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Ribosome Profiling

Ribosome Profiling

Ribosome profiling or ribo-sequencing is a deep sequencing technique that produces a snapshot of active translation in a cell. It selectively sequences the mRNAs protected by ribosomes to get an insight into a cell’s translation landscape at any given point in time.
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Ribosome profiling has many applications, including in vivo monitoring of translation inside a particular organ or tissue type and quantifying new protein synthesis levels.
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Mass Spectrometry-Guided Genome Mining as a Tool to Uncover Novel Natural Products

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Published on: March 12, 2020

Genome mining of RiPPs driven by highly efficient pathway reconstruction methods.

¹MOE Key Laboratory of Bio-Intelligent Manufacturing, School of Bioengineering, Dalian University of Technology, Dalian, China.

Methods in Enzymology

|July 12, 2025

Summary

Exploring cryptic RiPP biosynthetic gene clusters (BGCs) is now easier. New methods like pathway refactoring and CAPTURE enable efficient expression and cloning of these natural product BGCs for further study.

Keywords:

Direct cloning Genome mining Pathway refactoring RiPPs Synthetic biology

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Area of Science:

Natural Product Biosynthesis
Synthetic Biology
Genomics

Background:

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a growing class of natural products with therapeutic potential.
Genome mining has identified numerous RiPP biosynthetic gene clusters (BGCs), but their products are often cryptic due to complex regulation and diverse origins.

Purpose of the Study:

To describe methods for exploring the biosynthetic potential of cryptic RiPP BGCs.
To enable the expression and characterization of previously uncharacterized RiPPs.

Main Methods:

Pathway reconstruction using a plug-and-play refactoring workflow to rewire regulatory systems for expression in tractable hosts.
Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) for cloning large DNA fragments of complex BGCs.

Main Results:

The described methods facilitate the exploration of cryptic RiPP BGCs.
Pathway refactoring allows for efficient expression of target BGCs.
CAPTURE provides a robust method for cloning intricate BGCs.

Conclusions:

These efficient and robust methods are valuable for genome mining of RiPPs and other natural product families.
The described approaches advance the discovery of novel natural products from genomic data.