Rational reprogramming of fungal polyketide first-ring cyclization.

Resorcylic acid lactones and dihydroxyphenylacetic acid lactones represent important pharmacophores with heat shock response and immune system modulatory activities. The biosynthesis of these fungal polyketides involves a pair of collaborating iterative polyketide synthases (iPKSs): a highly reducing iPKS with product that is further elaborated by a nonreducing iPKS (nrPKS) to yield a 1,3-benzenediol moiety bridged by a macrolactone. Biosynthesis of unreduced polyketides requires the sequestration and programmed cyclization of highly reactive poly-ß-ketoacyl intermediates to channel these uncommitted, pluripotent substrates to defined subsets of the polyketide structural space. Catalyzed by product template (PT) domains of the fungal nrPKSs and discrete aromatase/cyclase enzymes in bacteria, regiospecific first-ring aldol cyclizations result in characteristically different polyketide folding modes. However, a few fungal polyketides, including the dihydroxyphenylacetic acid lactone dehydrocurvularin, derive from a folding event that is analogous to the bacterial folding mode. The structural basis of such a drastic difference in the way a PT domain acts has not been investigated until now. We report here that the fungal vs. bacterial folding mode difference is portable on creating hybrid enzymes, and we structurally characterize the resulting unnatural products. Using structure-guided active site engineering, we unravel structural contributions to regiospecific aldol condensations and show that reshaping the cyclization chamber of a PT domain by only three selected point mutations is sufficient to reprogram the dehydrocurvularin nrPKS to produce polyketides with a fungal fold. Such rational control of first-ring cyclizations will facilitate efforts to the engineered biosynthesis of novel chemical diversity from natural unreduced polyketides.

CYP287A1 is a carotenoid 2-ß-hydroxylase required for deinoxanthin biosynthesis in Deinococcus radiodurans R1.

The carotenoid deinoxanthin is a crucial resistance factor against various stresses in the radiation-resistant bacterium Deinococcus radiodurans. Disruption of the gene dr2473 encoding the cytochrome P450 CYP287A1 led to the accumulation of 2-deoxydeinoxanthin in D. radiodurans, demonstrating that CYP287A1 is a novel ß-carotene 2-hydroxylase. The dr2473 knockout mutant was shown to be more sensitive to UV radiation and oxidative stress than the wild-type strain D. radiodurans R1, indicating that the C2 alcohol of deinoxanthin is important for antioxidant activity.

Hymenobacter kanuolensis sp. nov., a novel radiation-resistant bacterium.

A Gram-reaction-negative, rod-shaped, non-motile, red-pigmented, radiation-resistant, aerobic bacterium designated T-3(T) was isolated from a soil sample from the Qinghai-Tibet Plateau in Tibet, China, after exposure to 10 kGy gamma radiation. Phylogenetic analysis based on 16S rRNA sequences indicated that this isolate represented a novel member of the genus Hymenobacter. Sequence identities of the 16S rRNA gene of strain T-3(T) with the type strains of species of the genus Hymenobacter with validly published names range from 89% to 97%, and the most closely related species is Hymenobacter psychrotolerans Tibet-IIU11(T) (97%). The DNA-DNA relatedness between strain T-3(T) and H. psychrotolerans is 59.10%. The major fatty acids of strain T-3(T) were iso-C(15 : 0) (27.66%), summed feature 4 (iso-C(17 : 1)I and/or anteiso-C(17 : 1)B, 15.84%), anteiso-C(15 : 0) (14.08%) and summed feature 3 (C(16 : 1)ω7c and/or C(16 : 1)ω6c, 12.38%). The major menaquinone of strain T-3(T) was MK-7. Phosphatidylethanolamine (PE) was predominant in the polar lipid profile. The G+C content of the DNA of strain T-3(T) was 69.17 mol%. On the basis of the results of the polyphasic characterization presented in this study, it is concluded that strain T-3(T) represents a novel species of the genus Hymenobacter, for which the name Hymenobacter kanuolensis is proposed. The type strain is T-3(T) ( = ACCC 05760(T) = KCTC 32407(T)).

Sphingobacterium arenae sp. nov., isolated from sandy soil.

A novel, Gram-stain-negative, non-motile, non-spore-forming, short rod-shaped, aerobic bacterium designated H-12(T) was isolated from a mixed sandy soil sample collected from Xinjiang, China. Strain H-12(T) grew at 20-37 °C (optimum, 30 °C), pH 7.0-11.0 (optimum, pH 8.5) on TGY medium with 0-5 % NaCl (w/v). 16S rRNA gene sequence analysis indicated that strain H-12(T) shared sequence similarities with Sphingobacterium composti DSM 18850(T) (90.0 %). Strain H-12(T) showed a low level of DNA-DNA relatedness to Sphingobacterium composti DSM 18850(T) (45.5 %). The predominant isoprenoid quinone of strain H-12(T) was MK-7 and the major polar lipid was phosphatidylethanolamine. The predominant cellular fatty acids were C16:1ω7c and/or C16 : 1ω6c, iso-C15 : 0, iso-C17 : 0 3-OH, C16 : 0, C16 : 0 3-OH and iso-C15 : 0 3-OH. DNA G+C content of strain H-12(T) was 44.15 mol%. On the basis of phenotypic, genetic and phylogenetic data, strain H-12(T) is proposed to be a representative of a novel species of the genus Sphingobacterium, for which the name Sphingobacterium arenae sp. nov. is suggested and the type strain is H-12(T) ( = ACCC 05758(T) = KCTC 32294(T)).

Whole-genome optical mapping and finished genome sequence of Sphingobacterium deserti sp. nov., a new species isolated from the Western Desert of China.

A novel Gram-negative bacterium, designated ZWT, was isolated from a soil sample of the Western Desert of China, and its phenotypic properties and phylogenetic position were investigated using a polyphasic approach. Growth occurred on TGY medium at 5-42°C with an optimum of 30°C, and at pH 7.0-11.0 with an optimum of pH 9.0. The predominant cellular fatty acids were summed feature 3 (C16:1ω7c/C16:1ω6c or C16:1ω6c/C16:1ω7c) (39.22%), iso-C15:0 (27.91%), iso-C17:0 3OH (15.21%), C16:0 (4.98%), iso-C15:0 3OH (3.03%), C16:0 3OH (5.39%) and C14:0 (1.74%). The major polar lipid of strain ZWT is phosphatidylethanolamine. The only menaquinone observed was MK-7. The GC content of the DNA of strain ZWT is 44.9 mol%. rDNA phylogeny, genome relatedness and chemotaxonomic characteristics all indicate that strain ZWT represents a novel species of the genus Sphingobacterium. We propose the name S. deserti sp. nov., with ZWT (= KCTC 32092T = ACCC 05744T) as the type strain. Whole genome optical mapping and next-generation sequencing was used to derive a finished genome sequence for strain ZWT, consisting of a circular chromosome of 4,615,818 bp in size. The genome of strain ZWT features 3,391 protein-encoding and 48 tRNA-encoding genes. Comparison of the predicted proteome of ZWT with those of other sphingobacteria identified 925 species-unique proteins that may contribute to the adaptation of ZWT to its native, extremely arid and inhospitable environment. As the first finished genome sequence for any Sphingobacterium, our work will serve as a useful reference for subsequent sequencing and mapping efforts for additional strains and species within this genus.