Abstract
5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. Here, we identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are also the major regulatory targets of the heme biosynthesis pathway. Interestingly, up-regulation of hemD and hemF benefited ALA accumulation whereas overexpression of hemB, hemG and hemH diminished ALA accumulation. Accordingly, by combinatorial overexpression of the hemA, hemL, hemD and hemF with different copy-number plasmids, the titer of ALA was improved to 3.25 g l−1. Furthermore, in combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX. This work has great potential to be scaled-up for microbial production of ALA and provides new important insights into the regulatory mechanism of the heme biosynthesis pathway.
Highlights
5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment
In combination with transcriptional and enzymatic analysis, we demonstrated that ALA dehydratase (HemB) encoded by hemB is feedback inhibited by the downstream intermediate protoporphyrinogen IX
The absolute production of ALA was low, distinct changes were observed with single overexpression of the above-mentioned genes (Fig. 2a)
Summary
5-Aminolevulinic acid (ALA), the committed intermediate of the heme biosynthesis pathway, shows significant promise for cancer treatment. We identified that in addition to hemA and hemL, hemB, hemD, hemF, hemG and hemH are the major regulatory targets of the heme biosynthesis pathway. Since chemical synthesis of ALA is complicated and low-yielding, studies focusing on alternative microbial production of ALA from renewable and inexpensive sources has received much attention[1]. In this regard, many native microbes including photosynthetic bacteria (Rhodobacter sphaeroides) have been isolated and randomly mutated to produce ALA2,9,10. After further overexpression of rhtA (encodes a membrane protein for threonine and homoserine exporting, E. coli) and optimization of minimal medium composition and cultivation process, the titer of ALA was improved to 4.13 g l21 in batch-fermentation with glucose as the sole carbon source. The heme biosynthesis pathway enzymes have been well studied[3,22,23], limited information on its regulation mechanism is available[3]
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