In Vitro Reconstitution of the dTDP-l-Daunosamine Biosynthetic Pathway Provides Insights into Anthracycline Glycosylation.

Many small molecule natural products are decorated with sugar moieties that are essential for their biological activity. A considerable number of natural product glycosides and their derivatives are clinically important therapeutics. Anthracyclines like daunorubicin and doxorubicin are examples of valuable glycosylated natural products used in medicine as potent anticancer agents. The sugar moiety, l-daunosamine (a highly modified deoxyhexose), plays a key role in the bioactivity of these molecules as evidenced by semisynthetic anthracycline derivatives such as epirubicin, wherein alteration in the configuration of a single stereocenter of the sugar unit generates a chemotherapeutic drug with lower cardiotoxicity. The nucleotide activated sugar donor that provides the l-daunosamine group for attachment to the natural product scaffold in the biosynthesis of these anthracyclines is dTDP-l-daunosamine. In an in vitro system, we have reconstituted the enzymes in the daunorubicin/doxorubicin pathway involved in the biosynthesis of dTDP-l-daunosamine. Through the study of the enzymatic steps in this reconstituted pathway, we have gained several insights into the assembly of this precursor including the identification of a major bottleneck and competing reactions. We carried out kinetic analysis of the aminotransferase that catalyzes a limiting step of the pathway. Our in vitro reconstituted pathway also provided a platform to test the combinatorial enzymatic synthesis of other dTDP-activated deoxyhexoses as potential tools for "glycodiversification" of natural products. To this end, we replaced the stereospecific ketoreductase that acts in the last step of dTDP-l-daunosamine biosynthesis with an enzyme from a heterologous pathway with opposite stereospecificity and found that it is active in the in vitro pathway, demonstrating the potential for the enzymatic synthesis of nucleotide-activated sugars with regio- and stereospecific tailoring.

A Genome-Wide Screen Identifies Genes in Rhizosphere-Associated Pseudomonas Required to Evade Plant Defenses.

Pseudomonas fluorescens and related plant root ("rhizosphere")-associated species contribute to plant health by modulating defenses and facilitating nutrient uptake. To identify bacterial fitness determinants in the rhizosphere of the model plant Arabidopsis thaliana, we performed a high-throughput transposon sequencing (Tn-Seq) screen using the biocontrol and growth-promoting strain Pseudomonas sp. WCS365. The screen, which was performed in parallel on wild-type and immunocompromised Arabidopsis plants, identified 231 genes that increased fitness in the rhizosphere of wild-type plants. A subset of these genes decreased fitness in the rhizosphere of immunocompromised plants. We hypothesized that these genes might be involved in avoiding plant defenses and verified 7 Pseudomonas sp. WCS365 candidate genes by generating clean deletions. We found that two of these deletion mutants, ΔmorA (encoding a putative diguanylate cyclase/phosphodiesterase) and ΔspuC (encoding a putrescine aminotransferase), formed enhanced biofilms and inhibited plant growth. We found that mutants ΔspuC and ΔmorA induced pattern-triggered immunity (PTI) as measured by induction of an Arabidopsis PTI reporter and FLS2/BAK1-dependent inhibition of plant growth. We show that MorA acts as a phosphodiesterase to inhibit biofilm formation, suggesting a possible role in biofilm dispersal. We found that both putrescine and its precursor arginine promote biofilm formation that is enhanced in the ΔspuC mutant, which cannot break down putrescine, suggesting that putrescine might serve as a signaling molecule in the rhizosphere. Collectively, this work identified novel bacterial factors required to evade plant defenses in the rhizosphere.IMPORTANCE While rhizosphere bacteria hold the potential to improve plant health and fitness, little is known about the bacterial genes required to evade host immunity. Using a model system consisting of Arabidopsis and a beneficial Pseudomonas sp. isolate, we identified bacterial genes required for both rhizosphere fitness and for evading host immune responses. This work advances our understanding of how evasion of host defenses contributes to survival in the rhizosphere.