摘要
Due to the selective permeability of the cytomembrane,high-yield fumaric acid strains form a steep difference between intra-and extracellular concentrations.Intracellular biosensors cannot detect the real concentration change of extracellular fumaric acid.To overcome this limitation,a two-component biosensor(TCB)that could respond to extracellular fumaric acid was designed based on the DcuS-DcuR two-component system.The two-component system consists of a histidine kinase(SK)and response regulator.SK is a transmembrane histidine kinase sensor that can detect concentration changes in extracellular compounds.To improve the dynamic range of the constructed fumaric acid TCB,we optimized the expression ratio and expression intensity of dcuS and dcuR.We found that the optimum expression ratio of dcuS:dcuR was 46:54.Under this ratio,the higher was the expression level,the greater the dynamic range.In addition,we modified the ATP-binding site on the DcuS,and the final dynamic range of the TCB reached 6.6-fold.Overall,the obtained fumaric acid-responsive TCB with a high dynamic range is reported for the first time,providing a synthetic biology tool for high-throughput screening and dynamic metabolic regulation of fumaric acid cell factories.
基金
supported by the National Key R&D Program of China(2019YFA0905502)
the National Natural Science Foundation of China(31900066 and 21877053)
the Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-015)
the Fundamental Research Funds for the Central Universities(JUSRP12056 and JUSRP51705A)
the China Postdoctoral Science Foundation(2021M690533).
