Pigment biosynthesis in chlorophototrophic bacteria: novel pathways, missing enzymes and new combinations

(Bacterio)chlorophylls and carotenoids are two of the major pigments involved in
photosynthesis; the former are responsible for light-harvesting and driving the photo-
chemical redox, while the later act as photoprotective accessory pigments.
Organisms are split by (bacterio)chlorophyll preference – broadly, oxygenic
phototrophs utilise chlorophylls, while anoxygenic phototrophs utilise lower energy
bacteriochlorophylls; the cyanobacterium Synechocystis sp. PCC 6803 and purple
bacterium Rhodobacter sphaeroides are model representatives of each group,
respectively. Contrastingly, carotenoid pathways are quite plastic and can vary
greatly between closely related phototrophs; consequentially, many carotenoid
pathways have yet to be fully elucidated, including that of the phototroph
Heliomicrobium modesticaldum. The focus of this work was split into three
independent chapters. Firstly, elucidation of the complete carotenoid pathway of H.
modesticaldum was attempted though the generation of a genomic library and
subsequent expression and colour complementation screening in E. coli, while the
uncertain structural role of this carotenoid within the heliobacterial reaction-centre
complex was investigated through the generation and spectroscopic
characterisation of a carotenoidless H. modesticaldum mutant. Secondly, the
plasticity of the R. sphaeroides carotenoid pathway and the effects of producing
carotenoids on the assembly of the light-harvesting complex 2 was investigated
through the plasmid-based expression of Staphylococcus aureus carotenoid
pathway genes; novel complexes incorporating a putative C35 carotenoid were
isolated and characterized by UV-Vis and fluorescence spectroscopy. During this
work we also identified a suppressor mutation that gives an interesting insight into
the function and evolution of some of the enzymes that act in the early stages of
carotenoid backbone biosynthesis. Finally, the prospect of combining chlorophyll
driven photosynthesis was explored though the attempted introduction of the
bacteriochlorophyll pathway into Synechocystis through recombinant genomic
engineering; in the second part of this chapter, an ‘in vivo assay’ is developed to
probe structure-function relationships in chlorophyll synthase, guided by a recent
cryo-EM structure of this enzyme.