Combinatorial CRISPR Interference Library for Enhancing 2,3-BDO Production and Elucidating Key Genes in Cyanobacteria

Area of Science:

• Metabolic Engineering
• Synthetic Biology
• Cyanobacterial Biotechnology

Background:

• Cyanobacteria offer a sustainable platform for converting carbon dioxide (CO2) into valuable chemicals like 2,3-butanediol (2,3-BDO).
• Current applications are hindered by low production titers, necessitating advanced engineering strategies.
• Optimizing metabolic pathways within cyanobacteria is crucial for efficient bio-production.

Purpose of the Study:

• To develop and implement a combinatorial CRISPR interference (CRISPRi) library strategy to enhance 2,3-butanediol (2,3-BDO) production in cyanobacteria.
• To identify and attenuate genes that negatively regulate 2,3-BDO biosynthesis.
• To achieve higher yields of 2,3-BDO for renewable chemical production.

Main Methods:

• Development of a combinatorial CRISPRi library targeting key metabolic genes in PCC7942 cyanobacteria.
• Integration of the 2,3-BDO pathway and CRISPRi library using an orthogonal CRISPR system.
• Systematic screening and statistical modeling to determine optimal gene inhibition levels for 2,3-BDO enhancement.

Main Results:

• Achieved a nearly twofold increase in 2,3-BDO production, reaching 1583.8 mg/L.
• Identified that inhibiting fructose-1,6-bisphosphatase (fbp) and pyruvate dehydrogenase (pdh) had the most significant impact.
• Demonstrated that simultaneous, differential inhibition of fbp, pdh, phosphoenolpyruvate carboxylase (ppc), and sucrose-phosphate synthase (sps) enhances 2,3-BDO synthesis without interference.

Conclusions:

• The combinatorial CRISPRi library strategy is effective for enhancing cyanobacterial 2,3-BDO production.
• Differential gene suppression offers a powerful tool for metabolic pathway optimization in cyanobacteria.
• This approach opens new avenues for combinatorial metabolic engineering in cyanobacteria for sustainable chemical manufacturing.