**4. Conclusions**

In this study, we developed an efficient method for producing crustacyanin subunits and used them to solubilize and concentrate carotenoids. Crustacyanin subunits with a NusA-tag attached to their N-terminus achieved soluble expression in *E. coli* and solubilized astaxanthin in aqueous solution. Astaxanthin could be concentrated by cleaving the NusA-tag from the crustacyanin subunit followed by its precipitation with astaxanthin. Furthermore, this method also could be applied to α-carotene and β-carotene. These results should indicate the potential of the attaching and releasing NusA-tag method to supply a solubilization process by attaching the NusA-tag, and a concentration process by releasing the NusA-tag, for the production of a broad range of carotenoids.

**Author Contributions:** Conceptualization, K.Y.H. and S.Y.; Methodology, K.Y.H., S.Y., Y.H.-H. and H.K.; Validation, K.Y.H. and S.Y.; Formal Analysis, S.Y.; Investigation, S.Y.; Data Curation, K.Y.H. and S.Y.; Writing—Original Draft Preparation, K.Y.H. and S.Y.; Writing—Review and Editing, Y.H.-H. and H.K.; Visualization, K.Y.H. and S.Y.; Project Administration, K.Y.H.; Funding Acquisition, K.Y.H. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was supported by JSPS KAKENHI Grant Numbers JP21H03645 and JP16K00616. This work was also supported by JST-Mirai Program Grant Number JPMJMI17EJ.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The article contains all the data produced in this study.

**Acknowledgments:** We thank N. Tanzawa for her technical assistance.

**Conflicts of Interest:** The authors declare no financial or commercial conflict of interest.
