Michael D. Leavell

High-level semi-synthetic production of the potent antimalarial artemisinin

In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths. The World Health Organization has recommended artemisinin-based combination therapies (ACTs) for the treatment of uncomplicated malaria caused by the parasite Plasmodium falciparum. Artemisinin is a sesquiterpene endoperoxide with potent antimalarial properties, produced by the plant Artemisia annua. However, the supply of plant-derived artemisinin is unstable, resulting in shortages and price fluctuations, complicating production planning by ACT manufacturers. A stable source of affordable artemisinin is required. Here we use synthetic biology to develop strains of Saccharomyces cerevisiae (baker’s yeast) for high-yielding biological production of artemisinic acid, a precursor of artemisinin. Previous attempts to produce commercially relevant concentrations of artemisinic acid were unsuccessful, allowing production of only 1.6 grams per litre of artemisinic acid. Here we demonstrate the complete biosynthetic pathway, including the discovery of a plant dehydrogenase and a second cytochrome that provide an efficient biosynthetic route to artemisinic acid, with fermentation titres of 25 grams per litre of artemisinic acid. Furthermore, we have developed a practical, efficient and scalable chemical process for the conversion of artemisinic acid to artemisinin using a chemical source of singlet oxygen, thus avoiding the need for specialized photochemical equipment. The strains and processes described here form the basis of a viable industrial process for the production of semi-synthetic artemisinin to stabilize the supply of artemisinin for derivatization into active pharmaceutical ingredients (for example, artesunate) for incorporation into ACTs. Because all intellectual property rights have been provided free of charge, this technology has the potential to increase provision of first-line antimalarial treatments to the developing world at a reduced average annual price.

Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin

Malaria, caused by Plasmodium sp, results in almost one million deaths and over 200 million new infections annually. The World Health Organization has recommended that artemisinin-based combination therapies be used for treatment of malaria. Artemisinin is a sesquiterpene lactone isolated from the plant Artemisia annua. However, the supply and price of artemisinin fluctuate greatly, and an alternative production method would be valuable to increase availability. We describe progress toward the goal of developing a supply of semisynthetic artemisinin based on production of the artemisinin precursor amorpha-4,11-diene by fermentation from engineered Saccharomyces cerevisiae, and its chemical conversion to dihydroartemisinic acid, which can be subsequently converted to artemisinin. Previous efforts to produce artemisinin precursors used S. cerevisiae S288C overexpressing selected genes of the mevalonate pathway [Ro et al. (2006) Nature 440:940–943]. We have now overexpressed every enzyme of the mevalonate pathway to ERG20 in S. cerevisiae CEN.PK2, and compared production to CEN.PK2 engineered identically to the previously engineered S288C strain. Overexpressing every enzyme of the mevalonate pathway doubled artemisinic acid production, however, amorpha-4,11-diene production was 10-fold higher than artemisinic acid. We therefore focused on amorpha-4,11-diene production. Development of fermentation processes for the reengineered CEN.PK2 amorpha-4,11-diene strain led to production of > 40 g/L product. A chemical process was developed to convert amorpha-4,11-diene to dihydroartemisinic acid, which could subsequently be converted to artemisinin. The strains and procedures described represent a complete process for production of semisynthetic artemisinin.

MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence

Mycobacterium tuberculosis, the causative agent of human tuberculosis, is unique among bacterial pathogens in that it displays a wide array of complex lipids and lipoglycans on its cell surface. One of the more remarkable lipids is a sulfated glycolipid, termed sulfolipid-1 (SL-1), which is thought to mediate specific host-pathogen interactions during infection. However, a direct role for SL-1 in M. tuberculosis virulence has not been established. Here we show that MmpL8, a member of a large family of predicted lipid transporters in M. tuberculosis, is required for SL-1 production. The accumulation of an SL-1 precursor, termed SL1278, in mmpL8 mutant cells indicates that MmpL8 is necessary for an intermediate step in the SL-1 biosynthesis pathway. We use a novel fractionation procedure to demonstrate that SL-1 is present on the cell surface, whereas SL1278 is found exclusively in more internal layers. Importantly, we show that mmpL8 mutants are attenuated for growth in a mouse model of tuberculosis. However, SL-1 per se is not required for establishing infection as pks2 mutants, which are defective in an earlier step in SL-1 biosynthesis, have no obvious growth defect. Thus, we hypothesize that either MmpL8 transports molecules in addition to SL-1 that mediate host-pathogen interactions or the accumulation of SL1278 in mmpL8 mutant cells interferes with other pathways required for growth during the early stages of infection.

PapA1 and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor Sulfolipid-1

Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL1278, has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL1278 and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL1278 and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2′-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL1278. Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the ΔpapA2 and ΔpapA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL1278) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection.

Conformational studies of Zn-ligand-hexose diastereomers using ion mobility measurements and density functional theory calculations.

Ion mobility studies and density functional theory calculations were used to study the structures of [Zn/diethylenetriamine/Hexose/Cl]+ complexes in an effort to probe differences in the three-dimensional conformations. This information allows us to gain insight into the structure of these complexes before collisional activation, which is the first step in understanding the stereoselective dissociations observed under collisionally activated conditions. The collision cross sections obtained from the ion mobility measurements showed that the mannose structure is more compact than the galactose and glucose complexes, respectively. Using density functional theory, candidate structures for each of the experimentally observed complexes were generated. Two criteria were used to determine the most likely structures of these complexes before activation: (1) The allowed relative energies of the molecules (between 0–90 kJ/mol) and (2) collision cross section agreement (within 2%) between the theoretically determined structures and the experimentally determined cross section. It was found that the identity of the monosaccharide made a difference in the overall conformation of the metal-ligand-monosaccharide complex. For glucose and galactose, metal coordination to O(6) was found to be favorable, with the monosaccharide occupying the 4C1 chair conformation, while for mannose, O(2) metal coordination was found with the monosaccharide in a B3,0 conformation. Coordination numbers varied between four and six for the Zn(II) metal centers. Given these results, it appears that the stereochemistry of the monosaccharide influences the conformation and metal coordination sites of the Zn(II)/monosaccharide/dien complex. These differences may influence the dissociation products observed under collisionally activated conditions.

Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach

The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell–cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{4}^{2-}\end{equation*}\end{document}). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

Stabilization and linkage analysis of metal-ligated sialic acid containing oligosaccharides

The dissociation of metal-ligated sialyllactose and sialyl-N-acetyllactosamine was investigated. Metal-ligand derivatization of the carbohydrate samples with the diethylenetriamine ligand and one of four transition metals [Co(II), Ni(II), Cu(II), Zn(II)] suppressed sialic acid loss in the collision-induced dissociation process. Suppression of sialic acid loss allows sialic acid linkage information to be gained through tandem mass spectrometry. Sialic acid stabilization is postulated to occur due to the doubly charged metal ion which allows for deprotonation of the sialic acid moiety. Furthermore, a connection between the metal center and the amount of sialic acid loss was found. These results were rationalized using the Irving-Williams series and a competition between different sites of deprotonation. Analysis of the product ion spectra showed a clear differentiation of sialic acid linkage. Linkage determination is proposed to be effective due to the available conformations allowed by the different linkages. A more flexible linkage will allow more coordination of the sialic acid residue with the metal center, whereas a less flexible linkage will make this interaction unlikely.

Investigation of ion/molecule reactions as a quantification method for phosphorylated positional isomers. an FT-ICR approach.

A rapid and accurate method of quantifying positional isomeric mixtures of phosphorylated hexose and N-acetylhexosamine monosacchrides by using gas-phase ion/molecule reactions coupled with FT-ICR mass spectrometry is described. Trimethyl borate, the reagent gas, reacts readily with the singly charged negative ions of phosphorylated monosaccharides to form two stable product ions corresponding to the loss of one or two neutral molecules of methanol from the original adduct. Product distribution in the ion/molecule reaction spectra differs significantly for isomers phosphorylated in either the 1- or the 6-position. As a result, the percents of total ion current of these product ions for a mixture of the two isomers vary with its composition. In order to determine the percentage of each isomer in an unknown mixture, a multicomponent quantification method is utilized in which the percents of total ion current of the two product ions for each pure monosaccharide phosphate and the mixture are used in a two-equation, two-unknown system. The applicability of this method is demonstrated by successfully quantifying mock mixtures of four different isomeric pairs: Glucose-1-phosphate and glucose-6-phosphate; mannose-1-phosphate and mannose-6-phosphate; galactose-1-phosphate and galactose-6-phosphate; N-acetylglucosamine-1-phosphate and N-acetylglucosamine-6-phosphate. The effects of mixture concentrations and ion/molecule reaction conditions on the quantification are also discussed. Our results demonstrate that this assay is a fast, sensitive, and robust method to quantify isomeric mixtures of phosphorylated monosaccharides.

Probing isomeric differences of phosphorylated carbohydrates through the use of ion/molecule reactions and FT-ICR MS.

Through the use of ion/molecule reactions and tandem mass spectrometry, phosphate position is assigned in both phosphorylated monosaccharides and oligosaccharides. In previous work [1, 2] phosphate moieties of monosaccharides were stabilized under collisional activation, by first derivatizing the deprotonated monosaccharide with trimethyl borate through an ion/molecule reaction, and the phosphate position determined through marker ions generated in tandem mass spectra. In this work, the methodology is extended to larger phosphorylated oligomers employing chlorotrimethylsilane (TMSCl) as the ion/molecule reagent. Phosphorylated monosaccharides were first investigated to determine diagnostic ions for phosphate linkage in monomeric standards. It was observed that the diagnostic ions showed both linkage and some monosaccharide stereochemical information. Furthermore, it was observed that TMS addition stabilized the phosphate moiety under collisionally activated conditions. Upon identification of the diagnostic ions, the methodology was applied to lactose-1-phosphate. It was found that TMSCl, stabilized the phosphate moiety upon collisional activation, and furthermore, the phosphate linkage could be determined through tandem mass spectrometric analysis. As a further extrapolation to biologically relevant problems, the methodology was applied to a lipophosphoglycan analog from the protozoan parasite Leishmania. This sample contains bridging phosphates which were converted to terminal phosphates through collision induced dissociation. The sample was then analyzed in the same manner as lactose-1-phosphate, yielding phosphate linkage information and stereochemical information. This study showed that, using the developed methodology, phosphate linkage can be determined from both monosaccharides and larger oligosaccharides; furthermore it is applicable to samples in which the phosphates are either terminating or bridging.

Determination of phosphate position in hexose monosaccharides using an FTICR mass spectrometer: ion/molecule reactions, labeling studies, and dissociation mechanisms

Abstract Determination of phosphate position in carbohydrates using mass spectrometry is difficult due to the low energy loss of the phosphate either as a neutral or as an ion in MS/MS experiments. A possible solution to this problem is proposed in this work, whereby we use ion/molecule reactions in combination with tandem mass spectrometry to determine the site of phosphorylation on phosphorylated monosaccharides. Singly charged negative ions from phosphorylated monosaccharides are reacted with trimethyl borate in an FTICR MS analyzer cell to produce ion/molecule reaction products with the loss of a neutral methanol molecule. This reaction product likely involves a covalent bond between one of the phosphate oxygen atoms and boron. Derivatization of the phosphate in this manner allows stabilization of the phosphate group under SORI-CID conditions, allowing generation of ions characteristic of the phosphate linkage. Ion structures and dissociation mechanisms explaining these results are presented and discussed. The mechanistic studies suggest that the extra degrees of freedom provided by the 6-linked phosphate allows formation of diagnostic ions in the 6-linked case that are not formed from the 1-linked isomer. The dissociation of the ion/molecule reaction products using infrared multi-photon dissociation (IRMPD) as an activation method was also investigated. While SORI-CID and IRMPD activation yield similar dissociation patterns, the characteristic differences in the product ion spectra between the monosaccharides phosphorylated in the 1- and 6-positions are not observed using IRMPD.