Iterative Genome Engineering Platform Enables Efficient Sucrose Biosynthesis From CO ₂ in Photosynthetic Synechococcus elongatus UTEX 2973

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 CO 2 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.