# Designer biosensors for directed evolution of macrolide biosynthetic enzymes

> **NIH NIH R01** · NORTH CAROLINA STATE UNIVERSITY RALEIGH · 2021 · $276,300

## Abstract

PROJECT SUMMARY
The complexity of polyketide biosynthetic machinery has hampered attempts to access macrolides and their
analogs via combinatorial biosynthesis. As part of our long-term goal of reprogramming the biosynthesis of
natural products for the synthesis of therapeutic leads, the overall objective here is to use genetically encoded
biosensors to enhance access to novel macrolides. Our hypotheses are (1) the established inducer promiscuity
of the MphR repressor protein can be manipulated to provide biosensors with new inducer specificities and
selectivities, (2) the specificity of macrolide tailoring enzymes can be manipulated by biosensor-guided directed
evolution, and (3) MphR can be used to identify hybrid assembly lines with improved activities. These
hypotheses are supported by (1) preliminary data that shows MphR variants with new specificities, selectivities,
and suitable detection capabilities can be generated, (2) the variety of macrolide tailoring enzymes available as
starting points for directed evolution and the success of directed evolution for altering substrate specificity of
other enzymes, and (3) preliminary data that demonstrates the feasibility of using trans-acting enzymes to
complement polyketide assembly lines. The rationale for the proposed research is that our approach of
leveraging designer biosensors offers the ability to report the activity of a variety of macrolide biosynthetic
enzymes, which can be applied to solving a broad range of problems related to macrolide biosynthesis, leading
to valuable new macrolides. To address these hypotheses, and to complete the overall objective of this
proposal, the following specific aims will be completed: (1) access novel macrolide O-alkyl derivatives, and (2)
rescue the activity of poorly active hybrid PKS assembly lines. Our approach is highly innovative because it
develops a set of screening tools that are currently not available and that can be applied to engineering the
biosynthesis of a broad range of macrolides in potentially any microbial host. The proposed research is
significant because it is expected to have broad positive impact in natural product biosynthesis and synthetic
biology by developing new strategies for producing macrolides, by expanding our understanding of biosensor
specificity, by developing new approaches for macrolide diversification, and by expanding the capabilities of
enzyme engineering and synthetic biology.

## Key facts

- **NIH application ID:** 10115752
- **Project number:** 5R01GM124112-04
- **Recipient organization:** NORTH CAROLINA STATE UNIVERSITY RALEIGH
- **Principal Investigator:** THOMAS ASHTON CROPP
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $276,300
- **Award type:** 5
- **Project period:** 2018-05-05 → 2023-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10115752

## Citation

> US National Institutes of Health, RePORTER application 10115752, Designer biosensors for directed evolution of macrolide biosynthetic enzymes (5R01GM124112-04). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10115752. Licensed CC0.

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