Phycocyanobilin (PCB) is a kind of light-harvesting pigment which naturally exists in algae and plays important roles in absorbing and transferring energy. Based on its antioxidant and optical properties, PCB has been applied in food, medicine, and cosmetics. Currently, PCB is mainly extracted from Spirulina through complicated steps; thus, the biosynthesis of PCB in Escherichia coli has attracted more attention. However, due to the lower catalytic efficiency of synthetic enzymes and the deficiency of precursors and cofactors, the titer of PCB remains at a low level. Here, we report the efficient synthesis of PCB by the expression of heme oxygenase-1 from Thermosynechococcus elongatus and PCB: ferredoxin oxidoreductase (PcyA) from Synechocystis sp. using a high-copy number plasmid with an inducible T7lac promoter and the assembly of these two enzymes at a suitable ratio of 2:1 with DNA scaffolds. Additionally, the synthesis of PCB was further enhanced by direct supplementation of 5-aminolevulinic acid (ALA), moderate overexpression of key enzymes in the heme biosynthetic pathway (hemB and hemH), and accelerated cycle of cofactors (NADPH) through the expression of NAD+ kinase and the addition of a reducing agent. Finally, based on the optimal conditions (Modified R medium with 200 mg/L ALA, 20 mg/L FeSO4·7H2O, and 5 g/L vitamin C induced by 0.8 mM isopropylthio-β-galactoside at 30 °C), the highest reported titer of PCB (28.32 mg/L) was obtained at the fermenter level by feeding glucose and FeSO4·7H2O. The strategies applied in this study will be useful for the synthesis of other natural pigments and PCB or heme derivatives in E. coli.
Keywords: 5-aminolevulinic acid; DNA scaffold; Escherichia coli; cofactor; fed batch; heme; phycocyanobilin; reducing agent.