Improved production of heme using metabolically engineered Escherichia coli

Abstract

Heme has recently attracted much attention due to its promising applications in the food and healthcare industries. However, the current titers and productivities of heme produced by recombinant microorganisms are not high enough for a wide range of applications. In this study, the process for the fermentation of the metabolically engineered Escherichia coli HAEM7 strain was optimized for the high‐level production of heme. To improve the production of heme, different carbon sources, iron concentration in the medium, pH control strategies, induction points, and iron content in the feeding solution were examined. Moreover, strategies of increasing cell density, regular iron supplementation, and supply of excess feeding solution were developed to further improve the production of heme. In the optimized fermentation process, the HAEM7 strain produced 1.03 g/L heme with productivity of 21.5 mg/L/h. The fermentation process and strategies reported here will expedite establishing industry‐level production of heme.

Production of zinc protoporphyrin IX by metabolically engineered Escherichia coli

“…Culture media used in this study were prepared as described in the accompanying paper (Choi et al, 2022). Briefly, lysogeny broth (LB; 10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl) supplemented with 10 g/L glucose was used for initial seed culture of the HAEM7 strain.…”

“…In addition, Escherichia coli genes responsible for the C4 pathway for ALA biosynthesis, the canonical heme biosynthesis pathway, and the heme transporter were overexpressed by introducing four plasmids pCDF-hemAL, pRSF-hemBCD, pET-hemEFGH, and pACYC-ccmABC (Supporting Information: Table S1). Next, the fermentation process of the HAEM7 strain was optimized in the accompanying paper (Choi et al, 2022). In short, MR-Fe50 and R-Fe100 media containing glycerol as a carbon source were used for fed-batch fermentation of the HAEM7 strain.…”

“…Supplementation of iron solution and excess supply of Feed2 were also exploited in the study. As a result, fed-batch culture of the HAEM7 strain produced 1.03 g/L heme in 48 h with a productivity of 21.5 mg/L/h (Choi et al, 2022).…”

“…In addition, the pH control mode 2 (i.e., initiation of pH control at 9.5-10 h when the decrease of pH value temporarily paused; Supporting Information: Figure S2) and IPTG induction at early stationary phase (i.e., OD 600 = 70-80), which are the conditions optimized in the accompanying paper (Choi et al, 2022) for heme production using R-Fe100 medium were applied together. For detailed information on the use of pH control mode 2 rather than the pH F I G U R E 2 Effects of different iron concentrations in the modified MR medium and zinc concentrations in the Feed2 during the fed-batch fermentation of the HAEM7 for ZnPPIX production.…”

“…The Feed2 containing 1 g/L (a-d), 2 g/L (e), or 4 g/L (f) of ZnSO 4 •7H 2 O was supplied after induction. control mode B, readers are encouraged to refer to section 3.8 of the accompanying paper (Choi et al, 2022). Unexpectedly, only 1547.2 and 1274.2 mg/L ZnPPIX were produced using the Feed2 containing 4 and 8 g/L ZnSO 4 •7H 2 O (rounds Z6 and Z7 of Table 1 and Figure 3c,d), respectively, which are similar to the ZnPPIX titers achieved using MR-Fe50 medium (1597.9 mg/L in 120 h and 1234.3 mg/L in 132 h; rounds Z4 and Z5 of Table 1).…”