Deborah A. Rathbone

The Genetic Map of Artemisia annua L. Identifies Loci Affecting Yield of the Antimalarial Drug Artemisinin

The Art of Artemisia As the malaria parasite, which is transmitted through mosquito vectors, develops resistance, previously useful control mechanisms are beginning to fail. Combination therapies based on the plant product artemisinin are a promising alternative. Graham et al. (p. 328; see the Perspective by Milhous and Weina) have now developed a genetic map of the plant Artemisia annua from which artemisinin is derived. The results lay the foundation for improving agricultural productivity of this natural product, which is becoming increasingly important in the fight against malaria. A linkage map for an important medicinal crop plant points to breeding targets for enhancing drug production. Artemisinin is a plant natural product produced by Artemisia annua and the active ingredient in the most effective treatment for malaria. Efforts to eradicate malaria are increasing demand for an affordable, high-quality, robust supply of artemisinin. We performed deep sequencing on the transcriptome of A. annua to identify genes and markers for fast-track breeding. Extensive genetic variation enabled us to build a detailed genetic map with nine linkage groups. Replicated field trials resulted in a quantitative trait loci (QTL) map that accounts for a significant amount of the variation in key traits controlling artemisinin yield. Enrichment for positive QTLs in parents of new high-yielding hybrids confirms that the knowledge and tools to convert A. annua into a robust crop are now available.

Biotransformation of Explosives by the Old Yellow Enzyme Family of Flavoproteins

ABSTRACT Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.

An explosive-degrading cytochrome P450 activity and its targeted application for the phytoremediation of RDX

The widespread presence in the environment of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), one of the most widely used military explosives, has raised concern owing to its toxicity and recalcitrance to degradation. To investigate the potential of plants to remove RDX from contaminated soil and water, we engineered Arabidopsis thaliana to express a bacterial gene xplA encoding an RDX-degrading cytochrome P450 (ref. 1). We demonstrate that the P450 domain of XplA is fused to a flavodoxin redox partner and catalyzes the degradation of RDX in the absence of oxygen. Transgenic A. thaliana expressing xplA removed and detoxified RDX from liquid media. As a model system for RDX phytoremediation, A. thaliana expressing xplA was grown in RDX-contaminated soil and found to be resistant to RDX phytotoxicity, producing shoot and root biomasses greater than those of wild-type plants. Our work suggests that expression of xplA in landscape plants may provide a suitable remediation strategy for sites contaminated by this class of explosives.

Microbial transformation of alkaloids

Alkaloids continue to provide mankind with a plethora of medicines, poisons and potions. Because many valuable drugs are derived from such natural compounds, there is much interest in their transformation to provide new compounds or intermediates for the synthesis of new or improved drugs. This review aims to provide a survey of alkaloid transformations, and concerns microbial transformations and microbially expressed recombinant plant enzymes and their biotechnological applications.

Cofactor regeneration by a soluble pyridine nucleotide transhydrogenase for biological production of hydromorphone.

ABSTRACT We have applied the soluble pyridine nucleotide transhydrogenase ofPseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP+-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.

The explosive-degrading cytochrome P450 system is highly conserved among strains of rhodococcus spp.

ABSTRACT Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is a widely used explosive and a serious environmental pollutant. Nineteen strains of Rhodococcus spp. capable of utilizing RDX as the sole nitrogen source have been isolated. The cytochrome P450 system XplA-XplB, which is responsible for RDX breakdown, is present in 18 of these strains.

Engineering novel biocatalytic routes for production of semisynthetic opiate drugs.

The morphine alkaloids and their semisynthetic derivatives provide a diverse range of important pharmaceutical drugs. Current production of semisynthetic opiate drugs is by chemical means from naturally occurring morphine, codeine and thebaine. Although various microbial transformations of morphine alkaloids have been identified since the 1960s, more recently there has been considerable effort devoted to engineering biocatalytic routes for producing these important compounds. Such biocatalytic routes are attractive, as they would provide an alternative to the chemical production processes which suffer from limited supply of precursors, often low yields and toxic wastes. The biotransformation of morphine and codeine to the potent analgesic hydromorphone and the mild analgesic/antitussive hydrocodone, respectively, by recombinant Escherichia coli has been demonstrated and the problems encountered when engineering such a system will be discussed.

Engineering pathways for transformations of morphine alkaloids

Semisynthetic opiates provide some of the most potent analgesic compounds currently in clinical use, the majority of which are synthesized from the naturally occurring alkaloids morphine, codeine and thebaine. The use of recombinant DNA technology to engineer pathways for the biological synthesis of semisynthetic opiate drugs could offer significant advantages over existing chemical methods and may, ultimately, provide novel biosynthetic routes for the development of new therapeutics.

Biotransformation of alkaloids.

Biotransformations of alkaloids over the last decade have continued to encompass a wide variety of substrates and enzymes. The elucidation of novel alkaloid biosynthetic and catabolic pathways will continue to furnish new biocatalysts for the synthetic organic chemist. Furthermore, an improved understanding of the genetic and biochemical basis of metabolic pathways will also permit the engineering of pathways in plants and other heterologous hosts for the production of therapeutically important alkaloids. The combination of increasing commercial interest and advances in molecular biology will facilitate the availability of robust biocatalysts which are a prerequsite to achieve economically feasible processes for the production of alkaloid-based therapeutics.

The Degradation of Nitrate Ester Explosives and TNT by Enterobacter Cloacae PB2

One of the major environmental problems facing the military establishment is the considerable amount of land and water that is contaminated with explosives, particularly TNT (2,4,6-trinitrotoluene). Explosives are found in the environment as a result of waste water produced by manufacturing plants and from the disposal of off-specification material or demilitarisation of out-of-date munitions. Concerns have arisen regarding the toxicity and environmental fate of TNT and nitrate ester explosives such as PETN (pentaerythritol tetranitrate) and GTN (glycerol trinitrate) due to their relative toxicity. PETN and GTN are produced in large amounts for use as explosives in blasting caps and detonators and as vasodilators for the control of angina. Nitrate esters are extremely rare in nature and multiply-substituted nitrate esters are not known to occur naturally. The environmental fate of such compounds which are produced in large quantities by the chemical industries, is therefore of considerable interest. In this paper we describe the isolation and characterisation of a strain of Enterobacter cloacae from explosives contaminated soilt that is capable of utilising nitrate ester explosives and TNT as sole nitrogen sources for growth.