The influence of 3 hydroxypropionic acid on the gene expression level of Halomonas bluephagenesis

Halomonas bluephagenesis TD has remarkably broad potential for industrial applications. Existing technologies for targeted integration and deletion of large DNA cargoes in H. bluephagenesis TD genome are constrained by low efficiency and dependence on homologous recombination. Here, we construct a versatile CRISPR-associated transposons system in H. bluephagenesis TD. Using multi-spacer crRNA arrays, we achieve simultaneous multiplexed insertions in two and four genomic loci at nearly 100 percent and 35 percent efficiency, respectively. By combining recombinase, VchCAST mediates up to 33 percent deletion of 119 kilobases genomic DNA sequences. We achieve the insertion of large DNA cargos up to 12 kilobases with an efficiency of 30 percent . Exploiting this powerful tool, we systematically identify beneficial gene targets that can be engineered to promote 3 hydroxypropionic acid production. We identify 23 beneficial gene targets from 52 targets related to 3-HP metabolism. The multicopy genome integration of 3 hydroxypropionic acidbiosynthesis pathway is achieved by designing crRNA arrays targeting multiple genomic loci. H. bluephagenesis TD strains carrying rate-limiting enzyme PanD in its genome with successively increased copy numbers, up to 14, are obtained. The combined rational engineering increases 3 HP production from 0.26 gram per liter to 13.82 gram per liter at a yield of 0.514 gram per gram glucose via the beta alanine pathway during shake flask fermentation. This study highlights the significance of discovering hidden genetic determinants and provides valuable insights into the high yield of 3 hydroxypropionic acid in next generation microbial chassis.