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Introduction and scope of the thesis

Introduction and scope

The research area Pharmaceutical Biology studies living cells as a source of biologically active compounds and applies them for production purposes. Traditionally the research mainly focused on the isolation and analysis of compounds from nature, generally from plant origin. The use of plants, plants extracts, isolated plant compounds and derivatives active against diseases was firmly rooted in healthcare. Nowadays still a significant part of the world population largely depends on the use of traditional medicine. Although in western societies pharmacy is no longer associated with plants, a significant part of the drugs used in contemporary medicine has (originally) been isolated from plants or is based on plant secondary metabolites. Due to the progress in science and chemical technology in the last century, the focus of the pharmaceutical industry shifted towards chemical synthesis for the discovery of new lead compounds. However, the awareness is alive that the pool of natural products and the knowledge of traditional medicine are still important for the development of new drugs. For relatively simple plant drugs chemical synthesis could replace the need for plant material. For example, the synthesis of salicylic acid, originally from Salix alba, was easily performed in the laboratory yielding the derivative Aspirin as a synthetic drug. However, due to the complexity and stereochemistry of many other biologically active plant compounds with pharmaceutical application, chemical synthesis is not economically or technically feasible and the pharmaceutical industry is fully dependent on plant resources. Plant cell biotechnology may overcome some of the problems related to the supply of plantderived substances needed for drug production. This will lessen the need for the harvesting of the plants from nature, which preserves rare species. Biotechnological production can be performed in a closed environment where conditions can easily be controlled. Drawbacks of the use of plant cell cultures often comprise a low bioconversion efficiency and low yields. The cytostatic agent paclitaxel is a nice example of the application of plant cell cultures for production purposes. The drug can be isolated from Taxus brevifolia, but the low yields and the intensive harvesting endanger this species with extinction. Other Taxus sp. serve as source for the precursor baccatin III which is more abundantly available. Plant cell cultures are used to convert the latter compound to the active drug and for the total biosynthesis of paclitaxel [1, 2]. In the past decades advances in biotechnology made it possible to elucidate various biosynthetic pathways, not only on a phytochemical level, but on an enzymatic and genomic level as well. Isolation and identification of genes involved in the biosynthetic pathway of natural products offers the opportunity to create microbial host cells for the production of natural products. This thesis focuses on two groups of natural compounds: terpenoids and lignans. Both include pharmaceutically interesting compounds. The major aims of the work described in this thesis were (1) to investigate an endogenous bacterial biosynthetic pathway for terpenoids to be used for the production of plant compounds using the bacterium as a host cell, (2) to describe the mechanism and structural characteristics of a plant terpene synthase, and (3) to explore the use of human cytochrome P450 enzymes for the derivatisation and production of plant secondary metabolites. Chapter 2 gives an overview of research using combinatorial biosynthesis strategies for the production of pharmaceutically interesting natural products originating from plants. This chapter presents comprehensive information about techniques and applications available for

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Chapter 1

metabolic pathway engineering and combinatorial biosynthesis as well as the future prospects in this area. Isoprene is emitted by several plants and bacteria. This phenomenon was used for the identification of Bacillus subtilis genes involved in the isoprenoid biosynthesis. This was done by measuring the amount of isoprene emitted by bacterial cultures of functional knock-out strains (chapter 3). Since bacteria are able to produce terpenoid structures, the endogenous precursor(s) in the organism can be used for the production of plant terpenes. The functional expression of a sesquiterpene synthase isolated from a plant in a microorganism was investigated in this respect. Chapter 4 describes the research performed to gain more insight in the mechanism of action of terpene synthases. The conversion of farnesyl diphosphate to amorphadiene synthase, a precursor of the antimalarial drug artemisinin, was investigated by single residue substitutions and purification of the protein expressed in Escherichia coli. In addition to that we investigated the use of an enzyme that is not related to the biosynthesis of a plant secondary metabolite for the bioconversion of natural products. The human liver cytochrome P450 monooxygenase 3A4, heterologously expressed in E. coli, was used to obtain new derivatives from rather common terpenoids (chapter 5). Cytochrome P450 monooxygenase 3A4 also served to replace the plant enzyme that catalyzes the bioconversion of deoxypodophyllotoxin into epipodophyllotoxin, a precursor for the cytostatic drugs teniposide and etoposide. This opens the possibility to use the abundantly available Anthriscus sylvestris as an alternative source for podophyllotoxin (chapter 6 and 7). This thesis describes several opportunities and approaches available for using heterologous host organisms for the production of pharmaceutically relevant natural products. The results may be applied in the near future in combination with metabolic pathway engineering techniques and combinatorial biosynthesis strategies.

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