Biosynthetic Pathway for γ-Cyclic Sarcinaxanthin in Micrococcus luteus : Heterologous Expression and Evidence for Diverse and Multiple Catalytic Functions of C 50 Carotenoid Cyclases

We report the cloning and characterization of the biosynthetic gene cluster (crtE ,crtB ,crtI ,crtE2 ,crtYg ,crtYh , andcrtX ) of the γ-cyclic C50 carotenoid sarcinaxanthin inMicrococcus luteus NCTC2665. Expression of the complete and partial gene cluster inEscherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C40 lycopene, C45 nonaflavuxanthin, C50 flavuxanthin, and C50 sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by thecrtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C50 carotenoid. Expression of the corresponding genes from the marineM. luteus isolate Otnes7 in a lycopene-producingE. coli host resulted in the production of up to 2.5 mg/g cell dry weight sarcinaxanthin in shake flasks. In an attempt to experimentally understand the specific difference between the biosynthetic pathways of sarcinaxanthin and the structurally related ε-cyclic decaprenoxanthin, we constructed a hybrid gene cluster with the γ-cyclic C50 carotenoid cyclase genescrtYg andcrtYh fromM. luteus replaced with the analogous ε-cyclic C50 carotenoid cyclase genescrtYe andcrtYf from the natural decaprenoxanthin producerCorynebacterium glutamicum . Surprisingly, expression of this hybrid gene cluster in anE. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C50 carotenoid sarprenoxanthin, described for the first time in this work. Together, these data contributed to new insight into the diverse and multiple functions of bacterial C50 carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.