A Versatile Marker-Free Genome Engineering Platform to Overcome Homologous Recombination Bias in Microbes: A Case Study in Synechococcus elongatus UTEX 2973

The single crossover occurring via homologous recombination is a common phenomenon exited among most microbes like Escherichia, Clostridium, Streptococcus, Lactobacillus , and cyanobacteria, threatening the stability of engineered strains. Among them, fast-growing cyanobacterium Synechococcus elongatus UTEX 2973 (Syn2973) is an attractive photosynthetic chassis for CO ₂ bioconversion. To address the challenge of homologous recombination, we constructed two endogenous plasmid-based shuttle vectors, pSES and pSEL, enabling stable DNA delivery without reliance on homologous recombination. In parallel, two robust counter-selection systems were developed based on sepT ₂ and rpsL , which, in combination with positive selection markers, significantly improved the screening efficiency of double-crossover mutants. Building upon these tools, we established three marker-free platforms: (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. All three methods successfully repaired mutations in the pilMNOQ pilus gene cluster in Syn2973, restoring natural competence with high efficiency and positive selection rates. 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 four days.