title: Assembly of functional photosystem complexes in Rhodobacter sphaeroides incorporating carotenoids from the spirilloxanthin pathway.
abstract: Carotenoids protect the photosynthetic apparatus against harmful radicals arising from the presence of both light and oxygen. They also act as accessory pigments for harvesting solar energy, and are required for stable assembly of many light-harvesting complexes. In the phototrophic bacterium Rhodobacter (Rba.) sphaeroides phytoene desaturase (CrtI) catalyses three sequential desaturations of the colourless carotenoid phytoene, extending the number of conjugated carbon-carbon double bonds, N, from three to nine and producing the yellow carotenoid neurosporene; subsequent modifications produce the yellow/red carotenoids spheroidene/spheroidenone (N=10/11). Genomic crtI replacements were used to swap the native three-step Rba. sphaeroides CrtI for the four-step Pantoea agglomerans enzyme, which re-routed carotenoid biosynthesis and culminated in the production of 2,2'-diketo-spirilloxanthin under semi-aerobic conditions. The new carotenoid pathway was elucidated using a combination of HPLC and mass spectrometry. Premature termination of this new pathway by inactivating crtC or crtD produced strains with lycopene or rhodopin as major carotenoids. All of the spirilloxanthin series carotenoids are accepted by the assembly pathways for LH2 and RC-LH1-PufX complexes. The efficiency of carotenoid-to-bacteriochlorophyll energy transfer for 2,2'-diketo-spirilloxanthin (15 conjugated C = C bonds; N=15) in LH2 complexes is low, at 35%. High energy transfer efficiencies were obtained for neurosporene (N=9; 94%), spheroidene (N=10; 96%) and spheroidenone (N=11; 95%), whereas intermediate values were measured for lycopene (N=11; 64%), rhodopin (N=11; 62%) and spirilloxanthin (N=13; 39%). The variety and stability of these novel Rba. sphaeroides antenna complexes make them useful experimental models for investigating the energy transfer dynamics of carotenoids in bacterial photosynthesis.
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Shuang C
authors.initials: SC
authors.lastname: Chi
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Mothersole
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130-4889, USA.
authors.forename: Preston
authors.initials: P
authors.lastname: Dilbeck
authors.affiliation: Photosynthetic Antenna Research Center, Washington University, St. Louis, MO 63130 USA.
authors.forename: Dariusz M
authors.initials: DM
authors.lastname: Niedzwiedzki
authors.affiliation: Photosynthetic Antenna Research Center, Washington University, St. Louis, MO 63130 USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Pu
authors.initials: P
authors.lastname: Qian
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Cvetelin
authors.initials: C
authors.lastname: Vasilev
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Katie J
authors.initials: KJ
authors.lastname: Grayson
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom; ChELSI Institute, Department of Chemical and Biological Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, United Kingdom.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Jackson
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom.
authors.forename: Elizabeth C
authors.initials: EC
authors.lastname: Martin
authors.affiliation: Department of Microbiology and Immunology, China Agricultural University, Haidian District, Beijing 100193, China.
authors.forename: Ying
authors.initials: Y
authors.lastname: Li
authors.affiliation: Department of Chemistry, Washington University, St. Louis, MO 63130-4889, USA.
authors.forename: Dewey
authors.initials: D
authors.lastname: Holten
authors.affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, United Kingdom. Electronic address: c.n.hunter@sheffield.ac.uk.
authors.forename: C
authors.initials: C
authors.lastname: Neil Hunter
mesh_terms: D001429:Bacteriochlorophylls / Q000378:metabolism; D002338:Carotenoids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004735:Energy Transfer; D013058:Mass Spectrometry; D010088:Oxidoreductases / Q000502:physiology; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry*; D012242:Rhodobacter sphaeroides / Q000378:metabolism*; D024341:Xanthophylls / Q000378:metabolism
chemical_list: D001429:Bacteriochlorophylls; D045322:Photosynthetic Reaction Center Complex Proteins; D024341:Xanthophylls; D002338:Carotenoids; D010088:Oxidoreductases; C022398:phytoene dehydrogenase; C055255:spirilloxanthin
keywords: Antenna; Bacterial photosynthesis; Carotenoid; Light harvesting; Membrane protein; Synthetic biology