Fig. 3

Generation of BC and KmR transformants and pigment analysis. a Schematic of p-BC and p-KmR DNA constructs. The first was designed for the replacement of the kmR gene in the bKT recipient strain by homologous recombination. The recombinant crtZ gene and the spectinomycin-resistance cassette (smR) were in an operon configuration, with the bKT gene as the leader gene, and under the control of the P_cpt constitutive promoter. The p-kmR construct was designed for replacing the acsA locus with the kmR gene only. b Genomic DNA PCR analysis of BC transformants and bKT recipient strain using primers bKT fw and acsA-fl3’ rv. The expected size of the PCR products was 1677 and 2181 bp in bKT and BC lines, respectively. c Genomic DNA PCR analysis of KmR lines and WT recipient strain using primers acsA-5’ fw and acsA-3’ rv. The expected sizes of the PCR products were 3767 and 2862 bp in WT and KmR lines, respectively. d Comparison of the pigmentation of WT, KmR, bKT, and BC cultures photoautotrophically grown in flasks. BC culture showed a brownish coloration, different from the blue-green WT and KmR cultures. e Absorption spectra in the visible range of pigment extracts from WT, KmR, bKT and BC lines. Spectra were normalized to the absorbance of chlorophyll a. f Absorption spectra in the visible range of pigment extracts from WT, KmR, bKT and BC lines. Spectra were normalized to the absorbance of carotenoids. Arrow indicates the shift of absorption attributed to the accumulation of Asta in the BC line. g Representative HPLC profiles of WT, bKT, and BC pigment extracts. 1, Mixoxanthophyll; 2, zeaxanthin; 3, chlorophyll a; 4, echinenone; 5, βcarotene; 6, 3S,3’S trans-astaxanthin; 7, mixoxanthophyll; 8, 3S,3’S 9-cis-astaxanthin