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Glycosylation is a prominent strategy to optimize the pharmacokinetic and pharmacodynamic properties of drug-like small-molecule scaffolds by modulating their solubility, stability, bioavailability, and bioactivity. Glycosyltransferases applicable for “sugarcoating” various small-molecule acceptors have been isolated and characterized from plants and bacteria, but remained cryptic from filamentous fungi until recently, despite the frequent use of some fungi for whole-cell biocatalytic glycosylations. Here, we use bioinformatic and genomic tools combined with heterologous expression to identify a glycosyltransferase–methyltransferase (GT–MT) gene pair that encodes a methylglucosylation functional module in the ascomycetous fungus Beauveria bassiana. The GT is the founding member of a family nonorthologous to characterized fungal enzymes. Using combinatorial biosynthetic and biocatalytic platforms, we reveal that this GT is a promiscuous enzyme that efficiently modifies a broad range of drug-like substrates, including polyketides, anthraquinones, flavonoids, and naphthalenes. It yields both O- and N-glucosides with remarkable regio- and stereospecificity, a spectrum not demonstrated for other characterized fungal enzymes. These glucosides are faithfully processed by the dedicated MT to afford 4-O-methylglucosides. The resulting “unnatural products” show increased solubility, while representative polyketide methylglucosides also display increased stability against glycoside hydrolysis. Upon methylglucosidation, specific polyketides were found to attain cancer cell linespecific antiproliferative or matrix attachment inhibitory activities. These findings will guide genome mining for fungal GTs with novel substrate and product specificities, and empower the efficient combinatorial biosynthesis of a broad range of natural and unnatural glycosides in total biosynthetic or biocatalytic formats. [ABSTRACT FROM AUTHOR]

Callose plays an important role in pollen development in flowering plants. In rice, 10 genes encoding putative callose synthases have been identified; however, none of them has been functionally characterized. In this study, a rice Glucan Synthase-Like 5 (GSL5) knock-out mutant was isolated that exhibited a severe reduction in fertility. Pollen viability tests indicated that the pollen of the mutant was abnormal while the embryo sac was normal. Further, GSL5-RNA interference transgenic plants phenocopied the gsl5 mutant. The RNA expression of GSL5 was found to be knocked out in the gsl5 mutant and knocked down in GSL5-RNA interference transgenic plants by real-time reverse transcripion-PCR (RT-PCR) analysis. The male sterility of the mutant was due to abnormal microspore development; an analysis of paraffin sections of the mutant anthers at various developmental stages revealed that abnormal microspore development began in late meiosis. Both the knock-out and knock-down of GSL5 caused a lack of callose in the primary cell wall of meiocytes and in the cell plate of tetrads. As a result, the callose wall of the microspores was defective. This was demonstrated by aniline blue staining and an immunogold labeling assay; the microspores could not maintain their shape, leading to premature swelling and even collapsed microspores. These data suggest that the callose synthase encoded by GSL5 plays a vital role in microspore development during late meiosis and is essential for male fertility in rice. [ABSTRACT FROM AUTHOR]

Background Aflatoxins (AFs) are potent carcinogenic compounds produced by several Aspergillus species, which pose serious threats to human health. As sugar is a preferred carbohydrate source for AF production, we examined the possibility of using sugar analogs to inhibit AF biosynthesis. Results We showed that although D-glucal cannot be utilized by A. flavus as the sole carbohydrate source, it inhibited AF biosynthesis and promoted kojic acid production without affecting mycelial growth when applied to a glucose-containing medium. The inhibition occurred before the production of the first stable intermediate, norsolorinic acid, suggesting a complete inhibition of the AF biosynthetic pathway. Further studies showed that exogenous D-glucal in culture led to reduced accumulation of tricarboxylic acid (TCA) cycle intermediates and reduced glucose consumption, indicating that glycolysis is inhibited. Expression analyses revealed that D-glucal suppressed the expression of AF biosynthetic genes but promoted the expression of kojic acid biosynthetic genes. Conclusions D-glucal as a non-metabolizable glucose analog inhibits the AF biosynthesis pathway by suppressing the expression of AF biosynthetic genes. The inhibition may occur either directly through interfering with glycolysis, or indirectly through reduced oxidative stresses from kojic acid biosynthesis. [ABSTRACT FROM AUTHOR]