Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.

Engineering the third wave of biocatalysis

Natural products have been a rich source of compounds for drug discovery. However, their use has diminished in the past two decades, in part because of technical barriers to screening natural products in high-throughput assays against molecular targets. Here, we review strategies for natural product screening that harness the recent technical advances that have reduced these barriers. We also assess the use of genomic and metabolomic approaches to augment traditional methods of studying natural products, and highlight recent examples of natural products in antimicrobial drug discovery and as inhibitors of protein-protein interactions. The growing appreciation of functional assays and phenotypic screens may further contribute to a revival of interest in natural products for drug discovery.

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The re-emergence of natural products for drug discovery in the genomics era

Saccharomyces cerevisiae is engineered to produce high concentrations of artemisinic acid, a precursor of the artemisinin used in combination therapies for malaria treatment; an efficient and practical chemical process to convert artemisinic acid to artemisinin is also developed. Artemisinin-based combination therapies are the treatment of choice for uncomplicated Plasmodium falciparum malaria, but the supply of plant-derived artemisinin can sometimes be unreliable, causing shortages and high prices. This manuscript describes a viable industrial process for the production of semisynthetic artemisinin, with the potential to help stabilize artemisinin supply. The process uses Saccharomyces cerevisiae yeast engineered to produce high yields of artemisinic acid, a precursor of artemisinin. The authors have also developed an efficient and scalable chemical process to convert artemisinic acid to artemisinin. In 2010 there were more than 200 million cases of malaria, and at least 655,000 deaths1. 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 manufacturers2. 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 acid3. 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.

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

Synthetic biology is bringing together engineers and biologists to design and build novel biomolecular components, networks and pathways, and to use these constructs to rewire and reprogram organisms. These re-engineered organisms will change our lives over the coming years, leading to cheaper drugs, 'green' means to fuel our cars and targeted therapies for attacking 'superbugs' and diseases, such as cancer. The de novo engineering of genetic circuits, biological modules and synthetic pathways is beginning to address these crucial problems and is being used in related practical applications.

Synthetic biology: applications come of age.

Synthetic biology is a research field that combines the investigative nature of biology with the constructive nature of engineering. Efforts in synthetic biology have largely focused on the creation and perfection of genetic devices and small modules that are constructed from these devices. But to view cells as true 'programmable' entities, it is now essential to develop effective strategies for assembling devices and modules into intricate, customizable larger scale systems. The ability to create such systems will result in innovative approaches to a wide range of applications, such as bioremediation, sustainable energy production and biomedical therapies.

http://fbae.org/2009/FBAE/website/images/pdf/the-Second-Wave-of-Synthetic-Biology-from-modules-to-systems.pdf

The second wave of synthetic biology: from modules to systems

Drug-resistant strains of the malaria parasite are widespread, and as a result mortality due to malaria has increased significantly in recent years. Artemisinin, isolated from the herb Artemisia annua (sweet wormwood), is one drug that shows a high efficacy in killing multi-resistant strains of the parasite. The drug is extremely expensive, and high demand has led to a shortage of artemisinin, available only by extraction from the plant source. Ro et al. now report the development of a yeast strain engineered to carry a cytochrome P450 monooxygenase from A. annua that can produce the drug precursor, artemisinic acid. Artemisinin can be synthesized from this precursor. If the efficiency of this process can be improved, this engineered yeast strain has the potential to alleviate the drug shortage. Through the bio-engineering of Saccharomyces cerevisiae high titres of artemisinic acid were produced using a novel cytochrome P450 monooxygenase. Optimization of this process on an industrial scale may significantly reduce the cost of artemisinin, which could then be used to combat malaria in resource-poor settings. Malaria is a global health problem that threatens 300–500 million people and kills more than one million people annually1. Disease control is hampered by the occurrence of multi-drug-resistant strains of the malaria parasite Plasmodium falciparum2,3. Synthetic antimalarial drugs and malarial vaccines are currently being developed, but their efficacy against malaria awaits rigorous clinical testing4,5. 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 sufferers6. Although total synthesis of artemisinin is difficult and costly7, 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 artemisinin8,9. 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.

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Terpenoids are a highly diverse class of natural products that have historically provided a rich source for discovery of pharmacologically active small molecules, such as paclitaxel (Taxol) and artemisinin. Unfortunately, these secondary metabolites are typically produced in low abundance in their host organism, and their isolation consequently suffers from low yields and high consumption of natural resources. Furthermore, chemical synthesis of terpenoids can also be difficult to scale for industrial production. For these reasons, an attractive alternative strategy is to engineer metabolic pathways for production of pharmaceuticals or their precursors in a microbial host such as Escherichia coli. A key step is developing methods to carry out cytochrome P450 (P450)-based oxidation chemistry in vivo. Toward this goal, we have assembled two heterologous pathways for the biosynthesis of plant-derived terpenoid natural products, and we present the first examples of in vivo production of functionalized terpenoids in E. coli at high titer using native plant P450s.

Eachus Rachel

Papers

Production of the antimalarial drug precursor artemisinic acid in engineered yeast

Engineering Escherichia coli for production of functionalized terpenoids using plant P450s

Isoprenoid-modifizierende enzyme codierende polynucleotide und anwendungsverfahren dafür