# Reverse Engineering High Yield Lasso Peptide Production by a Burkholderia Heterologous Host

> **NIH NIH F31** · UNIVERSITY OF ILLINOIS AT CHICAGO · 2024 · $48,974

## Abstract

ABSTRACT
Lasso peptides are bacterial metabolites characterized by a unique knotted configuration that confers thermal
stability, proteolytic resistance, and potent target affinity in biological applications. Known lasso peptides have a
diversity of biological functions, including as antibiotics. In terms of modification for drug development, they are
recognized for their ability to be engineered via epitope grafting and genetically encoded libraries. However, the
lack of a viable platform to produce lasso peptides has hindered the discovery and development of lasso-based
drug leads. Chemical synthesis of lasso peptides is impeded by the lasso fold, positioning microbial production
as a favorable alternative. However, heterologous expression of lasso peptide gene clusters (most commonly
using E. coli as the bacterial host) results in low yield (<15 mg/L). In an experiment to test the expression of the
model lasso peptide capistruin in an alternative Burkholderia bacterial host, a stochastic overproducer clone was
identified. Preliminary investigation into the underlying molecular mechanisms of capistruin overproduction led
to the recapitulation of high yield capistruin production (up to 130%) by way of plasmid copy number modulation.
Production of capistruin with this alternative Burkholderia host corresponds to a yield 800-fold greater than that
reported for production using E. coli. Our production platform was validated when Burkholderia was employed
to express an orphan gene cluster from the endosymbiont Mycetohabitans sp. B13 (Burkholderiaceae), leading
to the discovery of two lasso peptides. The objectives of this proposal are to further establish an effective
production platform for novel lasso peptides while simultaneously deepening our understanding of the molecular
determinants of compound overproduction by the Burkholderia host.

## Key facts

- **NIH application ID:** 10825799
- **Project number:** 1F31GM150250-01A1
- **Recipient organization:** UNIVERSITY OF ILLINOIS AT CHICAGO
- **Principal Investigator:** Hannah Naomi Fernandez
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $48,974
- **Award type:** 1
- **Project period:** 2024-01-16 → 2025-01-15

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10825799

## Citation

> US National Institutes of Health, RePORTER application 10825799, Reverse Engineering High Yield Lasso Peptide Production by a Burkholderia Heterologous Host (1F31GM150250-01A1). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10825799. Licensed CC0.

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