Production of diverse retinal analogues in engineered Escherichia coli through promiscuous carotenoid cleavage by Blh

Abstract Nature produces hundreds of carotenoids, yet only a handful of the apocarotenoids derived from them are accessible through microbial production. The best-known example is retinal, the chromophore of rhodopsins and a precursor of pharmaceutical retinoids, which is generated by the central cleavage of β-carotene. Whether the same cleavage chemistry can be extended to other carotenoids, yielding retinal analogues that differ in their ring structures, and potentially in their biological activities, has remained largely untested. In this study, we demonstrate a pathway engineering approach in E. coli for the biosynthesis of diverse retinal analogues by leveraging substrate promiscuity of Blh, a bacterial carotenoid cleavage enzyme originally identified in microbial rhodopsin gene clusters. While initial co-expression of Blh with carotenoid pathway genes often resulted in the production of retinal (by cleavage of β-carotene intermediate), we found that by optimizing the expression level of Blh, carotenoids such as astaxanthin or canthaxanthin were cleaved efficiently. Structure-guided engineering of Blh, informed by its predicted substrate-binding cavity, further improved the cleavage of zeaxanthin. This expanded catalytic activity suggests that Blh can serve as a versatile biocatalyst for the production of diverse retinal analogues, potentially yielding compounds with a range of biological activities. Furthermore, our findings raise the possibility of diverse biological roles for these enzymes in their native biological contexts. Importance This study demonstrated the successful biosynthesis of a diverse array of retinal analogues in engineered Escherichia coli through the heterologous expression of Blh, a β-carotene cleavage dioxygenase, together with several carotenoid pathways. Careful design of the Blh expression construct enabled modulation of retinoid proportions in the engineered pathway. This work uncovers previously unrecognized substrate promiscuity of Blh, revealing its capacity to accept carotenoids beyond β-carotene as substrates. For the first time, the predicted structure of Blh revealed the enzyme’s substrate cavity. Rational engineering by amino acid substitution designed to expand the cavity enabled the improved cleavage of hydroxylated carotenoids. These findings open new avenues for both fundamental research and biotechnological applications and have the potential to impact the microbial production of valuable retinoids.