Abstract
Heterologous expression of biosynthetic gene clusters (BGCs) represents an attractive route to the production of new natural products, but is often hampered by poor yields. It is therefore important to develop tools that enable rapid refactoring, gene insertion/deletion, and targeted mutations in BGCs. Ideally, these tools should be highly efficient, affordable, accessible, marker free, and flexible for use with a wide range of BGCs. Here, we present a one-step yeast-based method that enables efficient, cheap, and flexible modifications to BGCs. Using the BGC for the antibiotic bottromycin, we showcase multiple modifications including refactoring, gene deletions and targeted mutations. This facilitated the construction of an inducible, riboswitch-controlled pathway that achieved a 120-fold increase in pathway productivity in a heterologous streptomycete host. Additionally, an unexpected biosynthetic bottleneck resulted in the production of a suite of new bottromycin-related metabolites.
Highlights
Heterologous expression of biosynthetic gene clusters (BGCs) represents an attractive route to the production of new natural products, but is often hampered by poor yields
We integrate transformation-associated recombination (TAR) cloning in yeast,[12,17] yeast-mediated engineering,[20,21] and recently developed regulatory tools[22,23] into a BGC modification process that we applied to the bottromycin gene cluster.[24−27] We show how commercially available restriction enzymes, PCR products, and single stranded oligonucleotides can be used to simultaneously refactor, delete genes, make targeted mutations, and reorganize gene order in the bottromycin cluster
ACS Synthetic Biology refactoring and remodeling of this gene cluster led to a BGC that was 120-fold more productive in a heterologous host and yielded a variety of new bottromycins
Summary
Bottromycin is a heavily modified RiPP antibiotic characterized by a macrocyclic amidine and a terminal thiazole (Figure 1). It. has a unique architecture for an antibiotic, but biological studies have been hampered by low yields in native producing strains.[26] The bottromycin biosynthetic cluster in Streptomyces scabies consists of genes btmA to btmM (Figure 2), for which btmD encodes the precursor peptide, and the cluster has an overall GC content of 73.7%. Has a unique architecture for an antibiotic, but biological studies have been hampered by low yields in native producing strains.[26] The bottromycin biosynthetic cluster in Streptomyces scabies consists of genes btmA to btmM (Figure 2), for which btmD encodes the precursor peptide, and the cluster has an overall GC content of 73.7%
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