A two-component regulatory system involving StyS and StyR is involved in the regulation of indigo synthesis in Pseudomonas sp. However, the function of the styR gene in indigo synthesis and the detailed mechanisms through which StyR enhances the expression of the styAB operon are unclear. Accordingly, in this study, we constructed a styR/styS gene knockout mutant strain. By comparing the differences in indigo yields between the wild-type and mutant strains, we found that the styR gene mutant strain had no indigo synthesis ability, whereas the yield in the wild-type strain was 5.4 mg/L. Thus, these findings indicate that the styR gene plays a key role in the regulation of indigo synthesis. The interactions among StyS, StyR, and the styAB promoter were verified by electrophoresis mobility shift assays. The results showed that StyR interacts with the styAB promoter by binding to the palindrome in the styAB promoter. Moreover, the kinase function of StyS regulated StyR by transphosphorylating StyR during indigo biosynthesis in P. putida B3. Taken together, these findings provide important insights into the establishment of environmentally friendly indigo synthesis methods using P. putida. (C) 2019 Elsevier Inc. All rights reserved.