Genetically Modified Bacterial Strains and Novel Bacterial Artificial Chromosome Shuttle Vectors for Constructing Environmental Libraries and Detecting Heterologous Natural Products in Multiple Expression Hosts

Abstract

The enormous diversity of uncultured microorganisms in soil and other environments provides a potentially rich source of novel natural products, which is critically important for drug discovery efforts. Our investigators reported previously on the creation and screening of an Escherichia coli library containing soil DNA cloned and expressed in a bacterial artificial chromosome (BAC) vector. In that initial study, our group identified novel enzyme activities and a family of antibacterial small molecules encoded by soil DNA cloned and expressed in E. coli. To continue our pilot study of the utility and feasibility of this approach to natural product drug discovery, we have expanded our technology to include Streptomyces lividans and Pseudomonas putida as additional hosts with different expression capabilities, and herein we describe the tools we developed for transferring environmental libraries into all three expression hosts and screening for novel activities. These tools include derivatives of S. lividans that contain complete and unmarked deletions of the act and red endogenous pigment gene clusters, a derivative of P. putida that can accept environmental DNA vectors and integrate the heterologous DNA into the chromosome, and new BAC shuttle vectors for transferring large fragments of environmental DNA from E. coli to both S. lividans and P. putida by high-throughput conjugation. Finally, we used these tools to confirm that the three hosts have different expression capabilities for some known gene clusters.Natural products have been a rich source of pharmaceutical molecules, accounting for greater than 30% of all human therapeutics and more than 60% of antiinfective and anticancer drugs. Despite the advances in high-throughput screening technology and attempts to isolate and culture microorganisms from exotic environments, the discovery of novel natural products remains difficult. However, it has become clear that the vast majority of microorganisms in the environment are still unknown and that most of them are unculturable under standard laboratory conditions (15,38). Since the number of such "unculturable" microbial species in the soil represents at least 98% of the total population, these species constitute a potentially large untapped pool of novel natural products. To access their genetic information, the DNA of these microorganisms can be isolated directly from environmental samples, cloned into suitable vectors, and expressed in surrogate hosts that can be grown in the laboratory and manipulated genetically (9,17,18,26,29,36).Previously, our investigators and others reported on methods to isolate and clone environmental DNA and screen for novel bioactivities (9,17,18,26,29) using Escherichia coli strains and vectors. Although interesting and novel activities have been expressed and identified in this host, the potential advantage of expanding the range of bacterial hosts to capture additional expression capability is clear. We chose to extend our expression host range to include Streptomyces lividans an...

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Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism

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Phylogeny, classification and metagenomic bioprospecting of microbial acetyl xylan esterases

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