Elucidating the Role and Mechanism of Lycopene β-Cyclase from <i>Haloarcula marismortui</i> through Functional Characterization and Targeted Mutagenesis

Lycopene β-cyclase (LCYb) serves as a pivotal gatekeeper enzyme in carotenoid biosynthesis, catalyzing an enzymatic conversion of lycopene to β-carotene. While phylogenetically widespread across life domains, structural and functional characterization of archaeal LCYbs remains conspicuously understudied. Here, we report biochemical validation and mechanistic dissection of a putative LCYb from the hypersaline-adapted archaeon <i>Haloarcula marismortui</i> (Hma-LCYb). Heterologous expression of Hma-LCYb in the lycopene-accumulating <i>Escherichia coli</i> strain resulted in the efficient β-carotene biosynthesis (0.91 ± 0.01 mg/g DCW, dried cell weight), demonstrating cross-domain functionality. Remarkably, introducing the <i>Hma-LCYb</i> gene into the LCYb-deficient haloarchaeon <i>Haloferax volcanii</i> not only enabled β-carotene production but achieved the titers (0.21 ± 0.002 mg/g DCW) surpassing the endogenous bacterioruberin levels (0.06 ± 0.003 mg/g DCW), establishing the first archaeal platform for halo-adapted carotenoid engineering. Mutational analyses revealed that a combination of residues D55, W64, E82, Y140, R168, and E214 of Hma-LCYb is critical for catalyzing lycopene-to-β-carotene conversion. These findings resolve long-standing questions regarding archaeal carotenoid cyclization mechanisms and highlight biotechnological potential of extremophilic enzymes in industrial carotenoid production.