of six D-_NI strains.The 6×3×3=54 triple-gene dynamic control strains D-_NI_NO_NV were constructed.The fermentation results showed that the 4-HIL yield of 10 D-_NI_NO_NV strains was higher than that of the corresponding D-_NI strains.Among them,D-₀I₇O₇V exhibited the highest 4-HIL yield of 141.1±15.5 mmol×L^-1,27.0%higher than that of the best single-gene dynamic regulation strain D-₁I.The conversion of L-aspartic acid to Ile is the upstream of 4-HIL biosynthesis pathway.However,the activity of IDO is inhibited by excessive Ile.In order to continuously supply Ile and avoid inhibition of IDO by excess Ile,Ile attenuator P_{ilv BNC} was used to dynamically down-regulate the expression of ilv A,the key gene in Ile synthesis pathway.On the basis of six unidirectional dynamic regulation strains D-_NI,six bidirectional dynamic regulation strains D-_NIPA were constructed.The fermentation results showed that compared with the respective D-_NI strains,the 4-HIL production of four bidirectional regulation strains(D-₀IPA,D-₅IPA,D-_D5IPA,D-₇IPA)were improved.Among them,D-₇IPA showed the highest 4-HIL yield of 123.2±20.5 mmol×L^-1,which was 10.9%higher than that of the best unidirectional regulation strain D-₁I.In order to coordinate the activity of IDO,the synthesis of Ile and the supply ofα-KG and O₂ at the same time,the P_{ilv BNC}-controlled ilv A was expressed on the basis of 10 D-_NI_NO_NV strains with high yield of 4-HIL.And 7 D-_NI_NO_NVPA bidirectional regulation strains were successed constructed.The fermentation results showed that D-₇I₇O1VPA gave the highest 4-HIL yield of 127.1±20.2 mmol×L^-1,which was similar to that of D-₇IPA and D-₇I₇O₁V.The 4-HIL yield of other strains decreased greatly.Lys is the main by-product in the synthesis of 4-HIL.In order to reduce the synthesis of Lys,Lys-OFF riboswitch was integrated into the upstream sequence of dap A,the key gene of Lys synthesis pathway in SN01,and a D-RS strain was obtained.The expression plasmid with high yield of 4-HIL was transformed into this strain,generating the dual-channel dynamic regulation strains D-RS-_NIPA and D-RS-_NI_NO_NV.The 4-HIL yield of D-RS-₅IPA and D-RS-₀I₇O₇V strains reached 177.3±8.9mmol×L^-1 and 166.0±13.6 mmol×L^-1,respectively,and by-product Lys was reduced to 6.1±0.6mmol×L^-1.Therefore,the biosynthesis of 4-HIL in C.glutamicum can be effectively promoted by dynamically regulating the main pathway and branch pathway through three functional biosensors.