Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity.     

Fungal heme oxygenases: Functional expression and characterization of Hmx1 from Saccharomyces cerevisiae and CaHmx1 from Candida albicans

Heme oxygenases convert heme to free iron, CO, and biliverdin. Saccharomyces cerevisiae and Candida albicans express putative heme oxygenases that are required for the acquisition of iron from heme, a critical process for fungal survival and virulence. The putative heme oxygenases Hmx1 and CaHmx1 from S. cerevisiae and C. albicans, respectively, minus the sequences coding for C-terminal membrane-binding domains, have been expressed in Escherichia coli. The C-terminal His-tagged, truncated enzymes are obtained as soluble, active proteins. Purified ferric Hmx1 and CaHmx1 have Soret absorption maxima at 404 and 410 nm, respectively. The apparent heme binding Kd values for Hmx1 and CaHmx1 are 0.34 +/- 0.09 microM and 1.0 +/- 0.2 microM, respectively. The resonance Raman spectra of Hmx1 reveal a heme binding pocket similar to those of the mammalian and bacterial heme oxygenases. Several reductants, including ascorbate, yeast cytochrome P450 reductase (CPR), human CPR, spinach ferredoxin/ferredoxin reductase, and putidaredoxin/putidaredoxin reductase, are able to provide electrons for biliverdin production by Hmx1 and CaHmx1. Of these, ascorbate is the most effective reducing partner. Heme oxidation by Hmx1 and CaHmx1 regiospecifically produces biliverdin IXalpha. Spectroscopic analysis of aerobic reactions with H2O2 identifies verdoheme as a reaction intermediate. Hmx1 and CaHmx1 are the first fungal heme oxygenases to be heterologously overexpressed and characterized. Their heme degradation activity is consistent with a role in iron acquisition.     

Sulfated polysaccharides from marine green algae Ulva conglobata and their anticoagulant activity

Sulfated polysaccharides from the green algae Ulva conglobata were isolated and prepared by extraction in hot water, precipitation with ethanol and purification by ion-exchange and size-exclusion column chromatography. The characterizations of the sulfated polysaccharides were defined, and containing 23.04–35.20% sulfate ester groups, 10.82–14.91% uronic acid and 3.82–4.51% protein. Gas chromatography analysis shows that the sulfated polysaccharides from Ulva conglobata are mainly consisted of rhamnose with variable contents of glucose and fucose, trace amounts of xylose, glactose and mannose. The anticoagulant properties of the sulfated polysaccharides were compared with those of heparin by studying the activated partial thromboplastin time using normal human plasma. The sulfated polysaccharide from Ulva conglobata collected in Qingdao, China is the most potent among the sulfated polysaccharides tested. The mechanism of anticoagulant activity mediated by the sulfated polysaccharides is due to the direct inhibition of thrombin and the potentiation of heparin cofactor II.     

Effects of different fungal elicitors on growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous

Six fungal elicitors prepared from Rhodotorula rubra, Rhodotorula glutinis, Panus conchatus, Coriolus versicolor, Mucor mucedo, Mortieralla alpina M-23 were examined to determine their effects on the growth, total carotenoids and astaxanthin formation by Xanthophyllomyces dendrorhous. The results showed that different fungal elicitor could cause diversely stimulating effects. Among the fungal elicitors tested, the M. mucedo elicitor concentration of 30 mg l(-1) promoted the biomass and total carotenoids yield most remarkably, resulting in 69.81+/-6.00% and 78.87+/-4.15% higher than the control, respectively. At the concentration of 30 mg l(-1), R. glutinis elicitor stimulated the highest astaxanthin yield with a 90.60+/-5.98% increase compared to the control. The R. rubra elicitor concentration of 30 mg l(-1) resulted in the optimal total carotenoids and astaxanthin content to be 42.24+/-0.49% and 69.02+/-0.72% higher than the control, respectively. At the concentration of 30 mg l(-1), R. rubra elicitor gave the highest increase in the ratio of astaxanthin in total carotenoids by 18.85+/-0.11% of the control.     

Inhibitors of HCV NS5B polymerase: synthesis and structure-activity relationships of N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides

Substituted N-alkyl-4-hydroxyquinolon-3-yl-benzothiadiazine sulfamides were investigated as inhibitors of genotype 1 HCV polymerase. Structure-activity relationship patterns for this class of compounds are discussed.     

BREVIS RADIX is involved in cytokinin-mediated inhibition of lateral root initiation in <Emphasis Type="Italic">Arabidopsis</Emphasis>

In contrast to auxin, relatively little is known about the molecular mechanism of cytokinin (CTK) inhibition of lateral root initiation. Previous studies demonstrated that BREVIS RADIX (BRX), a protein of unknown biochemical function, maintains a rate-limiting brassinosteroid biosynthesis enzyme expression to keep brassinosteroid biosynthesis above a critical threshold. Here, we show that the brx-2 mutant is insensitive to exogenous CTK-induced inhibition of lateral root initiation and that this can be restored by embryonic brassinosteroid treatment. However post-embryonic brassinosteroid treatment can not rescue brx-2 mutant phenotype in the presence of CTK. Meanwhile the brassinosteroid receptor defective mutant bri1-6 shows normal CTK-mediated inhibition on LR growth. These results suggest the CTK-mediated inhibition of LR initiation is not directly dependent on brassinosteroid level. Furthermore, compared with wild type, brx-2 exhibits altered auxin response in presumptive founder cells, lateral root primodia and primary root tip in the presence of exogenous CTK. We concluded that CTK inhibition on lateral root initiation depend on specific auxin response loss in presumptive founder cell. The aberrant primary root growth caused by the embryonic brassinosteroid shortage can indirectly result in the lateral root phenotype of brx-2 in presence of CTK. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Release of developmental constraints on tetrad shape is confirmed in inaperturate pollen of Potamogeton

Background and Aims

Microsporogenesis in monocots is often characterized by successive cytokinesis with centrifugal cell plate formation. Pollen grains in monocots are predominantly monosulcate, but variation occurs, including the lack of apertures. The aperture pattern can be determined by microsporogenesis features such as the tetrad shape and the last sites of callose deposition among the microspores. Potamogeton belongs to the early divergent Potamogetonaceae and possesses inaperturate pollen, a type of pollen for which it has been suggested that there is a release of the constraint on tetrad shape. This study aimed to investigate the microsporogenesis and the ultrastructure of pollen wall in species of Potamogeton in order to better understand the relationship between microsporogenesis features and the inaperturate condition.

Methods

The microsporogenesis was investigated using both light and epifluorescence microscopy. The ultrastructure of the pollen grain was studied using transmission electron microscopy.

Key Results

The cytokinesis is successive and formation of the intersporal callose wall is achieved by centrifugal cell plates, as a one-step process. The microspore tetrads were tetragonal, decussate, T-shaped and linear, except in P. pusillus, which showed less variation. This species also showed a callose ring in the microsporocyte, and some rhomboidal tetrads. In the mature pollen, the thickening observed in a broad area of the intine was here interpreted as an artefact.

Conclusions

The data support the view that there is a correlation between the inaperturate pollen production and the release of constraint on tetrad shape. However, in P. pusillus the tetrad shape may be constrained by a callose ring. It is also suggested that the lack of apertures in the pollen of Potamogeton may be due to the lack of specific sites on which callose deposition is completed. Moreover, inaperturate pollen of Potamogeton would be better classified as omniaperturate.Key words: Alismatales, callose, microsporogenesis, pollen aperture, Potamogeton illinoensis, P. polygonus, P. pusillus, tetrad shape