Expanded synthetic small regulatory RNA expression platforms for rapid and multiplex gene expression knockdown

Area of Science:

• Synthetic biology
• Metabolic engineering
• Microbial biotechnology

Background:

• Synthetic small regulatory RNAs (sRNAs) are effective for downregulating gene expression at the translational level in metabolic engineering.
• Existing sRNA platforms face limitations in engineered strains with incompatible plasmids.

Purpose of the Study:

• To develop and demonstrate universally applicable synthetic sRNA expression platforms for rapid, multiplexed, and genome-scale gene knockdown in Escherichia coli.
• To overcome plasmid compatibility issues in engineered microbial hosts.

Main Methods:

• Development of expanded synthetic sRNA expression platforms.
• One-step co-transformation of sRNA expression vectors for multiplexed gene knockdown.
• Construction of a genome-scale sRNA library targeting 1,858 E. coli genes.
• High-throughput colorimetric screening for metabolite production.

Main Results:

• Rapid development of high-performance L-proline (54.1 g/L) and L-threonine (22.9 g/L) producing strains via combinatorial gene knockdown.
• Successful construction of crude violacein (5.19 g/L) and indigo (135 mg/L) producers using the genome-scale sRNA library.
• Demonstration of rapid chemical overproducer development irrespective of plasmid compatibility.

Conclusions:

• The expanded sRNA expression platforms provide a versatile tool for metabolic engineering.
• This technology enables efficient and rapid development of microbial cell factories for chemical production.
• The platform is applicable to genome-scale engineering and overcomes previous limitations in plasmid compatibility.