# Mechanistic Studies of the Functional Consequences of Heterologous Expression of Actinomycetal Megasynthases

> **NIH NIH R15** · UNIVERSITY OF TENNESSEE KNOXVILLE · 2022 · $431,821

## Abstract

PROJECT SUMMARY/ABSTRACT
Bacteria from the order Actinomycetales, especially those from the genus Streptomyces, are
some of the most prolific producers of bioactive natural products, including over 75% of
commercially available antibiotics. Although other orders of bacteria harbor biosynthetic
pathways, the metabolic giftedness of actinomycetes is the gold standard for pharmaceutical
discovery. In particular, they harbor many pathways that are modular such as type I polyketide
synthases (PKSs) and type I non-ribosomal peptide synthetases (NRPSs), which have been
noted for their considerable potential for engineered biosynthesis for well over 30 years. However,
actinomycetes present a number of challenges with their growth profiles and genetic systems that
serve as less than ideal as heterologous hosts, especially for rapid profiling of engineered
systems or for protein production for enzymology studies. Furthermore, their high GC genomes
(~75% GC) complicate routine molecular biology, genetic manipulations, and transcriptional and
translational efficiency. This is particularly the case with megasynthase enzymes such as PKSs
and NRPSs, which are large in size and frequently express poorly, as truncated products, or do
not fold correctly. While such issues are discussed in the natural products enzymology
community, few systematic studies exist in the literature to understand the functional
consequences of heterologous host choice and refactoring, especially at the level at which it
affects protein function. Furthermore, many of the systems that would be ideal to profile
megasynthases (such as in vitro transcription-translation; TXTL) create irregularities with regard
to truncation and folding, which are also poorly characterized. We will examine the functional
consequences of host and refactoring-dependent expression on such actinomycetal
megasynthases at the protein level. The characterization described in this proposal will lead to a
more systematic understanding of the benefits and drawbacks of different systems of
heterologous expression for different applications which include characterizing and profiling
engineered megasynthases and ultimately producing small molecule metabolites. This work will
afford the realization of the full potential of proteins from actinomycetes as a rich source for
synthetic biology parts for engineered metabolism.

## Key facts

- **NIH application ID:** 10439127
- **Project number:** 1R15GM146192-01
- **Recipient organization:** UNIVERSITY OF TENNESSEE KNOXVILLE
- **Principal Investigator:** Joshua A Baccile
- **Activity code:** R15 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $431,821
- **Award type:** 1
- **Project period:** 2022-04-01 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10439127

## Citation

> US National Institutes of Health, RePORTER application 10439127, Mechanistic Studies of the Functional Consequences of Heterologous Expression of Actinomycetal Megasynthases (1R15GM146192-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10439127. Licensed CC0.

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