Strategies Of Co-utilization Of Substrates In Biosynthesis Of 1,2,4-butanetriol And DO-stat Fed-batch Fermentation

D-1,2,4-butanetriol is a chiral polyhydric alcohol with four carbon.As an important and valuable intermediate in organic synthesis,BT has a lot of industrial applications,particularly in the military and the medical field.Currently industrialized production of BT mainly employs chemical synthesis,which has many drawbacks,such as harsh reaction conditions,low yield,more byproducts,severe environment contamination and so on.In recent years,with the development of synthetic biology,the safe and efficient biosynthesis method to produce chemicals has become a research hotspot.Recombinant E.coli conversion of D-xylose into BT has been reported,but the transformation efficiency is relatively low.In this study,based on the preliminary work,the following researches were carried out in order to improve the BT yield.The effects of the co-use of the substrates on the synthesis of1,2,4-butanetriol was investigated.As the carbon and energy source,glucose is essential for the synthesis of BT.By knockout the gene of pgi,mlc,mtfA,the reconstructed E.coli could convert xylose into BT,while grow on glucose.By knockout the gene of mtfA,the recombinant E.coli showed low consumption of glucose,but higher synthesis of BT,and the production of BT reached5.12 g/L.And the yield of BT per unit cell was also higher than the pre-constructed high-yield strain MG403K-1.The effects of the glucose transport mode on the synthesis of1,2,4-butanetriol was also explored.By overexpressing the glucose transport system composed by galactose transporter protein(GalP)and glucokinase(Glk),the cell could transport glucose independent on the PTS system.Although the co-utilization of glucose and xylose has been achieved,the BT yield of has declined,which implied that the glucose supply during the synthesis process need a more precise regulation.In addition,the relationship between strengthening of the xylose transportation and the biosynthesis of D-1,2,4-butenatriol was investigated.The results showed that strengthening xylose transport led to xylonate accumulation,which suggested that the transformation process from xylonate to 1,2,4-butenatriol need further optimized.The study also investigated the effect of DO-stat fed-batch fermentation on the synthesis of 1,2,4-butenatriol by the pre-constructed high-yield strain MG403K-1.Through the the online feedback by the dissolved oxygen value to control the glucose feeding,1,2,4-butanetriol final yield reached 8.79 g/L,which was improved nearly 14% compared with the fed-batch fermentation.The increasing of output indicates that the timing of glucose feeding is an important factor,which may have a significant influence on the final yield of the 1,2,4-butanetriol.