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

The single crossover occurring via homologous recombination is a common phenomenon existing among microbes like Escherichia coli, Bacillus subtilis, Vibrio natriegens, Gluconobacter oxydans and most cyanobacteria species, threatening the stability of engineered strains and challenging iterative genetic engineering. Among them, we take the fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) as a representative study due to its promising roles for CO₂ fixation and bioconversion. We established three marker-free platforms to achieve stable genome recombination: (i) T4CROSS, which employs two plasmids and four rounds of single crossover; (ii) TRIPLEARM, which uses a single plasmid containing three homologous arms for three rounds of single crossover; and (iii) CRISPRARM, which integrates CRISPR/Cpf1-mediated genome editing with homologous recombination. As proof of concept, we employed the CRISPRARM platform for a three-step sequential engineering of the sucrose biosynthetic pathway. The final engineered strain produced 7.12 g L^-1 of sucrose within 4 days.