Enhanced 1,2,4-butanetriol Bioproduction By Muti-strategy

D-1,2,4-butanetriol?BT?is an important non-natural four-carbon platform compound,which is widely used in military industry,medicine,tobacco and other fileds.In recent years,researchers have successfully constructed a biotransformation pathway to synthesize BT from xylose in recombinant Escherichia coli.Although multiple strategies,such as knocking-out branch pathways,over-expression of key enzymes and co-expression of molecular chaperone,were adopted to strengthen BT production,the production of BT was still low.To be noted,the disruption of xylose branch pathway is not conducive to biomass accumulation and the low efficiency of key decarboxylase is a rate-limiting step of BT synthesis.Based on that,we first selected high efficiency decarboxylases to overcome the rate-limiting step.Then the xylose branch pathway was fine-tuned with antisense RNA technology and the glucose effect was inactived by deleting the relative genes to improve cell growth and BT synthesis.Finally,the BT titer was further improved by optimizing the culture conditions.It is reported that decarboxylation is a rate-limiting step in BT pathway.To improve decarboxylation efficiency,the kivD gene encoding 2-keto isovalerate decarboxylase from L.lactis and the ipdC gene encoding indole-pyruvate decarboxylase from E.cloaca were cloned to construct the new recombinant strains for BT production,respectively.The substitute expression of the high efficient decarboxylase Kiv D led to a 53%increase of BT titer(1.3g·L^-1).However,no BT was detected in the fermentation broth of the recombinant strain harboring the ipdC gene.It was indicated that the 2-keto isovalerate decarboxylase KivD was more suitable for decarboxylation in BT production.In order to improve the growth ability of the recombinant strain,a fine-tuning system based on the asRNAs target the xylA gene were established to balance the carbon flux for cell growth and BT synthesis.The xylA gene transcriptions of all the four recombinant strains were repressed in different extent.Accordingly,the biomass and BT titers were increased.Compared with the control strain,the highest BT titer was increased by 200%,reaching 3.9g·L^-1.Additionally,the glucose effect of the recombinant strain was repressed by deleting ptsHI and mgsA genes to realize the simultaneous consumption of glucose and xylose.Compared with the start strain,the biomass and BT titer of the ptsHI and mgsA deficient strain were increased by 1.1 folds and 6.5 folds(6.0 g·L^-1),respectively.Then the ptsHI and mgsA deficient strain was used as the start strain for further study.In order to further improve the BT production,the concentrations of glucose,xylose and LB were optimized and the pH of fermentation broth was adjusted by adding Ca CO₃.The BT production of the recombinant strain was increased to 12.2 g·L^-1 with the optimum medium.Additionally,the culture conditions of fed-batch fermentation,such as the initial dosages of glucose and xylose,inoculum concentration,pH,stirring speed,ventilation,feeding time,were explored.Under the appropriate culture condition,the BT titer of the recombinant strain reached 14.9 g·L^-11 through fed batch fermentation in 5 L bioreactor.Finally,a 3.54 g·L^-11 BT titer was achieved from bagasse hydrolysate with 8.4 g·L^-11 of glucose and 16.8 g·L^-1 of xylose,accompany with 84.8%of molar conversion rate.