Engineering synthetic recursive pathways to generate non-natural small molecules.

Recursive pathways are broadly defined as those that catalyze a series of reactions such that the key, bond-forming functional group of the substrate is always regenerated in each cycle, allowing for a new cycle of reactions to begin. Recursive carbon-chain elongation pathways in nature produce fatty acids, polyketides, isoprenoids and α-keto acids (αKAs), which all use modular or iterative approaches for chain elongation. Recently, an artificial pathway for αKA elongation has been built that uses an engineered isopropylmalate synthase to recursively condense acetyl-CoA with αKAs. This synthetic approach expands the possibilities for recursive pathways beyond the modular or iterative synthesis of natural products and serves as a case study for understanding the challenges of building recursive pathways from nonrecursive enzymes. There exists the potential to design synthetic recursive pathways far beyond what nature has evolved.

MbtH-like proteins as integral components of bacterial nonribosomal peptide synthetases.

The biosynthesis of many natural products of clinical interest involves large, multidomain enzymes called nonribosomal peptide synthetases (NRPSs). In bacteria, many of the gene clusters coding for NRPSs also code for a member of the MbtH-like protein superfamily, which are small proteins of unknown function. Using MbtH-like proteins from three separate NRPS systems, we show that these proteins copurify with the NRPSs and influence amino acid activation. As a consequence, MbtH-like proteins are integral components of NRPSs.

Investigations into viomycin biosynthesis by using heterologous production in Streptomyces lividans.

Viomycin and capreomycin are members of the tuberactinomycin family of antituberculosis drugs. As with many antibacterial drugs, resistance to the tuberactinomycins is problematic in treating tuberculosis; this makes the development of new derivatives of these antibiotics to combat this resistance of utmost importance. To take steps towards developing new derivatives of this family of antibiotics, we have focused our efforts on understanding how these antibiotics are biosynthesized by the producing bacteria so that metabolic engineering of these pathways can be used to generate desired derivatives. Here we present the heterologous production of viomycin in Streptomyces lividans 1326 and the use of targeted-gene deletion as a mechanism for investigating viomycin biosynthesis as well as the generation of viomycin derivatives. Deletion of vioQ resulted in nonhydroxylated derivatives of viomycin, while strains lacking vioP failed to acylate the cyclic pentapeptide core of viomycin with beta-lysine. Surprisingly, strains lacking vioL produced derivatives that had the carbamoyl group of viomycin replaced by an acetyl group. Additionally, the acetylated viomycin derivatives were produced at very low levels. These two observations suggested that the carbamoyl group of the cyclic pentapeptide core of viomycin was introduced at an earlier step in the biosynthetic pathway than previously proposed. We present biochemical evidence that the carbamoyl group is added to the beta-amino group of L-2,3-diaminopropionate prior to incorporation of this amino acid by the nonribosomal peptide synthetases that form the cyclic pentapeptide cores of both viomycin and capreomycin.

Nonribosomal peptide synthetases involved in the production of medically relevant natural products.

Natural products biosynthesized wholly or in part by nonribosomal peptide synthetases (NRPSs) are some of the most important drugs currently used clinically for the treatment of a variety of diseases. Since the initial research into NRPSs in the early 1960s, we have gained considerable insights into the mechanism by which these enzymes assemble these natural products. This review will present a brief history of how the basic mechanistic steps of NRPSs were initially deciphered and how this information has led us to understand how nature modified these systems to generate the enormous structural diversity seen in nonribosomal peptides. This review will also briefly discuss how drug development and discovery are being influenced by what we have learned from nature about nonribosomal peptide biosynthesis.

Identification of the biosynthetic gene cluster and an additional gene for resistance to the antituberculosis drug capreomycin.

Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.