Engineering Of Corynebacterium Glutamicum For L-isoleucine And Polyhydroxyalkanoate Production

L-isoleucine is an essential branched-chain amino acid widely used in food,pharmaceutical,functional beverages and animal feeding.Corynebacterium glutamicum IWJ001 has been an excellent L-isoleucine producer for several years and throughly studied by several researchers until now.Based on the proteome and transcriptome data of C.glutamicum IWJ001,we did several studies on further engineering of this strain.For several interesting phenomena discovered during the studies,we tried a lot to explain the results and made a little innovations targeted on L-amino acid production.Our main conclusions is shown as follows:(1)Three genes gnd,pgl and fbp relevant to pentose phosphate pathway were overexpressed in Corynebacterium glutamicum IWJ001,leading to increase of L-isoleucine production.The transcriptional levels of gnd,pgl and fbp significantly increased in IWJ001/p DXW-8-gnd-fbp-pgl;intracellular NADPH were significantly increased at 24 h and then quickly consumed for L-isoleucine production.10.9 g/L and 28.96 g/L of L-isoleucine was produced in IWJ001/p DXW-8-gnd-fbp-pgl in shake flask cultivation and fed-batch fermentation,respectively.In addition,IWJ001/p DXW-8-gnd-fbp-pgl grew much better than the start strain,and its L-isoleucine yield on glucose reached 0.138 g/g.(2)Cysteine synthase A catalyzes the last reaction of L-cysteine synthesis in bacteria,but its moonlighting functions have been revealed recently.Cys K was overexpressed in Corynebacterium glutamicum IWJ001.Compared to the control IWJ001/p DXW-8,IWJ001/p DXW-8-cys K cells grew fast during log phase,and produced 26.5% more Lisoleucine in flask fermentation and 23.5% more L-isoleucine in fed-batch fermentation.The key genes asp C,lys C,hom,thr B,ilv A,and ilv BN involved in L-isoleucine biosynthesis were all up-regulated in IWJ001/p DXW-8-cys K,compared to IWJ001/p DXW-8.In addition,IWJ001/p DXW-8-cys K cells were longer and thicker than IWJ001/p DXW-8 cells.Compared to IWJ001/p DXW-8,the membrane permeability increased 15.8% and biofilm formation ability decreased 71.3% for IWJ001/p DXW-8-cys K cells.(3)Genome and transcriptome analysis showed that C.glutamicum IWJ001 has potential to accumulate L-isoleucine and propionyl-Co A pool.The recombinant strain IWJ001/p DXW-8-pha CAB produced high levels of PHBV with high 3-hydroxyvalerate fraction as well as Lisoleucine.The transcriptome results showed that compared to the wild-type C.glutamicum ATCC13869,the transriptional levels of the genes prp C,prp D,and prp B,which are key genes relevant to propionyl-Co A accumulation,increased 26.7,25.8,and 28.4-folds in IWJ001,respectively;and the intracellular level of propionyl-Co A increased 16.9-fold.(4)When the gene cluster pha CAB for polyhydroxyalkanoate(PHA)biosynthesis introduced into IWJ001,the recombinant strain IWJ001/p DXW-8-pha CAB produced 15.0 g/L PHBV with high percentage of 3-hydroxyvalerate as well as 30.1 g/L L-isoleucine after fedbatch fermentation.The maximum 3-hydroxyvalerate fraction in PHBV produced by IWJ001/p DXW-8-pha CAB using glucose as the sole carbon sourse could reach 72.5%,which is the highest fraction reported so far.Because of its high level of intracellular propionyl-Co A pool,IWJ001 could produce other propionyl-Co A derivatives.