Synthase-Selective Exploration of a Tunicate Microbiome by Activity-Guided Single-Cell Genomics.

While thousands of environmental metagenomes have been mined for the presence of novel biosynthetic gene clusters, such computational predictions do not provide evidence of their in vivo biosynthetic functionality. Using fluorescent in situ enzyme assay targeting carrier proteins common to polyketide (PKS) and nonribosomal peptide synthetases (NRPS), we applied fluorescence-activated cell sorting to tunicate microbiome to enrich for microbes with active secondary metabolic capabilities. Single-cell genomics uncovered the genetic basis for a wide biosynthetic diversity in the enzyme-active cells and revealed a member of marine Oceanospirillales harboring a novel NRPS gene cluster with high similarity to phylogenetically distant marine and terrestrial bacteria. Interestingly, this synthase belongs to a larger class of siderophore biosynthetic gene clusters commonly associated with pestilence and disease. This demonstrates activity-guided single-cell genomics as a tool to guide novel biosynthetic discovery.

In silico identification and in vitro evaluation of a protein-protein interaction inhibitor of Escherichia coli fatty acid biosynthesis.

To combat the rise in antibiotic resistance, new targets must be identified and probes against them developed. Protein-protein interactions (PPI) of bacterial type II fatty acid biosynthesis (FAS-II) represent an untapped, yet rich area for new antibiotic discovery. Here, we present a computational and in vitro workflow for the discovery of new inhibitors of PPI in Escherichia coli FAS-II. As part of this study, we identified suramin, an existing treatment for African sleeping sickness, to effectively block the interaction of E. coli dehydratase FabA and the acyl carrier protein EcACP, with an IC50 = 85 µΜ. This finding validates a workflow that combines in silico screening with in vitro PPI assays to identify probes appropriate for further optimization.

Quantifying protein-protein interactions of the acyl carrier protein with solvatochromic probes.

Protein-protein interactions (PPIs) are universal to life and their study and understanding is critical to drug discovery and bioengineering efforts. Historically, X-ray crystallography, isothermal titration calorimetry and other biophysical methods have been used to study PPIs, but can be costly and are low throughput, hindering progress towards rapid evaluation of these interactions. Recent interest in targeting PPIs and in engineering biosynthetic pathways in which PPIs play a critical role has driven innovation in their evaluation but a universal screen is still needed. One of the best characterized systems relying upon PPIs is Escherichia coli type II fatty acid biosynthesis in which the central acyl carrier protein (EcACP) shuttles substrates to a series of partner enzymes. Here we present a method by which EcACP is labeled with a solvatochromic dye, 4-DMN, and then allowed to interact with its various partner enzymes. Upon interaction, there is a large increase in fluorescence intensity which is easily monitored via fluorometer or plate reader. This method is useful in the study of known PPI, hypothetical PPI and in evaluation of inhibitors of both partner enzyme active site and of the PPI itself.

A Single Tool to Monitor Multiple Protein-Protein Interactions of the Escherichia coli Acyl Carrier Protein.

Protein-protein interactions are ubiquitous to all domains of life and have gained recent interest as drug targets. However, many current methods to study protein-protein interactions can be costly and are low-throughput. Here, we demonstrate a solvatochromic tool based on the natural post-translational modification of the Escherichia coli acyl carrier protein (EcACP) used to visualize protein-protein interactions between EcACP and 13 different partner enzymes from several biosynthetic pathways. We use this tool to confirm proposed interactions between EcACP and both catalytic and regulatory proteins. We also show the utility of this method toward detecting allosteric changes to partner enzyme structure and the validation of active site inhibitors. We anticipate the future adaptation of this assay into a high-throughput screen for antibiotic discovery.

Acyl carrier protein structural classification and normal mode analysis.

All acyl carrier protein primary and tertiary structures were gathered into the ThYme database. They are classified into 16 families by amino acid sequence similarity, with members of the different families having sequences with statistically highly significant differences. These classifications are supported by tertiary structure superposition analysis. Tertiary structures from a number of families are very similar, suggesting that these families may come from a single distant ancestor. Normal vibrational mode analysis was conducted on experimentally determined freestanding structures, showing greater fluctuations at chain termini and loops than in most helices. Their modes overlap more so within families than between different families. The tertiary structures of three acyl carrier protein families that lacked any known structures were predicted as well.