Research On Biological Synthesis Of 4-hydroxyisoleucine

4-Hydroxyisoleucine(4-HIL)is a natural nonproteinogenic amino acid possessing insulinotropic biological activity.Now 4-HIL is mainly produced by an extraction method from fenugreek seeds,but this method has a low extraction rate(0.091-0.6%)and,among the several isomers achieved,only(2S,3R,4S)-4-HIL has biological activity.It has been proved that L-isoleucine dioxygenase(IDO)catalyzes L-isoleucine to(2S,3R,4S)-4-HIL stereotively.Therefore,this article chose the ido gene,which belongs to Bacillus thuringiensis TCCC11826 and encodes IDO,as a target to research ways to synthsize 4-HIL by microorganism and develop more effective strategy.In this article,ido gene was cloned from B.thuringiensis TCCC11826,and its nucleotides and amino acids sequences shared 97.47%and 97.91%identities with that had been reported.Use Escherichia coli and Corynebacterium glut am icum as hos ts,W3110/pXMJido and ATCC13032/pXMJido were successfully constructed,respectively,and both of them were used for biotransformation to produce 4-HIL.As a result,on condition that 2-ketoglutaric acid and L-isoleucine were added,the L-iso leucine conversion rates of them were 89.28%and 56.7%,respectively.To synthsize 4-HIL,IDO needs L-isoleucine and 2-ketoglutaric acid as substrates.To decrease substrate addition and simply craft,YILW/pXMJido was constructed based on the L-iso leucine producer,C.glutamicum YILW,for 4-HIL fermentation,and the 4-HIL productivity was 2.01±0.04 g/L after fermentating for 50 hours.However,there had some L-iso leucine left,which implied that 2-ketoglutaric acid was not enough.Then experiments were carried out to research the way how 2-ketoglutaric acid adding influnenced 4-HIL production As data shown,to some extent,4-HIL productivity increased with the increase of 2-ketoglutaric acid added,and the highest was 4.85 g/L.So it was conclueded that methods that could increase the ability for 2-ketoglutaric acid synthsis should be adopted to decrease substrate additionTo decrease 2-ketoglutaric acid consumption,a-ketoglutarate dehydrogenase of YILW/pXMJido was knocked out by using gene recombination technology,but both growth and 4-HIL productivity reduced seriously.To increase 2-ketoglutaric acid synthesis,pyruvate carboxylase was overexpressed through a stronger promoter replacing the origin one of the pyc gene and YILW(Ppyc::Ptuf)/pXMJido was constructed,and the 4-HIL productivity increased by 40%.Based on this,YILW(Ppyc::Ptuf)(aceA::Ptuf-ido)/pXMJido was constructed by gene aceA deletion,which encoded the isocitrate lyase,and the 4-HIL productivity and convertion rate from glucose increased by 6.4%and 10%,respectively.These results indicated that enhancement of the CO2-fixed way and block of the glyoxylate cycle increased the 2-ketoglutaric acid supply,finally increased the 4-HIL production.This article researched biotransformationand fermentation methods for 4-HILproduce.The former has a short production period,high conversion rate and other characteristics,but need adding a-ketoglutarate and L-isoleucine,which is unfavorable for reducing production costs.The latter,without adding substrates,couples L-isoleucine fermentation with 4-HIL synthesis,and achieves direct fermentation for 4-HIL synthesis,and has certain advantages.This study could provide a theoretical basis for the extension of L-isoleucine metabolic pathway and of bio-manufacturing technology for 4-HIL production.