Construction And Preliminary Application Of Chassis Cell For Supplying 7-phosphate-Sedoheptulose

7-phosphate-sedoheptulose(7-P-S)is an important intermediate in the pentosephosphate pathway.It is also a nodal compound associated with a variety of natural secondary metabolites,which can be cyclized by different 7-P-S cyclases to participate in biosynthesis of many C7N-aminocyclitol natural products.As the intermediate of metabolic pathway,7-P-S together with 3-phosphate glyceraldehyde(3-P-G)is catalyzed by transaldolase to produce 4-phosphate erythritose and 6-phosphate fructose,so it is difficult for 7-P-S to accumulate in normal cells.In this study,we modified the pentose phosphate pathway of Escherichia coli MG1655 and constructed the chassis cell which accumulated enough precursor 7-P-S for biosynthesis of C7N-aminocyclitol natural products.we introduced the 2-epi-valiolone synthetase gene into this chassis cell and achieved the biosynthesis of 2-epi-valiolone.Firstly,we analyzed the transaldolase genes talA and talB which were responsible for catalyzing the tricarbon transfer reaction between 7-P-S and 3-P-G in Escherichia coli MG1655.These two genes were cloned and expressed.7-P-S and 3-P-G were served as substrates for analyzing their enzymatic properties.The results showed that TALA and TALB were isoenzymes those were responsible for catalyzing the carbon transfer reaction in MG1655.Under the optimum conditions,the specific enzyme activities of TALA and TALB were 2.46±0.12 U/mg and 1.94±0.71 U/mg,respectively.In order to reduce the consumption of 7-P-S by natural metabolic pathway and realize the accumulation of 7-P-S,we modified the pentose phosphate pathway by CRISPR-Cas9 gene traceless editing system.By constructed the working plasmids pTarget-talA29 and pTarget-talB43,we knocked out gene talA and talB and got the 7-P-S supply chassis cell MG1655?talA?talB successfully.The accumulation of 7-P-S in this mutant were 129.84 mg/L,which were 42.6-fold that of wild type.Therefore,This chassis cell can provided sufficient precursor for the biosynthesis of C7 Naminocyclitol natural products with 7-P-S as substrate.Based on the 7-P-S supply chassis cell mentioned above,we introduced the 7-P-S cyclase gene amirA from Actinosynnema mirum into it and identified its catalytic activity as 7-P-S cyclase to produce 2-epi-valiolone in vitro.Then we constructed two engineering strains for the biosynthesis of 2-epi-valiolone of MG1655?talA?talB(pTrc99a-amirA)(named MG-1)and MG1655?talA::amirA ?talB:: amirA(named MG-2)by pTrc99 A protein expression vector and CRISPR-Cas9 gene editing system.After fermentation for 24 hours,the production of 2-epi-valiolone in the fermentation broth of the strains MG-1 and MG-2 was 86.43±7.68 mg/L and 9.60±0.96 mg/L,respectively.In this work,we constructed the chassis cell and achieved the accumulation of 7-P-S through the modification of pentose phosphate pathway in E.coli MG1655,which provided precursor for biosynthesis of C7N-aminocyclitol natural products.The heterologous synthesis of 2-epi-valiolone was realized by introducing exogenous genes,which provided a reference for the subsequent use of the chassis cell to synthesize target product.