A flexible, modular and versatile functional part assembly toolkit for gene cluster engineering in Streptomyces

Area of Science:

• Microbiology
• Synthetic Biology
• Biotechnology

Background:

• Streptomyces bacteria possess vast potential for novel natural product (NP) discovery due to numerous silent biosynthetic gene clusters (BGCs).
• Inefficient gene cluster engineering strategies have historically limited the pace of NP and drug discovery from Streptomyces.
• Developing advanced genetic tools is crucial for unlocking the biosynthetic potential of these microorganisms.

Purpose of the Study:

• To create an accessible and versatile DNA assembly toolkit for efficient gene cluster engineering in Streptomyces.
• To facilitate the manipulation and optimization of BGCs for enhanced natural product production.
• To provide a platform for accelerating drug discovery through microbial natural product synthesis.

Main Methods:

• Development of a flexible DNA assembly toolkit compatible with multiple assembly methods (Biobrick, Golden Gate, CATCH, yeast homologous recombination, homing endonuclease-mediated assembly).
• Quantification of a library of modular regulatory parts.
• Engineering of the Streptomyces act gene cluster using characterized promoters.

Main Results:

• The toolkit demonstrated high flexibility in assembling multiple genetic parts and refactoring large gene clusters.
• Characterized promoters were successfully used to engineer the act gene cluster.
• The engineered gene cluster resulted in increased production of natural products.

Conclusions:

• The developed DNA assembly toolkit offers a powerful solution for natural product discovery and optimization in Streptomyces.
• This tool enhances the ability to engineer complex BGCs, paving the way for new drug candidates.
• The toolkit's versatility supports various genetic engineering approaches, accelerating innovation in microbial biotechnology.