Iterative Genome Engineering Platform Enables Efficient Sucrose Biosynthesis From CO₂ in Photosynthetic Synechococcus...

Area of Science:

• Synthetic Biology
• Microbial Engineering
• Genetics

Background:

• Homologous recombination via single crossover is common in microbes, posing challenges for engineered strain stability.
• The cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) is a promising platform for CO2 fixation and bioconversion.

Purpose of the Study:

• To establish robust, marker-free platforms for stable genome recombination in cyanobacteria.
• To overcome limitations in iterative genetic engineering of microbial strains.

Main Methods:

• Development of three distinct marker-free recombination platforms: T4CROSS (two plasmids, four crossovers), TRIPLEARM (single plasmid, three crossovers), and CRISPRARM (CRISPR/Cpf1 with homologous recombination).
• Application of the CRISPRARM platform for sequential, multi-step engineering of the sucrose biosynthetic pathway.

Main Results:

• Successful establishment of three stable genome recombination platforms.
• Demonstration of CRISPRARM for efficient three-step sequential engineering.
• Engineered Syn2973 strain produced 7.12 g/L of sucrose within 4 days.

Conclusions:

• The developed platforms, particularly CRISPRARM, offer efficient solutions for marker-free genome engineering in cyanobacteria.
• This advancement facilitates the development of robust engineered strains for applications like enhanced bioconversion.