Elucidating the Role and Mechanism of Lycopene β-Cyclase from Haloarcula marismortui through Functional...

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 Haloarcula marismortui (Hma-LCYb). Heterologous expression of Hma-LCYb in the lycopene-accumulating Escherichia coli 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 Hma-LCYb gene into the LCYb-deficient haloarchaeon Haloferax volcanii 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.