A multiplasmid approach to preparing large libraries of polyketides

Area of Science:

• Biotechnology
• Synthetic Biology
• Natural Product Chemistry

Background:

• Type I modular polyketide synthases (PKSs) are crucial for producing complex polyketide natural products.
• Heterologous expression of large PKS gene clusters can be challenging.
• Combinatorial biosynthesis offers a powerful approach to generate novel polyketide analogs.

Purpose of the Study:

• To develop a versatile three-plasmid system for the heterologous expression of 6-deoxyerythronolide B synthase (DEBS).
• To facilitate combinatorial biosynthesis of polyketides using modular PKS.
• To demonstrate the system's utility in generating a library of novel polyketide analogs.

Main Methods:

• Individual cloning of eryA PKS genes encoding DEBS subunits into compatible Streptomyces vectors.
• Transformation of Streptomyces lividans with the three-plasmid system.
• Generation of modified plasmids with defined mutations for combinatorial library production.

Main Results:

• Successful heterologous expression of DEBS using the three-plasmid system in Streptomyces lividans.
• Production of 6-deoxyerythronolide B at levels comparable to single-plasmid expression systems.
• Generation of a diverse library of 6-deoxyerythronolide B analogs through combinatorial assembly of mutated plasmids.

Conclusions:

• The developed three-plasmid system is effective for heterologous expression of DEBS and combinatorial biosynthesis of polyketides.
• This approach can be extended to other modular PKS, enabling the production of numerous novel natural products.
• The system holds significant potential for discovering new drug candidates through natural product analog generation.