Overexpression and Characterization of Lycopene Cyclase (CrtY) from Marine Bacterium Paracoccus haeundaensis

Abstract

Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

Cloning and Characterization of D-xylulose Kinase from Kocuria gwangalliensis Strain SJ2

“…Bacterial strains and growth conditions A pink-orange pigmented bacterial strain, K. gwangalliensis strain SJ2 [11], was isolated from seawater collected on the Gwangalli coast of Busan. This strain was isolated on was added to the tube and the mixture was inverted several times until mixed well.…”

“…The mixture was sonicated three times for 30 s each using an Ultra sonicator under 90~100% power and then centrifuged at 13,000 rpm for 10 min. The supernatant was filtered through 0.22 μM filter [11].…”

Research Progress in Heterologous Crocin Production

“…It is simultaneously converted into α-carotene and β-carotene in the carotenoid pathway, with the latter feeding into the crocin biosynthesis pathway. In order to obtain more β-carotene to improve the efficiency of crocin biosynthesis, strategies for genetic engineering have focused on lycopene β-cyclase (LCYB), which catalyzes lycopene to β-carotene, and β-carotene hydroxylase (CHYB), which generates zeaxanthin (Figure 3) [88,89]. For example, Eu-CrtZ, a gene encoding LCYB from the bacterium Pantoea ananatis (reclassified as Erwinia uredovora, hence Eu-CrtZ), can be expressed in Yarrowia lipolytica to obtain 21.98 ± 1.80 mg/L zeaxanthin [89].…”

Molecular Cloning and Overexpression of Phytoene Desaturase (CrtI) from Paracoccus haeundaensis

“…To analyze the activity of the phytoene desatruase expressed in E. coli, we expressed phytoene desaturase from BL21(DE3) and performed an enzyme assay after lyse the cells [28,29]. Overexpression was induced in BL21(DE3), which was transformed using pET-44(a)-CrtI plasmid, using IPTG, which was then lysed to analyze phytoene desaturase activity.…”