Global Genome Mining in Fungi for the Discovery of Hidden Families of Biosynthetic Enzymes and Associated Natural Products

Description

Natural products, defined as organic compounds synthesized by living organisms, have yielded many clinical drugs and their precursors. Thus, the discovery of natural products has been an important step in drug development. However, many natural products have been isolated and characterized over the last century, and thus, conventional approaches to natural product discovery have become less effective over time. One possible solution to this problem involves the use of microbial genome sequence big data, which contain a substantial number of unexploited natural product biosynthetic gene clusters (BGCs) that could potentially afford untapped metabolites. Nevertheless, this genome mining approach is hindered by uncertainty surrounding how available BGCs should be prioritized for the efficient discovery of natural products. In the proposed project, the concept of “hypothetical domainless protein-targeted global genome mining” will serve as a basis for the discovery of unprecedented natural products and biosynthetic reactions. Recent studies have led to the identification of many biosynthetic enzymes that do not exhibit sequence similarity with previously characterized proteins. Additionally, some of these enzymes are responsible for chemically intriguing reactions. Our preliminary study targeting approximately 2,000 fungal genomes revealed the existence of several BGCs that encode proteins with no protein sequence similarity to known proteins or detectable protein domains (i.e., “domainless”). We expect that the characterization of such BGCs will lead to the discovery of new families of biosynthetic enzymes, as well as the synthesis of natural products involving the activities of hidden enzymes. We first will seek to characterize selected fungal BGCs, discovered through global genome mining, that encode hypothetical domainless proteins. Characterization will be achieved primarily via the heterologous expression of biosynthetic genes in the fungusAspergillus oryzae. This process will lead to the identification of several natural products with novel scaffolds that are biosynthesized through reactions involving catalysis by domainless enzymes. These domainless enzymes will be further subjected to in-depth functional analyses, such as biochemical characterizations using purified enzymes. Homologues of some domainless enzymes identified in the proposed project will also be examined to gain a comprehensive understanding of the newly identified enzyme families. Collectively, the results of the proposed project will provide an efficient methodology for fungal global genome mining and the discovery of a series of novel natural products and biosynthetic enzymes, thus accelerating the diversification of natural products.