Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (Capsicum annuum), were produced in pathway-engineered Escherichia coli cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in E. coli. In a previous study, the levels of capsanthin synthesized were low despite the high expression of the CaCCS gene, which may have been due to the dual activity of CaCCS as a lycopene β-cyclase and CCS. An enhanced interaction between CaCCS and CaZEP that supplies antheraxanthin and violaxanthin, substrates for CaCCS, was considered to be crucial for an efficient reaction. To achieve this, we adapted S·tag and S-protein binding. The S·tag Thrombin Purification Kit (Novagen) is merchandized for in vitro affinity purification, and S·tag-fused proteins in the E. coli lysate are specifically trapped by S-proteins fixed on the agarose carrier. Furthermore, S-proteins have been reported to oligomerize via C-terminal swapping. In the present study, CaCCS and CaZEP were individually fused to the S·tag and designed to interact on oligomerized S-protein scaffolds in E. coli, which led to the biosynthesis of not only capsanthin and capsorubin but also cucurbitaxanthin A and capsanthin 3,6-epoxide. The latter reaction by CaCCS was assigned for the first time. This approach reinforces the scaffold's importance for multienzyme pathways when native biosynthetic systems are reconstructed in microorganisms.