Synthetic Biology - Pathways - Alkaloids

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1 Researchers
2 Project Description
3 Relevant Publications
4 Funding Resources

Researchers

Alex Juminaga, Clem Fortman, Sung Kuk Lee, Taek Soon Lee

Project Description

Alkaloids are nitrogen-containing, low-molecular-weight compounds found in approximately 20% of all plants. Approximately 12,000 alkaloid structures have been described, many of which serve as defense agents in plants against herbivores and pathogens. Many alkaloids have been used for the treatment of human disease: morphine and codeine as analgesics; vinblastine, vincristine, and camptothecin as anti-cancer agents; (+)-tubocurarine as a muscle relaxant; and ajmaline as an anti-arrhythmic, to name a few. In addition, some of the most significant drugs are alkaloids, including caffeine, nicotine, cocaine, and heroin. World-wide sales of alkaloid-containing drugs exceeded $US 4 billion in 2002. Currently, most, if not all, alkaloids that are used as drugs are obtained by extraction from the plants that produce them. Due to relatively low yields of the molecules from native plants, drugs can be extremely expensive. Although enhancement of the metabolic pathways responsible for the production of drugs from native plants has met with some success, the production of drugs in a heterologous host, particularly a microorganism, would allow not only the cost-effective production of drugs, but also the manipulation of the products' structures by the engineering of the enzymes responsible for their biosynthesis.

1. Benzylisoquinoline alkaloids

Benzylisoquinoline alkaloids (BIAs) are one of the largest and most diverse alkaloid families, having approximately 2,500 compounds. Examples of BIAs are morphine, the microtubule disruptor colchicines, the antibiotic berberine, the muscle relaxant (+)-tubocurarine, and sanguinarine, which is currently added to toothpastes and oral rinses due to its anti-plaque properties. Many BIAs have found use as analgesics (namely codeine and morphine). Several analgesics (buprenorphine and oxycodone) are produced using thebaine, an advanced intermediate of BIA biosynthetic pathway, as a starting material. An intermediate in the BIA pathway, reticuline, is also used as a starting material for the synthesis of several anti-cancer and anti-malarial molecules, and reticuline itself has been known to have hair growth activity.

BIAs are biosynthesized from tyrosine as a building block through a series of decarboxylation and deamination reactions. With the exception of berberine, which is isolated from cell suspension cultures of Coptis japonica, most BIAs are isolated from the plants. Due to the complex structure of BIAs, few high-yielding chemical synthesis of BIAs have been reported so far. The goal of our research is to reconstitute the BIA biosynthetic pathway in E. coli for the production of (S)-reticuline, a key intermediate of BIA pathway, as the first target molecule, and eventually we will explore the biosynthesis of morphine in E. coli.

2. Terpene indole alkaloids

The terpene indole alkaloids are a large group of chemically related compounds with a broad range of bioactivities. Among the approximately 3000 molecules thus far described from this class are the clinically proven anticancer compounds vincristine and vinblastine. These important anticancer compounds are too complex to be produced commercially by traditional chemical synthesis. They are instead still isolated from their native source, the tropical plant Catharanthus roseus. Unfortunately, these therapeutic agents are only present in minute quantities with yields of 2x10-4% of dry weight for vinblastine. Due to this paucity of production other options have been explored most notably cell and tissue culture. Unfortunately, this methodology results in lower yields of vinblastine and no vincristine. In fact, less than 50% of the full metabolite complement of approximately 130 indole alkaloids described from C. roseus have been isolated from cultured cells.

Strictosidine is a key intermediate of terpene indole alkaloids. It is produced by the condensation of tryptamine and secologanin by the enzyme strictosidine synthase. Strictosidine is a stable intermediate subsequently processed in several enzymatic steps to yield vincristine and vinblastine. While isolation and fundamental understanding of all enzymes involved in this biosynthesis is the ultimate goal of this work, a strictosidine-producing host will be an essential first step towards this objective.

The goal of heterologous strictosidine production represents a crucial first step toward efficient and economical production of the terpene indole alkaloids. This includes not only the proven cancer fighting agents derived from C. roseus and other proven therapeutics such as the anti-hypertensive ajmalicine, but also rare alkaloids that are obtained in quantities that have thus far prohibited rigorous exploration of their clinical potential. In addition, an increase in the availability of these natural products provides much needed materials for structure activity relationship studies as well as investigations of semi synthetic derivatives. In summary, establishing of the proposed system has the potential to facilitate the production of literally thousands of members from this medically important class of compounds.

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