Fig. 3
Generation and analysis of engineered Synechocystis sp. PCC 6803 strains with a complete native 2-keto acid pathway integration. A Schematic diagram of plasmids used to generate strains in Fig. 3B-E. P1 and P2 are integrative plasmids targeting ddh (slr1556) site of Synechocystis chromosome. P3 and P4 are integrative plasmids targeting slr0168 site. P5 is a self-replicating plasmid. B The IB titer and IB production per cell on day 10 of engineered Synechocystis strains HX88, HX89 and HX91. Strain HX88 was generated by transformation with plasmids P1, P4 and P5; strain HX89 was generated by transformation with plasmids P1, P3 and P5; strain HX91 was generated by transformation with plasmids P2, P3 and P5. C The IB and 3M1B titers on day 10 of engineered Synechocystis strains HX88, HX89 and HX91. D SDS-PAGE (top) and Western-immunoblot (bottom). L, ladder (in kDa). For SDS-PAGE, 10 μg of total soluble proteins were loaded for each strain. For Western-immunoblot, 10 μg, 20 μg, and 20 μg of total soluble proteins were loaded for each strain to detect Strep-tagged, Flag-tagged, and His-tagged proteins, respectively. Protein size: KivdS286T, 61 kDa; Sll1363, 40 kDa; Slr0452, 59 kDa; Slr1192OP, 36 kDa. E Growth profile of engineered Synechocystis strains HX88, HX89 and HX91. Results are the mean of three biological replicates, each with three technical replicates. Error bars represent standard deviation. Asterisk represents significant difference between strains HX88 and HX89, or strains HX89 and HX91 (one-way ANOVA, * p < 0.05, ** p < 0.005)