Genetic platforms for heterologous expression of microbial natural products

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Abstract

This review covers current genetic technologies for accessing and manipulating natural product biosynthetic gene clusters through heterologous expression.

Abstract

Covering: 2005 up to 2019

Natural products are of paramount importance in human medicine. Not only are most antibacterial and anticancer drugs derived directly from or inspired by natural products, many other branches of medicine, such as immunology, neurology, and cardiology, have similarly benefited from natural product-based drugs. Typically, the genetic material required to synthesize a microbial specialized product is arranged in a multigene biosynthetic gene cluster (BGC), which codes for proteins associated with molecule construction, regulation, and transport. The ability to connect natural product compounds to BGCs and vice versa, along with ever-increasing knowledge of biosynthetic machineries, has spawned the field of genomics-guided natural product genome mining for the rational discovery of new chemical entities. One significant challenge in the field of natural product genome mining is how to rapidly link orphan biosynthetic genes to their associated chemical products. This review highlights state-of-the-art genetic platforms to identify, interrogate, and engineer BGCs from diverse microbial sources, which can be broken into three stages: (1) cloning and isolation of genomic loci, (2) heterologous expression in a host organism, and (3) genetic manipulation of cloned pathways. In the future, we envision natural product genome mining will be rapidly accelerated by de novo DNA synthesis and refactoring of whole biosynthetic pathways in combination with systematic heterologous expression methodologies.

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Discovery of microbial natural products by activation of silent biosynthetic gene clusters.

Peter Rutledge, Gregory Challis (2015)

Microorganisms produce a wealth of structurally diverse specialized metabolites with a remarkable range of biological activities and a wide variety of applications in medicine and agriculture, such as the treatment of infectious diseases and cancer, and the prevention of crop damage. Genomics has revealed that many microorganisms have far greater potential to produce specialized metabolites than was thought from classic bioactivity screens; however, realizing this potential has been hampered by the fact that many specialized metabolite biosynthetic gene clusters (BGCs) are not expressed in laboratory cultures. In this Review, we discuss the strategies that have been developed in bacteria and fungi to identify and induce the expression of such silent BGCs, and we briefly summarize methods for the isolation and structural characterization of their metabolic products.

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Targets for cell cycle arrest by the immunosuppressant rapamycin in yeast

Robert Heitman, N. Movva, J. Hall (1991)

FK506 and rapamycin are related immunosuppressive compounds that block helper T cell activation by interfering with signal transduction. In vitro, both drugs bind and inhibit the FK506-binding protein (FKBP) proline rotamase. Saccharomyces cerevisiae cells treated with rapamycin irreversibly arrested in the G1 phase of the cell cycle. An FKBP-rapamycin complex is concluded to be the toxic agent because (i) strains that lack FKBP proline rotamase, encoded by FPR1, were viable and fully resistant to rapamycin and (ii) FK506 antagonized rapamycin toxicity in vivo. Mutations that conferred rapamycin resistance altered conserved residues in FKBP that are critical for drug binding. Two genes other than FPR1, named TOR1 and TOR2, that participate in rapamycin toxicity were identified. Nonallelic noncomplementation between FPR1, TOR1, and TOR2 alleles suggests that the products of these genes may interact as subunits of a protein complex. Such a complex may mediate nuclear entry of signals required for progression through the cell cycle.