Biosynthetic pathway for γ-cyclic sarcinaxanthin in Micrococcus luteus: heterologous expression and evidence for diverse and multiple catalytic functions of C(50) carotenoid cyclases

Abstract

We report the cloning and characterization of the biosynthetic gene cluster (crtE, crtB, crtI, crtE2, crtYg, crtYh, and crtX) of the γ-cyclic C(50) carotenoid sarcinaxanthin in Micrococcus luteus NCTC2665. Expression of the complete and partial gene cluster in Escherichia coli hosts revealed that sarcinaxanthin biosynthesis from the precursor molecule farnesyl pyrophosphate (FPP) proceeds via C(40) lycopene, C(45) nonaflavuxanthin, C(50) flavuxanthin, and C(50) sarcinaxanthin. Glucosylation of sarcinaxanthin was accomplished by the crtX gene product. This is the first report describing the biosynthetic pathway of a γ-cyclic C(50) carotenoid. Expression of the corresponding genes from the marine M. luteus isolate Otnes7 in a lycopene-producing E. 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 C(50) carotenoid cyclase genes crtYg and crtYh from M. luteus replaced with the analogous ε-cyclic C(50) carotenoid cyclase genes crtYe and crtYf from the natural decaprenoxanthin producer Corynebacterium glutamicum. Surprisingly, expression of this hybrid gene cluster in an E. coli host resulted in accumulation of not only decaprenoxanthin, but also sarcinaxanthin and the asymmetric ε- and γ-cyclic C(50) 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 C(50) carotenoid cyclases as key catalysts for the synthesis of structurally different carotenoids.

FIG. 1.

(A to D) HPLC elution profiles of carotenoids extracted from M. luteus strains Otnes7 (A), E. coli(pAC-LYC)(pCRT-E2YgYh-O7) (B), E. coli(pAC-LYC)(pCRT-E2YgYhX-O7) (C), and E. coli(pAC-LYC)(pCRT-E2-O7) (D). Peak 1, sarcinaxanthin diglucoside; peak 2, sarcinaxanthin monoglucoside; peak 3, sarcinaxanthin; peak 4, lycopene; peak 5, flavuxanthin; peak 6, nonaflavuxanthin; Peaks 4′, 5′, and 6′ are the cis isomers of 4, 5, and 6, respectively. (E) Absorption spectra of carotenoids from peaks 1, 2, and 3 (solid line) and peaks 4, 5, and 6 (dashed line). AU, arbitrary units.

FIG. 2.

Chromosomal organization of the M. luteus sarcinaxanthin biosynthetic gene cluster presented in this study. The analogous C. glutamicum and Dietzia sp. biosynthetic gene clusters for the C₅₀ carotenoids decaprenoxanthin and C.p.450, respectively, are included for comparison. Genes indicated by white arrows are suggested not to be involved in carotenoid biosynthesis.

FIG. 3.

Relative carotenoid abundances in extracts from E. coli(pAC-LYC)(pCRT-E2YgYh-O7) and E. coli(pAC-LYC)(pCRT-E2YgYh-2665) overexpressing crtE2, crtYg, and crtYh genes from M. luteus strains Otnes7 and NCTC2665 (Table 1) cultivated in the presence of various Pm inducer concentrations (0, 0.002, 0.01, and 0.5 mM m-toluic acid). The fractions of sarcinaxanthin, lycopene, and intermediates are indicated. Samples (three replicates) were analyzed after 48 h of cultivation to ensure maximum sarcinaxanthin production levels (see Materials and Methods).

FIG. 4.

Elucidated biosynthetic pathway for the individual steps in the formation of sarcinaxanthin and its glucosides from lycopene. CrtEBI, GGPP synthase, phytoene synthase, and phytoene desaturase; CrtE2, lycopene elongase; CrtYg plus CrtYf, C₅₀ carotenoid γ-cyclase; CrtX, C₅₀ carotenoid glycosyl transferase.

FIG. 5.

HPLC elution profiles of the carotenoids extracted from E. coli(pAC-LYC)(pCRT-E2 ml-YeYfcg) (A); purified peak 1, sarcinaxanthin (B); peak 2, sarprenoxanthin (C); and peak 3, decaprenoxanthin (D).

FIG. 6.

Diverse biochemical functions of the M. luteus and the C. glutamicum C₅₀ carotenoid cyclases. Biosynthesis of both sarcinaxanthin and decaprenoxanthin involves conversion of lycopene to flavuxanthin catalyzed by the lycopene elongases CrtEb and CrtE2 in M. luteus and C. glutamicum, respectively. The M. luteus CrtYgYh polypeptides constitute a γ-cyclase specifically converting flavuxanthin into sarcinaxanthin. In contrast, the C. glutamicum CrtYgYh polypeptides constitute both γ-cyclase and ɛ-cyclase activities and can convert flavuxanthin into three different C₅₀ carotenoids; decaprenoxanthin, sarcinaxanthin, and sarprenoxanthin.