A study of iterative type II polyketide synthases, using bacterial genes cloned from soil DNA: a means to access and use genes from uncultured microorganisms

Abstract

To examine as randomly as possible the role of the ␤-ketoacyl and acyl carrier protein (ACP) components of bacterial type II polyketide synthases (PKSs), homologs of the chain-length-factor (CLF) genes were cloned from the environmental community of microorganisms. With PCR primers derived from conserved regions of known ketosynthase (KS ␣ ) and ACP genes specifying the formation of 16-to 24-carbon polyketides, two CLF (KS ␤ ) genes were cloned from unclassified streptomycetes isolated from the soil, and two were cloned from soil DNA without the prior isolation of the parent microorganism. The sequence and deduced product of each gene were distinct from those of known KS ␤ genes and, by phylogenetic analysis, belonged to antibiotic-producing PKS gene clusters. Hybrid PKS gene cassettes were constructed with each novel KS ␤ gene substituted for the actI-ORF2 or tcmL KS ␤ subunit genes, along with the respective actI-ORF1 or tcmK KS ␣ , tcmM ACP, and tcmN cyclase genes, and were found to produce an octaketide or decaketide product characteristic of the ones known to be made by the heterologous KS ␣ gene partner. Since substantially less than 1% of the microorganisms present in soil are thought to be cultivatable by standard methods, this work demonstrates a potential way to gain access to a more extensive range of microbial molecular diversity and to biosynthetic pathways whose products can be tested for biological applications.The polyketide metabolites are a large group of structurally complex and diverse natural products, many of which are clinically valuable antibiotics or chemotherapeutic agents or have other useful pharmacological activities (48). Polyketides are synthesized by polyketide synthases (PKSs) which, like the related fatty acid synthases, are multifunctional enzyme assemblies that catalyze repeated decarboxylative condensations between enzyme-bound acylthioesters (52). The structural diversity of polyketides is a result of the different numbers and types of acyl units involved, coupled with various possibilities for enzyme-mediated reduction, dehydration, cyclization, and aromatization of the initial ␤-ketoacyl condensation products (52). Manipulation of the sequence or specificity of the PKS enzymes can lead to the production of novel secondary metabolites (28,29,33). The key to further exploitation of this approach to molecular diversity lies in an increased understanding of the genetics and biochemistry of the various PKS systems.Recent progress in the cloning and sequencing of microbial PKS genes has led to the identification of at least two architecturally different types of PKSs (28, 33). Modular type I PKSs, represented by erythromycin A (11, 17), avermectin (41), rapamycin (58), and soraphen (57), are discrete multifunctional enzymes sporting numerous unique active site domains. The domains are grouped into distinct modules that in turn control the sequential addition of acylthioester units to the growing polyketide chain and the subsequent modification of the respective condensation prod...

Construction of soil environmental DNA cosmid libraries and screening for clones that produce biologically active small molecules

“…Culture-independent analysis of the genetic and biochemical diversity present in naturally occurring bacterial communities, which began in the 1990s (see refs. [14][15][16][17][18][19]) is now a rapidly growing area of research and has been given the name metagenomics 20 .…”

Functional analysis of environmental DNA-derived type II polyketide synthases reveals structurally diverse secondary metabolites

“…The minimal PKS is responsible for the iterative condensation of malonylCoAs into a nascent polyketide chain that is then cyclized, aromatized, reduced, oxidized, rearranged, and functionalized in pathway-specific ways to generate the extraordinary structural diversity that is known to arise from these systems (8,9). PCRbased studies as well as shotgun-sequencing efforts indicate that eDNA samples are rich in unique minimal PKS genes (10)(11)(12)(13). Through the functional characterization of eDNA-derived type II PKS containing clones, we have identified PKS systems that encode structurally diverse metabolites including compounds with unique and rare carbon skeletons.…”

Bacteria in agricultural soils: Diversity, role and future perspectives

“…Canadian scientists have contributed to the development and implementation of both nucleic acid-based and chemical biomarker-based methods now widely used for assessing soil microbial biodiversity without the need for isolation and cultivation. Some examples include methods for soil DNA extraction (Berthelet et al 1996), achieving taxa-specific (Watson et al 1995) and quantitative (Leung et al 1997) PCR, resolving mixed PCR products by denaturant gradient gel electrophesis (Vallaeys et al 1997), detecting specific signature DNA sequences in soil DNA by reverse genome probing (Telang et al 1994), and sequencing genes cloned directly from soil DNA (Seow et al 1997). have explored the use of signature phospholipids, extracted directly from soil and identified by chemical methods, for assessing the global biodiversity of soil microbial communities.…”