Pathway engineering in yeast for synthesizing the complex polyketide bikaverin.

Fungal polyketides display remarkable structural diversity and bioactivity, and therefore the biosynthesis and engineering of this large class of molecules is therapeutically significant. Here, we successfully recode, construct and characterize the biosynthetic pathway of bikaverin, a tetracyclic polyketide with antibiotic, antifungal and anticancer properties, in S. cerevisiae. We use a green fluorescent protein (GFP) mapping strategy to identify the low expression of Bik1 (polyketide synthase) as a major bottleneck step in the pathway, and a promoter exchange strategy is used to increase expression of Bik1 and bikaverin titer. Then, we use an enzyme-fusion strategy to directly couple the monooxygenase (Bik2) and methyltransferase (Bik3) to efficiently channel intermediates between modifying enzymes, leading to an improved titer of bikaverin at 202.75 mg/L with flask fermentation (273-fold higher than the initial titer). This study demonstrates that the biosynthesis of complex fungal polyketides can be established and efficiently engineered in S. cerevisiae, highlighting the potential for natural product synthesis and large-scale fermentation in yeast.

Natural Product Discovery through Improved Functional Metagenomics in Streptomyces.

Because the majority of environmental bacteria are not easily culturable, access to many bacterially encoded secondary metabolites will be dependent on the development of improved functional metagenomic screening methods. In this study, we examined a collection of diverse Streptomyces species for the best innate ability to heterologously express biosynthetic gene clusters. We then optimized methods for constructing high quality metagenomic cosmid libraries in the best Streptomyces host. An initial screen of a 1.5 million-membered metagenomic library constructed in Streptomyces albus, the species that exhibited the highest propensity for heterologous expression of gene clusters, led to the identification of the novel natural product metatricycloene (1). Metatricycloene is a tricyclic polyene encoded by a reductive, iterative polyketide-like gene cluster. Related gene clusters found in sequenced genomes appear to encode a largely unexplored collection of structurally diverse, polyene-based metabolites.

Antibacterial enzymes from the functional screening of metagenomic libraries hosted in Ralstonia metallidurans.

Phenotype-based screening of bacterial metagenomic libraries provides an avenue for the discovery of novel genes, enzymes, and metabolites that have a variety of potential clinical and industrial uses. Here, we report the identification of a functionally diverse collection of antibacterially active enzymes from the phenotypic screening of 700 000 cosmid clones prepared from Arizona soil DNA and hosted in Ralstonia metallidurans. Environmental DNA clones surrounded by zones of growth inhibition in a bacterial overlay assay were found, through bioinformatics and functional analyses, to encode enzymes with predicted peptidase, lipase, and glycolytic activities conferring antibiosis. The antibacterial activities observed in our R. metallidurans-based assay could not be replicated with the same clones in screens using Escherichia coli as a heterologous host, suggesting that the large-scale screening of metagenomic libraries for antibiosis using phylogenetically diverse hosts should be a productive strategy for identifying enzymes with functionally diverse antibacterial activities.

Biocatalysts and small molecule products from metagenomic studies.

The vast majority of bacteria present in environmental samples have never been cultured and therefore have not been exploited for the ability to produce useful biocatalysts or collections of biocatalysts generating interesting small molecules. Metagenomic libraries constructed using DNA extracted directly from natural bacterial communities offer access to the genetic information present in the genomes of these as yet uncultured bacteria. This review highlights recent efforts to recover both discrete enzymes and small molecules from metagenomic libraries.