Biology subjects

Budimir, Z. L.

Publications and source records attributed to Budimir, Z. L..

2 recordsLinked to original sources

bioRxiv2025

Streptomyces Autoregulator Biosensors from Natural Product Cluster-Situated Regulators

The soil dwelling bacteria Streptomyces is an abundant producer of numerous anticancer, antifungal, and antibiotic compounds (i.e. Natural Products, NPs). The sophisticated cellular machinery required to produce NPs is frequently regulated by quorum-sensing systems, consisting of cluster situated regulators (CSRs), such as TetR-like repressors, and small-molecule autoregulator (AR) ligands. Only a small fraction of bioinformatically predicted quorum-sensing AR circuits have been experimentally determined, and fewer still have been engineered as inducible expression systems for synthetic biology. This research details the development of eight CSR-based AR biosensors and the synthetic routes to their AR ligands. Overall, the AR biosensors exhibit a range of maximum activation, AR affinity, and AR selectivity. We examined crosstalk between noncognate CSRs and ARs, as well as the ability of CSRs to regulate alternative operators. Additionally, we establish these biosensors can be cocultured with Streptomyces for rapid analysis of AR production. Finally, we demonstrate the CSR-based biosensor vectors can be combined to create orthogonal signaling systems in bacterial coculturing or multi-input genetic circuits. Longterm, these Streptomyces AR biosensors will contribute to the elucidation of small molecule quorum sensing circuits employed by Streptomyces as well as increasing the complexity of genetic circuits used in industrial or agricultural settings.

synthetic biology↗

bioRxiv2024

Biocatalytic Tetrapeptide Macrocyclization by Cryptic Penicillin-binding Protein-type Thioesterases

Cyclic tetrapeptides (CTPs) are a diverse class of natural products with a broad range of biological activities. However, they are challenging to synthesize due to the ring strain associated with their small ring size. While chemical methods have been developed to access CTPs, they generally require the presence of certain amino acids, limiting their substrate scopes. Herein, we report the first bioinformatics-guided discovery of a thioesterase from a cryptic biosynthetic gene cluster for peptide cyclization. Specifically, we hypothesized that predicted penicillin-binding type thioesterases (PBP-TEs) from cryptic nonribosomal peptide synthetase (NRPS) gene clusters containing four adenylation domains would catalyze tetrapeptide cyclization. We found that one of the predicted PBP-TEs, WP516, efficiently cyclizes a wide variety of tetrapeptide substrates. To date, it is only the second stand-alone enzyme capable of cyclizing tetrapeptides, and its substrate scope greatly surpasses that of the only other reported tetrapeptide cyclase Ulm16. AlphaFold modeling, covalent docking, molecular dynamics, and mutational analyses were used to rationalize the broad substrate scope of WP516. Overall, the bioinformatics guided workflow outlined in this paper, and the discovery of WP516, represent promising tools for the biocatalytic production of head-to-tail CTPs, as well as a more general strategy for discovery of enzymes for peptide cyclization.

biochemistry↗