The Effect Of Overexpressing Key Genes Of NADPH And Substrates Supply On 4-hydroxyisoleucine Biosynthesis

4-Hydroxyisoleucine(4-HIL)exhibits unique insulinotropic and insulin-sensitizing activities,so it has potential medical value in treating diabetes.In our previous study,L-isoleucine(Ile)dioxygenase gene ido was overexpressed in an Ile-producing strain Corynebacterium glutamicum SN01,and 4-HIL was de novo synthesized by the resultant SN02 strain.To increase the 4-HIL production,NADPH and substrates supply were further performed in this study.The main results are listed as follows.1.A preliminary insight into the mechanism of 4-HIL production in SN02 with NADPH and 2-oxoglutarate(OG)supply was achieved by studying the effects of overexpressing the NADPH related genes or deletion of serine/threonine protein kinase gene pknG.1)The zwf,zwf-ppnK and POS5 were co-expressed with ido,resulting in strains SL01,SL02 and SL05,respectively.After 144 h of fermentation,4-HIL production and the total amounts of 4-HIL and Ile of SL01 and SL02 were higher than the control strain SN02.This indicates that co-expression of zwf and zwf-ppnK with ido are beneficial for biosynthesis of Ile as well as 4-HIL.However,the 4-HIL production of SL05 was lower than SN02,and its conversion ratio of Ile to 4-HIL was 53.6%after 144 h of fermentation.This indicates that co-expression of POS5 with ido is not beneficial for 4-HIL biosynthesis.2)To increase OG supply and the conversion ratio of Ile to 4-HIL,pknG gene was deleted and replaced by ido gene in SL02 and SL05.The 4-HIL production of the resultant strains SL03 and SL06 were higher than SN02 and the conversion ratio of Ile to 4-HIL reached 98.5%in SL06.This indicates that?pknG::ido substitution combined with POS5 overexperssion is beneficial for 4-HIL biosynthesis.2.To study the effects of OG and O2 supply on 4-HIL production,odhl and odhl-vgb were co-expressed with ido separately.The resultant strains SL07 and SL08 only produced 41.05±0.77 mmol/L and 50.09±4.22 mmol/L 4-HIL,respectively after 144 h of fermentation.Compared with SN02,the concentration of L-glutamate,L-alanine and L-lysine(Lys)reduced by 67.0%,48.4%and 37.4%,respectively in SL08.3.To reduce the concentration of the main by-product Lys,the ddh gene was deleted in SN02 and SL05 separately,resulted in strains SL09 and SL10.After 144 h of fermentation,the 4-HIL yields of SL09 and SL10 were 63.31 ± 1.50 mmol/L and 25.49±2.51 mmol/L,respectively,and the Lys concentrations were 22.37±0.88 mmol/L and 25.80±2.61 mmol/L,respectively,20.4%and 8.2%lower than those of SN02.