Alanine Scanning of YbdZ, an MbtH-like Protein, Reveals Essential Residues for Functional Interactions with Its Nonribosomal Peptide Synthetase Partner EntF.

Nonribosomal peptide synthetases (NRPSs) are megasynthetases that require complex and specific interactions between multiple domains and proteins to functionally produce a metabolite. MbtH-like proteins (MLPs) are integral components of many NRPSs and interact directly with the adenylation domain of the megasynthetases to stimulate functional enzymology. All of the MLP residues that are essential for functional interactions between the MLP and NRPS have yet to be defined. Here we probe the interactions between YbdZ, an MLP, and EntF, an NRPS, from Escherichia coli by performing a complete alanine scan of YbdZ. A phenotypic screen identified 11 YbdZ variants that are unable to replace the wild-type MLP, and these YbdZ variants were characterized using a series of in vivo and in vitro assays in an effort to explain why functional interactions with EntF were disrupted. All of the YbdZ variants enhanced the solubility of overproduced EntF, suggesting they were still capable of direct interactions with the megasynthase. Conversely, we show that EntF also influences the solubility of YbdZ and its variants. In vitro biochemical analyses of EntF function with each of the YbdZ variants found the impact that an amino acid substitution will have on NRPS function is difficult to predict, highlighting the complex interaction between these proteins.

Discovery of a lagriamide polyketide by integrated genome mining, isotopic labeling, and untargeted metabolomics.

Microorganisms from the order Burkholderiales have been the source of a number of important classes of natural products in recent years. For example, study of the beetle-associated symbiont Burkholderia gladioli led to the discovery of the antifungal polyketide lagriamide; an important molecule from the perspectives of both biotechnology and chemical ecology. As part of a wider project to sequence Burkholderiales genomes from our in-house Burkholderiales library we identified a strain containing a biosynthetic gene cluster (BGC) similar to the original lagriamide BGC. Structure prediction failed to identify any candidate masses for the products of this BGC from untargeted metabolomics mass spectrometry data. However, genome mining from publicly available databases identified fragments of this BGC from a culture collection strain of Paraburkholderia. Whole genome sequencing of this strain revealed the presence of a homologue of this BGC with very high sequence identity. Stable isotope feeding of the two strains in parallel using our newly developed IsoAnalyst platform identified the product of this lagriamide-like BGC directly from the crude fermentation extracts, affording a culturable supply of this interesting compound class. Using a combination of bioinformatic, computational and spectroscopic methods we defined the absolute configurations for all 11 chiral centers in this new metabolite, which we named lagriamide B. Biological testing of lagriamide B against a panel of 21 bacterial and fungal pathogens revealed antifungal activity against the opportunistic human pathogen Aspergillus niger, while image-based Cell Painting analysis indicated that lagriamide B also causes actin filament disruption in U2-OS osteosarcoma cells.

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.