Product Speculation from Carotenogenic Gene Cluster of Nonlabens spongiae Genome, and Identification of Myxol and Functional Analysis of Each Gene

<b>Background:</b> Myxol, a monocyclic carotenoid with β- and ψ-end groups, has been identified in only a limited number of bacteria, such as flavobacteria and cyanobacteria. Despite its biological significance, the biosynthetic pathway of myxol is not well understood, and studies on its physiological functions and biological activities are limited because of its rarity. <b>Methods:</b> BLAST homology searches for carotenoid biosynthesis genes in the genome of <i>Nonlabens</i> were performed. The carotenogenesis-related genes in the genome of the marine flavobacteria <i>Nonlabens spongiae</i> were individually cloned and functionally characterized using a heterologous <i>Escherichia coli</i> expression system. Carotenoids from <i>N. spongiae</i> were identified using an LC-MS analysis. <b>Results:</b> We identified a gene cluster involved in carotenoid biosynthesis in the genome of <i>N. spongiae</i>. This cluster includes genes encoding phytoene synthase (CrtB), phytoene desaturase (CrtI), lycopene cyclase (CrtY), carotenoid 1,2-hydratase (CruF), carotenoid 3,4-desaturase (ψ-end group) (CrtD), carotenoid 2-hydroxylase (ψ-end group) (CrtA-OH), and carotene hydro-xylase (CrtZ). Based on the characteristics of these enzymes, the primary products were predicted to be myxol and/or zeaxanthin. A spectroscopic analysis confirmed that myxol was the primary carotenoid. Furthermore, a plasmid containing a reconstructed gene cluster and geranylgeranyl pyrophosphate synthase (CrtE) located outside the cluster was introduced into <i>E. coli</i>. This system predominantly accumulated myxol, indicating that the reconstructed gene cluster enabled efficient myxol production in <i>E. coli</i>. <b>Conclusions:</b> This study highlighted the potential biotechnological applications of the carotenoid biosynthesis gene clusters for myxol production.