Heterologous Expression of a Myxobacterial Natural Products Assembly Line in Pseudomonads via Red/ET Recombineering

Abstract

Natural products of microbial origin are widely used as pharmaceuticals and in agrochemistry. These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown. We describe a straightforward strategy that combines the power of advanced DNA engineering (recombiogenic cloning) in Escherichia coli with the utility of pseudomonads as the heterologous host for the analysis and mutagenesis of known and unknown secondary metabolite pathways. The myxochromide S biosynthetic gene cluster from Stigmatella aurantiaca was rebuilt and engineered in E. coli to contain the elements required for expression in pseudomonads. The successful production in Pseudomonas putida, at unprecedented levels, demonstrates the feasibility of the new approach to the analysis and mutagenesis of these important pathways.

Heterologous Expression of the Oxytetracycline Biosynthetic Pathway in Myxococcus xanthus

“…An enormous and currently inaccessible reservoir of new natural products is located in the biosynthetic pathways found in the genomes of uncultivated bacteria (18). Heterologous expression of these biosynthetic gene clusters represents a powerful tool for discovering new natural products (20,21). Herein, we demonstrate that the deltaproteobacterium Myxococcus xanthus is an effective host for heterologous expression of aromatic polyketide biosynthetic pathways.…”

“…The observed titer of 10 mg/liter of culture broth is comparable to titers reported for the heterologous expression of myxobacterial polyketide biosynthetic pathways in myxobacteria (11) and streptomycete-derived polyketide biosynthetic pathways in Streptomyces (14,23) and is sufficient for characterization of the polyketide product. Pseudomonas putida, which has a more favorable growth profile, has been shown to be a good host for heterologous expression of myxobacterial polyketide biosynthetic pathways, with product titers in the range of 0.6 to 40 mg/liter of culture broth (14,21,23). The observed breadth of polyketide pathways accessible and the titers of the polyketide products produced make M. xanthus an attractive potential candidate for a "universal" host for facilitating heterologous expression of polyketide biosynthetic pathways derived from environmental samples of metagenomic DNA.…”

Industrial biotechnology of Pseudomonas putida and related species

“…These compounds are often biosynthesized by multifunctional megasynthetases whose genetic engineering and heterologous expression offer considerable promise, especially if the natural hosts are genetically difficult to handle, slow growing, unculturable, or even unknown (Wenzel et al 2005). In this regard, P. putida has shown to be capable to express and activate biosynthetic proteins of complex natural products from myxobacteria (Gross et al 2005;Gross et al 2006;Wenzel et al 2005). This has enabled production of high-value pharmaceuticals in this bacterium (Table 3) More recently, transposition was established for genetic engineering of P. putida allowing efficient transfer of extremely large gene clusters which in general build the basis for biosynthesis of natural products (Fu et al 2008).…”

Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A

“…Although innovative methods have been developed to stitch together overlapping clones into a functional single clone harboring the complete gene cluster (15), this approach can be very time-consuming (16,17). Even with bacterial artificial chromosomes capable of cloning large genomic DNA fragments up to 200 kb, the need to identify desired clones that stably maintain the complete cluster remains.…”