Enhanced Production of High-Value Porphyrin Compound Heme by Metabolic Engineering Modification and Mixotrophic Cultivation of Synechocystis sp. PCC6803

Abstract

Heme, as an essential cofactor and source of iron for cells, holds great promise in various areas, e.g., food and medicine. In this study, the model cyanobacteria Synechocystis sp. PCC6803 was used as a host for heme synthesis. The heme synthesis pathway and its competitive pathway were modified to obtain an engineered cyanobacteria with high heme production, and the total heme production of Synechocystis sp. PCC6803 was further enhanced by the optimization of the culture conditions and the enhancement of mixotrophic ability. The co-expression of hemC, hemF, hemH, and the knockout of pcyA, a key gene in the heme catabolic pathway, resulted in a 3.83-fold increase in the heme production of the wild type, while the knockout of chlH, a gene encoding a Mg-chelatase subunit and the key enzyme of the chlorophyll synthesis pathway, resulted in a 7.96-fold increase in the heme production of the wild type; further increased to 2.05 mg/L, its heme production was 10.25-fold that of the wild type under optimized mixotrophic culture conditions. Synechocystis sp. PCC6803 has shown great potential as a cell factory for photosynthetic carbon sequestration for heme production. This study provides novel engineering targets and research directions for constructing microbial cell factories for efficient heme production.

Keywords: Synechocystis sp. PCC6803; cell factory; heme; metabolic transformation.

Figure 1

Heme synthesis pathway in microorganisms. 3-PG, 3-phosphoglycerate; 3-HP, 3-Hydroxypyruvate; PS, phosphatidyl serine; O-KG, α-ketoglutarate; PA, pyruvic acid; PEP, phosphoenolpyruvate; OAA, oxaloacetic acid; CA, citric acid; Glu-tRNA, glutamate-transfer RNA; GSA, glutamate-1-semialdehyde; 5-ALA, 5-aminolevulinic acid; PBG, porphobilinogen synthase; MeCh, methylocholine; UroIII, uroporphyrinogen III; CPO III, coproporphyrinogen III; CP III, coproporphyrin III; CP, coproporphyrin; PPG IX, protoporphyrinogen IX; PP IX, protoporphyrin IX; HM B, heme B; BV, biliverdin; HemB, bilirubinogen synthase; HemC, cholesterol deaminase; HemD, uroporphyrin III synthase; HemE, uroporphyrinogen decarboxylase; HemY, protoporphyrinogen oxidase; HemH, iron chelating enzyme; HemQ, fecal heme decarboxylase; HemF/HemN, fecal porphyrinogen oxidase; HemL, glutamate-1-semialdehyde 2,1-aminomutase; Ho1/Ho2, heme oxygenase; PcyA, phycocyanobilin:ferredoxin oxidoreductase; CysG, uroporphyrin-III C-methyltransferase; ChlI, magnesium chelatase subunit I; ChlH, magnesium chelatase subunit H; ChlD, magnesium chelatase subunit D. These are the siroheme-dependent (SHD) pathway, which is the most ancient but least common of the three; the coproporphyrin-dependent (CPD) pathway, which, with one known exception, is found only in Gram-positive bacteria; and the protoporphyrin-dependent (PPD) pathway, which is found in Gram-negative bacteria and all eukaryotes.

Figure 2

Heme contents of wild-type (WT) and five mutant strains of Synechocystis sp. PCC6803. Symbols “**” indicate significant difference compared with WT based on p < 0.01.

Figure 3

Growth and chemical characterization of wild-type (WT) and mutant strains of Synechocystis sp. PCC6803. (A) Growth curves. (B) Phycocyanin content. (C) Chlorophyll a content. (D) Carotenoid content. Symbols “*” and “**” indicate significant difference compared with WT based on p < 0.05 and p < 0.01, respectively.

Figure 4

Growth of wild-type and mutant strain ptsG of Synechocystis sp. PCC6803, showing (A) growth curves and (B) residual amount of glucose in the culture medium containing glucose of different concentration. For example, “G 0.5” indicates transformant medium containing 0.5 g/L glucose and “W 0.5” stands for wild-type medium containing 0.5 g/L glucose.

Figure 5

Heme content of wild-type (WT) and three transformant strains of Synechocystis sp. PCC6803. Symbols “**” indicate the significant difference compared with WT based on p < 0.01.

Figure 6

Growth and chemical characterization of wild-type (WT) and mutant strains of Synechocystis sp. PCC6803. (A) Growth curves. (B) Phycocyanin content. (C) Chlorophyll a content. (D) Carotenoid content. Symbols “**” indicate the significant difference compared with WT based on p < 0.01.

Figure 7

Expression profiles of nine heme synthesis-related genes in three mutant strains of Synechocystis sp. PCC6803, i.e., Glbn-ptsG-CFH, CFH-PcyA, and PcyA-ChIH.

Figure 8

Scanning electron micrographs of wild-type (WT) and three combined transformants of Synechocystis sp. PCC6803.

Figure 9

Heme content of wild-type (WT) and three combined transformants of Synechocystis sp. PCC6803 under optimized fermentation conditions. Symbols “**” indicate the significant difference compared with WT based on p < 0.01.

Figure 10

Growth and chemical characterization of wild-type (WT) and three mutant strains of Synechocystis sp. PCC6803. (A) Growth curves. (B) Phycocyanin content. (C) Chlorophyll a content. (D) Carotenoid content. Symbols “*” and “**” indicate the significant difference compared with WT based on p < 0.05 and p < 0.01, respectively.