Manipulating Intracellular Oxidative Conditions to Enhance Porphyrin Production in Escherichia coli

Abstract

Being essential intermediates for the biosynthesis of heme, chlorophyll, and several other biologically critical compounds, porphyrins have wide practical applications. However, up till now, their bio-based production remains challenging. In this study, we identified potential metabolic factors limiting the biosynthesis of type-III stereoisomeric porphyrins in Escherichia coli. To alleviate this limitation, we developed bioprocessing strategies by redirecting more dissimilated carbon flux toward the HemD-enzymatic pathway to enhance the production of type-III uroporphyrin (UP-III), which is a key precursor for heme biosynthesis. Our approaches included the use of antioxidant reagents and strain engineering. Supplementation with ascorbic acid (up to 1 g/L) increased the UP-III/UP-I ratio from 0.62 to 2.57. On the other hand, overexpression of ROS-scavenging genes such as sod- and kat-genes significantly enhanced UP production in E. coli. Notably, overexpression of sodA alone led to a 72.9% increase in total porphyrin production (1.56 g/L) while improving the UP-III/UP-I ratio to 1.94. Our findings highlight the potential of both antioxidant supplementation and strain engineering to mitigate ROS-induced oxidative stress and redirect more dissimilated carbon flux toward the biosynthesis of type-III porphyrins in E. coli. This work offers an effective platform to enhance the bio-based production of porphyrins.

Keywords: Escherichia coli; antioxidant; porphyrin; reactive oxygen species; strain engineering.

Figure 1

The Shemin/C4 pathway for porphyrin biosynthesis from succinyl-CoA and glycine. 5-ALA, 5-aminolevulinic acid; CPG-I, coproporphyrinogen I; CPG-III, coproporphyrinogen III; CP-I, coproporphyrin I; CP-III, coproporphyrin III; HemA, 5-aminolevulinate synthase; HemB, porphobilinogen synthase; HemC, porphobilinogen deaminase; HemD, uroporphyrinogen III synthase; HemE, uroporphyrinogen decarboxylase; HemF, coproporphyrinogen III oxidase; HemG, protoporphyrinogen oxidase; HemH, protoporphyrin ferrochelatase; HemN, oxygen-independent coproporphyrinogen III oxidase; HMB, Hydroxymethylbilane; PBG, porphobilinogen; PP-IX, protoporphyrin IX; PPG-IX, protoporphyrinogen IX; UP-I, uroporphyrin I; UP-III, uroporphyrin III; UPG-I, uroporphyrinogen I; UPG-III, uroporphyrinogen III; YfeX, porphyrinogen peroxidase.

Figure 2

Aerobic bioreactor cultivation of DMB for UP biosynthesis with ascorbic acid supplementation. Time profiles of (a–d) cell density (OD₆₀₀), glycerol consumption, and acetate/succinate formation, (e–h) 5-ALA and PBG biosynthesis, and (i) UP biosynthesis at 144 h. All values are reported as means ± SD (n = 2).

Figure 3

Aerobic bioreactor cultivation of SOD2 and SOD3 for UP biosynthesis with ascorbic acid supplementation. Time profiles of (a–c) cell density (OD₆₀₀), glycerol consumption, and acetate/succinate formation, (d–f) 5-ALA and PBG biosynthesis, and (g) UP biosynthesis at 144 h. All values are reported as means ± SD (n = 2).

Figure 4

Aerobic bioreactor cultivation of KAT1, KAT2, KAT3, KAT4, and KAT5 for UP biosynthesis. Time profiles of (a–e) cell density (OD₆₀₀), glycerol consumption, and acetate/succinate formation, (f–j) 5-ALA and PBG biosynthesis, and (k) UP biosynthesis at 144 h. All values are reported as means ± SD (n = 2).