# Construction and Diversification of Natural Product Biosynthetic Gene Clusters at High Efficiency and Accuracy

## TL;DR

A new method for accurately cloning and modifying natural product biosynthetic gene clusters is developed, enabling efficient pathway engineering and discovery of new molecules.

## Contribution

A hierarchical Golden Gate Assembly strategy for high-efficiency and accurate BGC cloning and refactoring is introduced.

## Key findings

- A 23 kb actinorhodin BGC and 23 mutant derivatives were constructed with 100% efficiency.
- Nine genes were identified as essential for actinorhodin production in Streptomyces coelicolor M1152.
- GNPS molecular networking revealed numerous unidentified molecules from ACT biosynthesis.

## Abstract

Biosynthetic gene clusters (BGCs) encode the biosynthesis
of natural
products, which serve as the foundation for therapeutics such as antibiotics,
anticancer agents, antifungals, and immunosuppressants. The vast majority
of the BGCs remain uncharacterized due to lack of expression or inability
to cultivate the native host, making refactoring and expression of
BGCs in optimized hosts a prerequisite for genome-based drug discovery.
Transformation-associated recombination (TAR) cloning and Gibson assembly
are error prone due to the use of homologous recombination. Here,
we present a BGC cloning and refactoring strategy based on a hierarchical
Golden Gate Assembly (GGA), which enables systematic pathway engineering
and mutagenesis with unprecedented accuracy and efficiency. We constructed
the 23 kb actinorhodin (ACT) BGC and 23 mutant derivatives with either
one of the act genes inactivated, within the same
experiment and with 100% efficiency. Introduction of the BGCs in the
ACT-nonproducer Streptomyces coelicolor M1152 revealed that nine genes are essential for ACT production,
while inactivation of others led to significant rewiring of the biosynthetic
pathway. Global Natural Products Social (GNPS) molecular networking
thereby revealed a surprisingly large number of unidentified molecules,
significantly expanding the chemical space associated with ACT biosynthesis.
Additionally, we refactored the act cluster through
promoter engineering and evaluated expression outcomes across multiple Streptomyces strains. Together, our work establishes a GGA-based
platform for BGC construction, refactoring, and functional dissection,
accelerating synthetic-biology-driven natural product discovery.