Metabolic Engineering Of Recombinant Corynebacterium Glutamicum To Enhance The Precursor Supply For Producing 4-hydroxyisoleucine

4-hydroxyisoleucine?4-HIL?,which can exhibit unique insulinotropic and insulin-sensitizing activities,has potential medical value in treating diabetes.In our previous study,L-isoleucine dioxygenase gene?ido?from Bacillus thuringiensis YBT 1520 was cloned and overexpressed in an L-isoleucine-producing strain,Corynebacterium glutamicum ssp.lactofermentum SN01,and 4-HIL was produced from the endogenous L-isoleucine?Ile?.In this study,metabolic engineering strategies are used to further enhance the 4-HIL production.The main results are as follows.?1?In order to improve Ile supply,ilvA was overexpressed and the ido-ilvA-expressing strain SZ02 was constructed.After 144 h of fermentation,SZ02 produced 10.68±0.10 mmol/L4-HIL,and ido-mere expressing strain SZ01 produced 52.79±1.04 mmol/L 4-HIL.The total amount of 4-HIL and Ile were 60.39±0.33 mmol/L and 114.60±0.24 mmol/L,while the conversion ratio of Ile to 4-HIL was 87%and 9%in SZ01 and SZ02,respectively.This indicates that overexpression of ilvA improves the total amount of 4-HIL and Ile but was not beneficial for 4-HIL conversion.?2?In order to improve?-ketoglutarate??-KG?supply,lgox and cat were overexpressed and the ido-lgox-expressing strain SZ03 and ido-lgox-cat-expressing strain SZ04 were constructed.After 120 h of fermentation,the production of 4-HIL in SZ03 and SZ04 were5.15±0.03 mmol/L and 16.81±0.30 mmol/L,respectively,obviously lower than that in ido-mere expressing strain SZ01?46.36±1.82 mmol/L?.The production of 4-HIL in SZ04 was higher than that in SZ03,indicating that coexpression of lgox and cat could partially alleviate the deficiency of mere expression of lgox.?3?In order to enhance oxaloacetate supply,mqo was overexpressed and the ido-mqo-expressing strain SZ05 was constructed.After 144 h of fermentation,the production of 4-HIL in SZ05 was 60.86±2.24 mmol/L,slightly lower than that in SZ01?72.06±5.60mmol/L?,while the production of Ile in SZ05 was 101.39±2.20 mmol/L,obviously higher than that in SZ01?5.00±1.43 mmol/L?.Finally,the conversion ratio of Ile to 4-HIL was 94%and 38%in SZ01 and SZ05,respectively.The RT-PCR analysis of some key genes in the metabolic pathway of SZ05 showed that the carbon flowing into the TCA cycle was diverted into the glyoxylic acid cycle by isocitrate lyase,resulting in the insufficient supply of?-KG,another substrate in IDO reaction,and excessive accumulation of Ile.By adding?-KG in the fermentation medium of SZ05,the yield and conversion ratio of 4-HIL was increased.?4?In order to enhance O₂ supply,vgb was expressed and the ido-vgb-expressing strain SZ06 was constructed.The IDO activity of SZ06 was slightly higher than that of ido-mere expressing strain SZ01.After 144 h of fermentation,OD and 4-HIL production increased to91.3 and 108.10±9.91 mmol/L,respectively.Therefore,expression of vgb improved the cell growth and 4-HIL production by 13%and 50%,respectively as compared with SZ01.?5?In order to enhance the supply of O₂ and oxaloacetate or Ile at the same time,vgb and mqo or ilvA were overexpressed and the ido-mqo-vgb-expressing strain SZ07 and ido-vgb-ilvA-expressing strain SZ08 were constructed.The two recombinant strains grew slowly.After 144 h of fermentation,SZ08 hardly produced 4-HIL,while SZ07 produced30.52±1.21 mmol/L 4-HIL,obviously lower than that in ido-vgb-expressing strain SZ06?98.57±3.44 mmol/L?.The intracellular concentration of?-KG in SZ07 and SZ08 was lower than that in SZ06.This indicates that coexpression of vgb and mqo or ilvA is not benefit to cell growth and make the supply of?-KG insufficient.These may result in the slower conversion of Ile to 4-HIL and lower ability to synthesize 4-HIL.However,the reasons for this phenomenon need to be further explored.