Repression of hemB expression for extracellular 5-ALA accumulation
We first implemented the Shemin pathway by episomal expression ofhemA from R. sphaeroides in BW∆ldhA , deriving DMH. The effects of the implemented Shemin pathway were observed by comparing the two cultures of BW∆ldhA and DMH in screw-cap shake-flasks containing 20 g l-1 glycerol as the sole carbon source. While BW∆ldhA generated no detectable levels of 5-ALA or porphyrin, DMH produced 0.44 g l-1 5-ALA (5.22% yield) with significant porphyrin biosynthesis (Fig. 3), suggesting that the Shemin pathway was active in DMH. In particular, the dark red color of the DMH culture (Fig. 3) suggests that most glycerol was converted to porphyrin pigments with limited 5-ALA accumulation.
We then aimed to reduce the carbon flux toward porphyrin biosynthesis in DMH by repressing the expression of hemB (Fig. 1), leading to a limited conversion of two 5-ALA molecules into porphobiliongen (PBG) and extracellular 5-ALA accumulation. We applied CRISPRi by designing fourhemB -targeting gRNAs with different relative expression efficiencies, generating four corresponding strains of DMH-L1, DMH-L2, DMH-L3, and DMH-L4 (Fig. 3). Note that DMH-CT is the control strain without a hemB -specific gRNA for CRISPRi. While cell growth and glycerol consumption were somewhat affected by the presence of the triple-plasmid system, all hemB -repressed strains had significant extracellular 5-ALA accumulation compared to the control strain DMH-CT upon shake-flask cultivation. The minimal physiological impacts suggest that the hemB -repressed strains still had sufficient biosynthesis of essential porphyrins. In particular, DMH-L1 and DMH-L4 had both titers and yields up to 4-5 fold higher than the control strain DMH-CT (Fig. 3). This significant increase in 5-ALA accumulation in DMH-L1 (1.26 g l-1) and DMH-L4 (1.61 g l-1) occurred simultaneous with considerably reduced levels of the relativehemB expression at 67% and 40%, respectively (Fig. 2). Additional supporting evidence for the repressed hemB expression was reflected by the decrease in porphyrin biosynthesis in all fourhemB -repressed strains as the degrees of pigmentation and the corresponding absorbances (in 405 and 495 nm) of the cell-free media were significantly lower than that of the control strain DMH-CT (Fig. 3). Given the lowest relative hemB expression and the most superior extracellular 5-ALA accumulation in DMH-L4 among allhemB -repressed strains, we used this strain for all subsequent experiments.