Karyn L. Newman

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Malaria is a global health problem that threatens 300–500 million people and kills more than one million people annually. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing. Artemisinin, a sesquiterpene lactone endoperoxide extracted from Artemisia annua L (family Asteraceae; commonly known as sweet wormwood), is highly effective against multi-drug-resistant Plasmodium spp., but is in short supply and unaffordable to most malaria sufferers. Although total synthesis of artemisinin is difficult and costly, the semi-synthesis of artemisinin or any derivative from microbially sourced artemisinic acid, its immediate precursor, could be a cost-effective, environmentally friendly, high-quality and reliable source of artemisinin. Here we report the engineering of Saccharomyces cerevisiae to produce high titres (up to 100 mg l-1) of artemisinic acid using an engineered mevalonate pathway, amorphadiene synthase, and a novel cytochrome P450 monooxygenase (CYP71AV1) from A. annua that performs a three-step oxidation of amorpha-4,11-diene to artemisinic acid. The synthesized artemisinic acid is transported out and retained on the outside of the engineered yeast, meaning that a simple and inexpensive purification process can be used to obtain the desired product. Although the engineered yeast is already capable of producing artemisinic acid at a significantly higher specific productivity than A. annua, yield optimization and industrial scale-up will be required to raise artemisinic acid production to a level high enough to reduce artemisinin combination therapies to significantly below their current prices.

Use of a Green Fluorescent Strain for Analysis of Xylella fastidiosa Colonization of Vitis vinifera

ABSTRACT Xylella fastidiosa causes Pierces disease of grapevine as well as several other major agricultural diseases but is a benign endophyte in most host plants. X. fastidiosa colonizes the xylem vessels of host plants and is transmitted by xylem sap-feeding insect vectors. To understand better the pattern of host colonization and its relationship to disease, we engineered X. fastidiosa to express a green fluorescent protein (Gfp) constitutively and performed confocal laser-scanning microscopic analysis of colonization in a susceptible host, Vitis vinifera. In symptomatic leaves, the fraction of vessels colonized by X. fastidiosa was fivefold higher than in nearby asymptomatic leaves. The fraction of vessels completely blocked by X. fastidiosa colonies increased 40-fold in symptomatic leaves and was the feature of colonization most dramatically linked to symptoms. Therefore, the extent of vessel blockage by bacterial colonization is highly likely to be a crucial variable in symptom expression. Intriguingly, a high proportion (>80%) of colonized vessels were not blocked in infected leaves and instead had small colonies or solitary cells, suggesting that vessel blockage is not a colonization strategy employed by the pathogen but, rather, a by-product of endophytic colonization. We present evidence for X. fastidiosa movement through bordered pits to neighboring vessels and propose that vessel-to-vessel movement is a key colonization strategy whose failure results in vessel plugging and disease.

Cell-to-Cell Signaling in Xylella fastidiosa Suppresses Movement and Xylem Vessel Colonization in Grape

Cell-to-cell signaling mediated by a fatty acid diffusible signaling factor (DSF) is central to the regulation of the virulence of Xylella fastidiosa. DSF production by X. fastidiosa is dependent on rpfF and, although required for insect colonization, appears to reduce its virulence to grape. To understand what aspects of colonization of grape are controlled by DSF in X. fastidiosa and, thus, those factors that contribute to virulence, we assessed the colonization of grape by a green fluorescent protein-marked rpfF-deficient mutant. The rpfF-deficient mutant was detected at a greater distance from the point of inoculation than the wild-type strain at a given sampling time, and also attained a population size that was up to 100-fold larger than that of the wild-type strain at a given distance from the point of inoculation. Confocal laser-scanning microscopy revealed that approximately 10-fold more vessels in petioles of symptomatic leaves harbored at least some cells of either the wild type or rpfF mutant when compared with asymptomatic leaves and, thus, that disease symptoms were associated with the extent of vessel colonization. Importantly, the rpfF mutant colonized approximately threefold more vessels than the wild-type strain. Although a wide range of colony sizes were observed in vessels colonized by both the wild type and rpfF mutant, the proportion of colonized vessels harboring large numbers of cells was significantly higher in plants inoculated with the rpfF mutant than with the wild-type strain. These studies indicated that the hypervirulence phenotype of the rpfF mutant is due to both a more extensive spread of the pathogen to xylem vessels and unrestrained multiplication within vessels leading to blockage. These results suggest that movement and multiplication of X. fastidiosa in plants are linked, perhaps because cell wall degradation products are a major source of nutrients. Thus, DSF-mediated cell-to-cell signaling, which restricts movement and colonization of X. fastidiosa, may be an adaptation to endophytic growth of the pathogen that prevents the excessive growth of cells in vessels.

Virulence of Plant Pathogenic Bacteria Attenuated by Degradation of Fatty Acid Cell-to-Cell Signaling Factors

Diffusible signal factor (DSF) is a fatty acid signal molecule involved in regulation of virulence in several Xanthomonas species as well as Xylella fastidiosa. In this study, we identified a variety of bacteria that could disrupt DSF-mediated induction of virulence factors in Xanthomonas campestris pv. campestris. While many bacteria had the ability to degrade DSF, several bacterial strains belonging to genera Bacillus, Paenibacillus, Microbacterium, Staphylococcus, and Pseudomonas were identified that were capable of particularly rapid degradation of DSF. The molecular determinants for rapid degradation of DSF in Pseudomonas spp. strain G were elucidated. Random transposon mutants of strain G lacking the ability to degrade DSF were isolated. Cloning and characterization of disrupted genes in these strains revealed that carAB, required for the synthesis of carbamoylphosphate, a precursor for pyrimidine and arginine biosynthesis is required for rapid degradation of DSF in strain G. Complementation of carAB mutants restored both pyrimidine prototrophy and DSF degradation ability of the strain G mutant. An Escherichia coli strain harboring carAB of Pseudomonas spp. strain G degrades DSF more rapidly than the parental strain, and overexpression of carAB in trans increased the ability of Pseudomonas spp. strain G to degrade as compared with the parental strain. Coinoculation of X. campestris pv. campestris with DSF-degrading bacteria into mustard and cabbage leaves reduced disease severity up to twofold compared with plants inoculated only with the pathogen. Likewise, disease incidence and severity in grape stems coinoculated with Xylella fastidiosa and DSF-degrading strains were significantly reduced compared with plants inoculated with the pathogen alone. Coinoculation of grape plants with a carAB mutant of Pseudomonas spp. strain G complemented with carAB in trans reduced disease severity as well or better than the parental strain. These results indicate that overexpression of carAB in other endophytes could be a useful strategy of biocontrol for the control of diseases caused by plant pathogens that produce DSF.

Contribution of rpfB to Cell-to-Cell Signal Synthesis, Virulence, and Vector Transmission of Xylella fastidiosa

In Xylella fastidiosa the fatty acid signal molecule diffusible signaling factor (DSF) is produced and sensed by components of the regulation of pathogenicity factors (rpf) cluster; lack of DSF production in RpfF mutants results in a non-vector-transmissible phenotype yet cells are hypervirulent to grape. rpfB has not been characterized in Xylella fastidiosa, although its homolog has been suggested to be required for DSF synthesis in Xanthomonas campestris pv. campestris. We show that RpfB is involved in DSF processing in both Xylella fastidiosa and Xanthomonas campestris, affecting the profile of DSF-like fatty acids observed in thin-layer chromatography. Although three fatty acids whose production is dependent on RpfF were detected in Xylella fastidiosa and Xanthomonas campestris wild-type strains, their respective rpfB mutants accumulated primarily one chemical species. Although no quantifiable effect of rpfB on plant colonization by Xylella fastidiosa was found, insect colonization and transmission was reduced. Thus, RpfB apparently is involved in DSF processing, and like Xanthomonas campestris, Xylella fastidiosa also produces multiple DSF molecules. It is possible that Xylella fastidiosa coordinates host vector and plant colonization by varying the proportions of different forms of DSF signals via RpfB.