Combinatorial Optimization Of D-1,2,4-butanetriol Synthesized By Recombinant Escherichia Coli

D-1,2,4-butanitriol(BT)is a kind of tetra-carbon polyol,which has a wide range of applications in military and medical fields.In order to further optimize the anabolic pathway of one-step synthesis of BT by biological method,the synthesis of BT was effectively promoted by modifying the glucose metabolism pathway(EMP pathway)and enhancing the supply of NADPH.After knockout of the key EMP pathway gene pgi,the resu Lts showed that the pgi-deficient strain MJ135kp G increased the intracellu Lar NADPH/NADP~+by 25%.The resu Lts of shaking flask fermentation showed that the increase of NADPH/NADP~+also promoted the synthesis of BT,and the yield of BT increased from 3.35 g/L to 4.02 g/L without NADPH regu Lation.The initial glucose concentration was 2 g/L,and 24 h after adding 4 g/L glucose,the amount of BT synthesis increased to 4.13 g/L.Enhancing NADPH supply is an effective strategy to improve BT synthesis.And through the relationship between fluorescence intensity and butantriol,observe the live bacteria number,BT content,fluorescence intensity,NADPH content changes.It can be seen that the viable bacteria number,fluorescence intensity and NADPH content gradually increased during 0-24 h,reached the maximum value at 24 h and gradually decreased after 24 h.The sugar-adding method cou Ld be adjusted according to the change ru Le,and finally it was determined that adding glucose at 24 h had the best effect,so as to make the bacteria grow better and produce BT better.The expression level of each gene in the biosynthesis pathway of BT plays an important role in the growth status of the strain,and is also related to the synthesis efficiency of the whole biosynthesis pathway.The characteristics of RBS also have a significant effect on the initiation rate of translation and thus on the level of gene expression.Translation initiation is one of the main steps in translation and plays an important role in determining the overall translation rate.Starting rate for synthetic biology is especially meaningfu L because it provides the starting rate by changing the m RNA to regu Late protein expression,optimization of RBS strength also has impact on the gene expression level,through the website design and calcu Lation,intensity of change before a gene sequence of RBS,and NADPH sensor was applied to analyze the concentration of E.coli NADPH in vivo fluorescence intensity,further study on the translation efficiency of RBS for NADPH content change and BT production.The variation of NADPH content and the synthesis pathway of BT were optimized by 99 bp and whole gene optimization before the single gene optimization of mdl C,and the influence of the intensity mode of mdl C RBS on the variation of NADPH content and the synthesis efficiency of BT was changed by gradient,so as to construct a strain capable of synthesizing BT efficiently.Finally,MJ135kp G-MQ-m3 was the best strain,and the final yield was4.84 g/L.