decaprenoxanthin and sarcinaxanthin

While the majority of the natural carotenoid pigments are based on 40-carbon (C

Topics: Biosynthetic Pathways; Carotenoids; Corynebacterium; Escherichia coli; Fatty Acid Elongases; Lycopene; Micrococcus luteus; Multigene Family; Protein Engineering; Xanthophylls

The yellow-pigmented soil bacterium Corynebacterium glutamicum ATCC13032 is accumulating the cyclic C50 carotenoid decaprenoxanthin and its glucosides. Carotenoid pathway engineering was previously shown to allow for efficient lycopene production. Here, engineering of C. glutamicum for production of endogenous decaprenoxanthin as well as of the heterologous C50 carotenoids C.p.450 and sarcinaxanthin is described. Plasmid-borne overexpression of genes for lycopene cyclization and hydroxylation from C. glutamicum, Dietzia sp., and Micrococcus luteus, in a lycopene-producing platform strain constructed here, resulted in accumulation of these three C50 carotenoids to concentrations of about 3-4 mg/g CDW. Chromosomal deletion of a putative carotenoid glycosyltransferase gene cg0730/crtX in these strains entailed production of non-glucosylated derivatives of decaprenoxanthin, C.p.450, and sarcinaxanthin, respectively. Upon introduction of glucosyltransferase genes from M. luteus, C. glutamicum, and Pantoea ananatis, these hydroxylated C50 carotenoids were glucosylated. We here also demonstrate production of the C40 carotenoids β-carotene and zeaxanthin in recombinant C. glutamicum strains and co-expression of the P. ananatis crtX gene was used to obtain glucosylated zeaxanthin. Together, our results show that C. glutamicum is a potentially valuable host for production of a wide range of glucosylated C40 and C50 carotenoids.

Topics: Actinomycetales; Corynebacterium glutamicum; Glycosylation; Metabolic Engineering; Micrococcus; Pantoea; Xanthophylls

Carotenoid cleavage oxygenases are nonheme iron enzymes that specifically cleave carbon-carbon double bonds of carotenoids. Their apocarotenoid cleavage products serve as important signaling molecules that are involved in various biological processes. A database search revealed the presence of putative carotenoid cleavage oxygenase genes in the genomes of Sphingopyxis alaskensis RB2256 and Plesiocystis pacifica SIR-1. The four genes sala_1698, sala_1008, ppsir1_15490 and ppsir1_17230 were cloned and heterologously expressed in carotenoid-producing Escherichia coli JM109 strains. Two of the four encoded proteins exhibited carotenoid cleavage activity. S. alaskensis RB2256 carotenoid cleavage oxygenase (SaCCO), which is encoded by sala_1698, was shown to cleave acyclic and monocyclic substrates. Coexpression of sala_1698 in carotenoid-producing E. coli JM109 strains revealed cleavage activity for lycopene, hydroxylycopene, and dihydroxylycopene. The monocyclic substrate apo-8'-carotenal was cleaved in vitro by purified SaCCO at the 9'/10' and 11'/12' double bonds. The second enzyme, P. pacifica SIR-1 carotenoid cleavage oxygenase (PpCCO), is encoded by ppsir1_15490. PpCCO-mediated carotenoid cleavage requires the presence of either hydroxy or keto groups. PpCCO cleaved zeaxanthin, hydroxylycopene, and dihydroxylycopene, and also the C(50) carotenoids decaprenoxanthin, sarprenoxanthin and sarcinaxanthin, in carotenoid-producing E. coli JM109 strains. Whole cells of E. coli JM109 overexpressing ppsir1_15490mut, a mutant of ppsir1_15490 with enhanced gene expression, were applied for the conversion of carotenoids. Analysis of the carotenoid cleavage products revealed a single cleavage site at the 13'/14' double bond for astaxanthin, and two cleavage sites at the 11'/12' or 13'/14' double bond for zeaxanthin, nostoxanthin, and canthaxanthin.

Topics: Bacterial Proteins; Binding Sites; Biocatalysis; Carotenoids; Chromatography, Liquid; Escherichia coli; Isoenzymes; Kinetics; Lycopene; Mass Spectrometry; Molecular Structure; Mutation; Oxygenases; Proteobacteria; Retinoids; Species Specificity; Substrate Specificity; Xanthophylls