Benjamin N. Mijts, Pyung Cheon Lee, Claudia Schmidt-Dannert

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Presentation transcript:

Identification of a Carotenoid Oxygenase Synthesizing Acyclic Xanthophylls Benjamin N. Mijts, Pyung Cheon Lee, Claudia Schmidt-Dannert Chemistry & Biology Volume 12, Issue 4, Pages 453-460 (April 2005) DOI: 10.1016/j.chembiol.2005.02.010 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Biosynthesis of Oxygenated C30 and C40 Carotenoids Biosynthetic pathways to oxygenated, linear C30 (A) and C40 (B) carotenoids. Solid arrows represent natural biosynthetic pathways suggested for staphyloxanthin in S. aureus (A) and lycopene (B). Biosynthetic pathway steps in engineered recombinant E. coli are indicated by dashed arrows. Carotenoids identified in recombinant E. coli are underlined. Chemistry & Biology 2005 12, 453-460DOI: (10.1016/j.chembiol.2005.02.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 S. aureus Carotenoid Operon Region of the S. aureus genome sequence showing open reading frames upstream of the known S. aureus carotenoid genes crtM (dehydrosqualene synthase) and crtN (dehydrosqualene desaturase) in a typical bacterial operon structure. NP_373089, crtOX; NP_373089, no known functional homology; NP_373087, conserved glycosyl transferase motif. Chemistry & Biology 2005 12, 453-460DOI: (10.1016/j.chembiol.2005.02.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Carotenoid Production in Recombinant E. coli Cells LB media cultures (top), cell pellets (center), and TLC analysis (bottom) of recombinant C30 (A) and C40 (B) carotenoid-producing E. coli strains. Background plasmids strains are JM109 pAC-ispA-crtM-crtN (A) and pAC-crtE-crtB-crtI14 (B) cotransformed with 1. pUCMod, 2. pUC-crtOx, 3. pUC-crtOxmut1, 4. pUC-crtOxmut2, and 5. pUC-crtOxmut3. Identified compounds from Figure 1 are indicated. Chemistry & Biology 2005 12, 453-460DOI: (10.1016/j.chembiol.2005.02.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Analysis of E. coli Cells Producing Oxygenated C30 Carotenoids HPLC profiles of recombinant E. coli expressing the C30 carotenoid background plasmid pAC-ispA-crtN-crtM with (A) pUC-crtOx, (B) pUC-crtOxmut1, (C) pUC-crtOxmut2, (D) pUC-crtOxmut3, and E. coli expressing the C40 carotenoid background plasmid pAC-crtE-crtB-crtI14 with (E) pUC-crtOx, (F) pUC-crtOxmut1, (G) pUC-crtOxmut2, and (H) pUC-crtOxmut3. Chemistry & Biology 2005 12, 453-460DOI: (10.1016/j.chembiol.2005.02.010) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 Carotenoid Profiles of Lycopene-Producing Recombinant E. coli Cells with and without CrtOx Present HPLC profiles at wavelengths of 300 and 500 nm of recombinant E. coli expressing the C40 carotenoid lycopene background plasmid pAC-crtE-crtB-crtI with (A) pUCMod and (B) pUC-crtOx. Based on mass spectrometry, HPLC retention time, and UV-Vis spectra, the peaks were identified as L: lycopene (500 nm) and P: phytoene (300 nm). Chemistry & Biology 2005 12, 453-460DOI: (10.1016/j.chembiol.2005.02.010) Copyright © 2005 Elsevier Ltd Terms and Conditions