Unveiling the critical role of K⁺ for xanthorhodopsin expression in E. coli

Xanthorhodopsin (XR), a retinal-binding 7-transmembrane protein isolated from the eubacterium Salinibacter ruber, utilizes two chromophores (retinal and salinixanthin (SAL)) as an outward proton pump and energy-donating carotenoid. However, research on XR has been impeded owing to limitations in achieving heterogeneous expression of stable forms and high production levels of both wild-type and mutants. We successfully expressed wild-type and mutant XRs in Escherichia coli in the presence of K⁺. Achieving XR expression requires significant K⁺ and a low inducer concentration. In particular, we highlight the significance of Ser-159 in helix E located near Gly-156 (a carotenoid-binding position) as a critical site for XR expression. Our findings indicate that replacing Ser-159 with a smaller amino acid, alanine, can enhance XR expression in a manner comparable to K⁺, implying that Ser-159 poses a steric hindrance for pigment formation in XR. In the presence of K⁺, the proton pumping and photocycle of the wild-type and mutants were characterized and compared; the wild-type result suggests similar properties to the first reported XR isolation from the S. ruber membrane fraction. We propose that the K⁺ gradient across the cell membrane of S. ruber serves to uphold the membrane potential of the organism and plays a role in the expression of proteins, such as XR, as demonstrated in our study. Our findings deepen the understanding of adaptive protein expression, particularly in halophilic organisms. We highlight salt selection as a promising strategy for improving protein yield and functionality.