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A novel carotenoid 1,2-hydratase (CruF) responsible for the C-1',2' hydration of [gamma]-carotene was identified in the non-photosynthetic bacteria Deinococcus radiodurans R1 and Deinococcus geothermalis DSM 11300. Gene expression and disruption experiments demonstrated that drO091 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.

Deinococcus radiodurans strain R1 synthesizes deinoxanthin, a unique carotenoid product, which contributes to cell resistance following various stresses. The biosynthetic pathway of deinoxanthin is unclear, although several enzymes are presumed to be involved. The gene (dr2250) predicted by gene homologue analysis to encode carotenoid 3',4'-desaturase (CrtD) was deleted to investigate its function. A mutant deficient in the gene homologue of crtLm (dr0801) was also constructed to verify the catalytic function of the gene product in the native host. Oarotenoid analysis of the resultant mutants verified that DR2250 encodes carotenoid 3',4'-desaturase, which catalyses the C-3',4'-desaturation of the monocyclic precursor of deinoxanthin but not acyclic carotenoids. Mutation of the gene homologue of crtLm (dr0801) resulted in accumulation of lycopene, confirming that it encodes the lycopene cyclase in the native host. The lack of CrtD decreased the antioxidant capacity of the mutant deficient in dr2250 compared with the wild-type, indicating that the C-3',4'-desaturation step contributes to the antioxidant capacity of deinoxanthin in D. radiodurans.

Plasmid DNA (pDNA) is used as an important vector for gene therapy, and its wide application is restricted by the purity and yield. To obtain high-purity pDNA, a chromatographic method based on anion-exchange supermacroporous cryogel was explored. The anion-exchange cryogel was prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrix and pUC19 plasmid was used as a target to test the method. The plasmid was transferred into Escherichia coli DH5alpha, cultivated, harvested and lysed. The obtained culture was centrifuged and the supernatant was used as the plasmid feedstock, which was loaded into the anion-exchange cryogel bed for chromatographic separation. By optimizing the pH of running buffer and the elution conditions, high-purity pDNA was obtained by elution with 0.5 mol/L sodium chloride solution at pH 6.6. Compared to the traditional methods for purification of pDNA, animal source enzymes and toxic reagents were not involved in the present separation process, ensuring the safety of both the purification operations and the obtained pDNA.

To examine the substrate specificity of carotenoid 3',4'-desaturase (DR2250) from Deinococcus radiodurans, we amplified the dr2250 gene by using PCR methods. The PCR products were digested by Hind III-BamH I and ligated into the vector pUC19, yielding recombinant vector pUC-CRTD. We analyzed the carotenoids of E. coli transformants containing pACCRT-EBI(Eu) and (or) pRK-CRTC and (or) pUC-CRTD. Our results demonstrated that DR2250 had substrate specificity on the carotenoids with hydroxyl group at C1 (1').

Deinococcus radiodurans strain R1 synthesizes a unique ketocarotenoid product named deinoxanthin. The detailed steps involved in the biosynthesis of deinoxanthin remain unresolved. 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 R1DeltacrtO demonstrated that dr0093 encodes gamma-carotene ketolase (CrtO) catalysing the introduction of one keto group into the C-4 position of gamma-carotene derivatives to form ketolated carotenoids. The mutant R1DeltacrtO became more sensitive to H(2)O(2) 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 R1DeltacrtO exhibited lower 2,2-diphenyl-1-picrylhydrazyl 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.