Biosynthetic Enzymes For The Active Groups Of Carotenoids In Deinococcus Radiodurans

Deinococcus radiodurans is a red-pigmented and nonphotosynthetic bacterium well known for its resistance to y-ray, ultraviolet radiation and desiccation. Its extremely resistance was attributted to its highly efficient antioxidative effect and the complex network of DNA repair mechanism. Most of the damaging effects of ionizing radiation on biological macromolecules are due to the reactive oxygen species (ROS) produced by water radiolysis. Among non-enzymic antioxidants, carotenoids are efficient scavengers of ROS, especially of singlet oxygen. The major carotenoid in D. radiodurans is a unique hydroxylated ketocarotenoid (deinoxanthin), which shows stronger ROS scavenging ability than lycopene andβ-carotene and contributes to the cell resistance of D. radiodurans under oxidative stress. The active groups of deinoxanthin, including C-3',4'double bond, C-1'hydroxyl group and C-4 keto group, may be important for the antioxidant ability of deinoxanthin. So, investigations of the carotenoid synthetases for these active groups can help us to reveal the biosynthetic pathway of deinoxanthin and understand the role of carotenoids in the cell resistance of DR. This Ph.D thesis focuses on identification of carotenoid synthetases for the active groups. The main results are as follows:1. The biosynthetic pathway of deinoxanthin is unclear, although several enzymes are presumed to be involved. The gene (dr2250) was predicted by gene homologue analysis to encode carotenoid 3',4'-desaturase (CrtD). This putative gene was deleted to investigate its function. Carotenoid analysis of the resultant mutant verified that DR2250 encodes the carotenoid 3',4'-desaturase, which catalyses the C3',4'-desaturation of the monocyclic precursor of deinoxanthin but not acyclic carotenoids. The co-transformed plamids experiments confirmed that DR2250 can not catalyze the non-modifiedψend, but only catalyze theψend with hydroxylation at C1'. The lack of CrtD decreased the antioxidant capacity of the mutant of dr2250 compared with the wild-type, indicating that the C3',4'-desaturation step contributes to the antioxidant capacity of deinoxanthin in D. radiodurans.2. Carotenoid 1,2-hydratase is required to catalyse the synthesis of deinoxanthin by hydration at the C-1',2'double bond. A novel carotenoid 1,2-hydratase (CruF) responsible for the C-1',2'hydration of y-carotene was identified in the non-photosynthetic bacteria D. radiodurans R1 and D. geothermalis DSM 11300. Gene expression and disruption experiments demonstrated that dr0091 and dgeo2309 encode CruF in D. radiodurans and D. geothermalis, respectively. Their homologues were also found in the genomes of cyanobacteria, and exhibited little homology to the hydroxyneurosporene synthase (CrtC) proteins found mainly in photosynthetic bacteria. Phylogenetic analysis showed that CruF homologues form a separate family, which is evolutionarily distant from the known CrtC family.3. D. radiodurans strain R1 synthesizes the unique ketocarotenoid deinoxanthin. It was reported that expression of DR0093 in the E. coli strain which accumulated P-carotene resulted in the synthesis of canthaxanthin and echinenone. However, the bicyclic carotenoids were not the intermediate product of the deinoxanthin biosynthetic pathway, in which only monocyclic carotenoids were detected. Thus, the function of DR0093 and its catalyzing step need to be elucidated in the native host. A carotene ketolase homologue encoded by dr0093 was inactivated by gene mutation to verify its function in the native host D. radiodurans. Analysis of the carotenoids in the resultant mutant RIΔcrtO demonstrated that dr0093 encodesγ-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position ofγ-carotene derivatives to form ketolated carotenoids. The mutant R1ΔcrtO became more sensitive to H2O2 treatment than the wild-type strain R1, indicating that the C-4 keto group is important for the antioxidant activity of carotenoids in D. radiodurans. Carotenoid extracts from mutant R1ΔcrtO exhibited lower DPPH radical-scavenging activity than those from the wild-type strain R1. The enhanced antioxidant ability of ketocarotenoids in D. radiodurans might be attributed to its extended conjugated double bonds and relative stability by the C-4 keto group substitution.In sum, our results demonstrated that the active groups of deinoxanthin were important for its antioxidant capability. The carotenoid biosynthetic enzymes identified from D. radiodurans can also be used, via genetic engineering, for the production of novel carotenoids with high activities.