Abstract
A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding because of the lack of specific functions or substrates in the host cell, the cell's sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. Specifically, we synthesize indigoidine and rhabdopeptides with the in vitro produced multidomain nonribosomal peptide synthetases BpsA and KJ12ABC from the organisms Streptomyces lavendulae and Xenorhabdus KJ12.1, respectively. These in vitro produced proteins are analyzed in yield, post-translational modification and in their ability to synthesize the natural compounds, and compared to recombinantly produced proteins. Our study highlights cell-free PURE system as suitable setting for the characterization of biosynthetic gene clusters that can potentially be harnessed for the rapid engineering of biosynthetic pathways.
Highlights
Genome mining has become the main driver in the discovery of new natural products 1-2.while the “orphan clusters” become available by the advent of generation sequencing technologies and bioinformatics tools, the substances can often neither be identified nor extracted under laboratory conditions
Such an approach is beneficial for the analysis and design of biosynthetic pathways for mainly three reasons: First, it prevents the biosynthetic pathway from interfering with physiological processes of a host cell, second, it is of minimal complexity allowing for simple and direct read-out of enzymatic properties, and, third, it is an open system that is not constrained to canonical compounds
We focused on the protein class of megasynthases, which are responsible for the synthesis of non-ribosomal peptides (NRPs) and polyketides (PKs)
Summary
Genome mining has become the main driver in the discovery of new natural products 1-2.while the “orphan clusters” become available by the advent of generation sequencing technologies and bioinformatics tools, the substances can often neither be identified nor extracted under laboratory conditions. System for the cell-free synthesis of natural compounds from genomic DNA, we worked with the commercially available E. coli-based PURExpress In Vitro Protein Synthesis (IVPS) Kit as a “reaction solution” for gene expression and product formation
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