II. MOLECULAR BIOLOGY AND HIGH-THROUGHPUT SCREENING (HTS)
Modern molecular biology has significantly changed the practice of natural products drug discovery through the introduction of high-throughput screening (HTS). Genetic manipulation is routinely employed to provide defined molecular and cell-based assay targets. These targets can be produced cheaply and in such abundance that it is possible to engage in HTS. If an assay is dependent upon a difficult to isolate enzyme, then one need only clone and overexpress the DNA that encodes the enzyme of interest to render the once precious enzyme quite abundant. It is now a matter of routine to generate essentially unlimited amounts of desired enzymes, receptors, and other screening targets in isolated molecular form or in an engineered cellular environment for HTS.
Over the past 15 years, the field of molecular biology has dramatically advanced our genetic, molecular, and biochemical understanding of natural product biosynthesis. The approaches described in this chapter embrace and apply these advances to the field of natural products drug discovery. This new approach to natural products discovery is called combinatorial biology [7,8]. In this process, new sets of biosynthetic genes are generated by deliberately ''shuffling'' genes from distinct biosynthetic pathways. In addition, it is now possible to obtain biosynthetic genes from previously inaccessible microbial sources, such as uncul-tivable organisms or symbionts. These gene sets are cloned and heterologously expressed to yield libraries of recombinant organisms bearing novel sets of bio-synthetic enzymes. This genetic process is called combinatorial biology. Complemented by an integrated bioassay system to identify and isolate recombinant clones efficiently, we can produce natural products with a wide range of biological activities and a high probability of structural novelty.
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