| Ectoine is a high value-added humectant and protective agent,widely used in medicine,cosmetics and other fields.It can be naturally synthesized by some moderate halophiles and also be heterologously producted by Escherichia coli,but the enzyme reaction of heterologous synthesis pathway often faces problems such as rapid diffusion of intermediate metabolites and low substrate utilization efficiency.In this study,the DNA scaffold system was used to locate and assemble key enzymes of the ectoine synthesis pathway in Escherichia coli to promote the efficient delivery of intermediate metabolites between different enzymes,thereby improving the conversion rate of the ectoine synthesis pathway and product yield.The main results of our study are as follows:（1）Constructing expression plasmid with fusions between enzymes in ectoine biosynthesis pathway and ZF domain.The recombinant plasmid p FT24-ect ABC was obtained by using p FT24 as the starting plasmid,and the expression of ect ABC,an ectoine synthesis gene cluster,was controlled by using the thermosensitive circuit c Its-p R-p L.The G4S linker sequence was further used to fuse and express the enzymes involved in the ectoine synthesis pathway（Ect A,Ect B,and Ect C）with the corresponding ZF domains（ZFa,ZFb,and ZFc）to obtain the recombinant plasmid p FT24-ect ABC-ZFabc.The three genes of Eclys C*,asp DH,and ppc in the precursor supply pathway were overexpressed by attaching to the plasmid p FT24-ect ABC-ZFabc to obtain the recombinant plasmid p FV1,and the yield of the corresponding strain MWZ003/p FV1 was 1.26 g·L-1.（2）Replacing recombinant plasmid replicons regulates the expression of key enzymes for ectoine synthesis in Escherichia coli.The p15A replicons of plasmid p FV1 were replaced with Col A,RSF1030,and p MB1 replicons respectively to obtain the new recombinant plasmids p FV2,p FV3,and p FV4.The results of shaker fermentation showed that p MB1replicons with the highest copy number were more suitable for the production of ectoine,and the yield of the corresponding strain MWZ003/p FV4 reached 11.85 g·L-1,which was 8.4times higher than that of MWZ003/p FV1.（3）Design and optimization of DNA scaffolds,localization,assembly of ectoine synthesis pathway enzymes for cascade reaction.A DNA scaffold structure is inserted into the recombinant plasmid p FV4 for precise immobilization of ZF fusion enzymes.By adjusting the enzyme binding site spacing and enzyme binding direction on the DNA scaffold,the DNA scaffold repeat unit was optimized,and the stoichiometry number of the enzyme binding site on the DNA scaffold was changed,among which the strain MWZ003/p FV20 with enzyme reverse binding,11-bp spacing,4 repeat units and 1:1:2 stoichiometric ratio had the best ectoine conversion efficiency,and its yield reached 17.97 g·L-1,which was 51.65%higher than that of the control strain MWZ003/p FV4 without DNA scaffold.（4）Increasing the expression of the rate-limiting enzyme Ect B and optimizing the fermentation temperature conditions.The recombinant plasmid p FV24 is obtained by inserting the PRpromoter before the Ect B-ZFb fusion enzyme expression gene of plasmid p FV4.The DNA scaffold structure was inserted into recombinant plasmid p FV24 to further explore the optimal enzyme binding site stoichiometry of DNA scaffold after increasing the expression of rate-limiting enzyme Ect B,and it was found that the strain MWZ003/p FV30with enzyme reverse binding,11-bp spacing,four repeat units and 1:2:2 stoichiometric ratio had the best ectoine conversion efficiency,and its yield reached 19.95 g·L-1.In this study,a temperature control switch was used to switch the strain growth and production mode with three different temperature shift time points（3 h,4 h,and 5 h）,and the ectoine titer of MWZ003/p FV30 was increased to 22.79 g·L-1when the temperature was changed from 37℃to 42℃4 h after the start of fermentation,which was nearly 1.92 times higher than that of the initial strain MWZ003/p FV4,and its conversion was 0.65 g·g-1glucose. |
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