Uncovering the antibiotic apramycin's biosynthesis to reduce antibiotic resistance

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About This Project

Antibiotic resistance is rising, but nature still holds secrets to new treatments. I am studying AprL (aminotransferase encoded in the apramycin biosynthetic gene cluster), a key enzyme in apramycin biosynthesis. This project focuses on expressing and purifying AprL and understanding how it binds its essential cofactor, pyridoxal 5'-phosphate (PLP). I will uncover how this enzyme works with your support, paving the way for better antibiotic design and a stronger fight against deadly superbugs.

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What is the context of this research?

Many antibiotics we rely on today come from natural compounds made by bacteria. One such antibiotic is apramycin, which fights tough infections in animals, including humans. To make apramycin, bacteria use special enzymes to build their complex structure step-by-step. One important enzyme in this process is called AprL. AprL helps add an important chemical group needed for apramycin's activity. However, AprL is tricky to study because it requires a helper molecule called pyridoxal 5'-phosphate (PLP) that acts as a cofactor, allowing the enzyme to transfer amino groups during the biosynthetic process. Without PLP, the enzyme cannot function properly. Understanding how AprL binds PLP and functions is the first step toward potentially improving apramycin or creating new antibiotics inspired by it.

What is the significance of this project?

Antibiotic resistance is a growing global health threat that was directly responsible for 1.27 million deaths globally in 2019, making it harder to treat infections. Finding new or improved antibiotics is critical. By studying AprL, we explore a unique enzyme involved in apramycin production, a promising antibiotic that works against resistant bacteria. This project uncovers how AprL binds its helper molecule and functions, information that can guide future engineering of better antibiotics. Also, by overcoming the challenges in producing and studying AprL, we build the foundation for deeper research into this enzyme and related proteins. This foundational work will enable further studies on AprL's structure and function, advancing our understanding of antibiotic biosynthesis and opening doors to new therapeutic developments.

What are the goals of the project?

The main goal is to produce and purify the AprL enzyme from Streptoalloteichus hindustanus (MW ~42 kDa), retaining its essential PLP cofactor for structural studies. AprL will be cloned into pET-28a(+) and expressed in E. coli BL21(DE3) using ZYM-5052 auto-induction medium with a C-terminal His₆-tag. To maintain critical PLP binding, media and purification buffers will be supplemented with 0.1 mM pyridoxal 5'-phosphate. The enzyme will be purified using Ni-NTA affinity chromatography in HEPES/phosphate buffers, followed by ultra-centrifugation for concentration and size-exclusion chromatography for homogeneity. PLP binding will be verified using UV-Visible spectroscopy (characteristic 420 nm peak) and protein quantification via A₂₈₀. The primary objective is to determine the enzyme's 3D structure using X-ray crystallography and/or cryo-EM to understand PLP binding architecture. A key milestone is presenting findings at a scientific conference for expert feedback.

Budget

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This funding enables crucial early-stage work to produce and study AprL, an enzyme key to developing new antibiotics. It covers essential supplies to express and purify the enzyme and analyze its cofactor binding, foundational steps needed to unlock its function. Presenting these initial results at a major scientific conference will bring valuable expert feedback, help overcome technical challenges, and build collaborations that accelerate progress. Each budget item directly supports launching a promising research path with the potential to advance antibiotic discovery and innovation.

Endorsed by

Oluwaseun's current work on AprL represents a critical piece of the antibiotic resistance puzzle. Her methodical approach to understanding enzyme-cofactor interactions demonstrates exactly the kind of rigorous thinking needed to unlock new therapeutic pathways against superbugs.
Oluwaseun was one of my most dedicated mentees, and her current research on AprL exemplifies her ability to identify and pursue high-impact questions in biochemistry and analytical chemistry. This project addresses a genuine gap in our understanding of antibiotic biosynthesis at a time when the world desperately needs new weapons against resistant pathogens, and her careful experimental design gives me confidence she'll generate the necessary foundational knowledge for future antibiotic development.
Having mentored Oluwaseun, I can attest to her tenacity and scientific insight, which make her uniquely suited for this ambitious project on apramycin biosynthesis. She's diving into uncharted territory with AprL, an enzyme that could hold keys to combating antibiotic resistance, and her systematic plan to unravel its PLP-binding mechanisms shows the kind of innovative thinking that leads to breakthrough discoveries.

Project Timeline

Over six months, this project will optimize production and purification of the active AprL enzyme, characterize its cofactor binding and stability using spectroscopy and chromatography techniques, and present initial findings at a leading scientific conference. Supporters will receive regular updates, data insights, and a digital poster, fostering engagement and advancing antibiotic biosynthesis research.

Jun 14, 2025

Project Launched

Jul 01, 2025

Expression and Purification of Active AprL Enzyme

Sep 01, 2025

Characterization of PLP Cofactor Binding and Enzyme Stability

Nov 01, 2025

Presentation of Initial Results at Scientific Conference

Meet the Team

Oluwaseun Ajayi
Oluwaseun Ajayi

Affiliates

University of Georgia, Athens
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Oluwaseun Ajayi

I am Oluwaseun Ajayi, a bioanalytical chemist with a strong focus on enzyme biochemistry and natural product biosynthesis. Throughout my academic and research career, I have been driven by a deep curiosity about how living organisms produce complex molecules, particularly antibiotics, which have critical applications in medicine. My training in bioanalytical chemistry has equipped me with advanced skills in protein expression, purification, and spectroscopic analysis, enabling me to study the molecular details of enzyme function and cofactor interactions.

My current research centers on AprL, a pyridoxal phosphate (PLP)-dependent aminotransferase involved in the biosynthesis of apramycin, an important antibiotic. By investigating AprL’s expression, purification, and cofactor binding, I aim to uncover fundamental biochemical properties that could lead to engineering improved antibiotic pathways. This work has the potential to contribute to combating antibiotic resistance, one of the most pressing global health challenges.

I became interested in this field during my graduate studies when I recognized the power of combining analytical chemistry techniques with molecular biology to address real-world problems. I am passionate about bridging fundamental science with applied research, working at the interface of chemistry and biology to drive innovation.

Beyond the lab, I am committed to sharing my findings through conferences and publications, fostering collaborations that accelerate progress in antibiotic discovery. This project marks an important foundation in my research journey, where I seek not only to generate new scientific knowledge but also to contribute solutions that can improve human health worldwide.

Lab Notes

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Additional Information

This project is part of a larger effort to explore natural enzymes that bacteria use to build complex antibiotics. AprL is just one piece of this puzzle, but it's a vital one because it helps create a key structural component of apramycin. By funding this early-stage research, you're helping us overcome the technical challenges that often block progress in enzyme studies, like producing stable, active proteins with their essential cofactors in the lab. Your support will not only advance scientific knowledge but also help train the next generation of researchers passionate about fighting antibiotic resistance. Thank you for being part of this important journey toward discovering new therapeutic solutions.


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