Figure captions
  1. Schematic representation of the natural metabolism and the implemented Shemin pathway for 5-ALA and porphyrin biosynthesis inE. coli from glycerol. Metabolic pathways outlined: glycolysis, glycine biosynthesis, pyruvate carboxylation, and oxidative TCA cycle (in black); glyoxylate shunt in the TCA cycle (in light brown); reductive branch of TCA cycle (in blue); Shemin pathway (in green); porphyrin biosynthesis (in red). Colored proteins: mutations (in red); overexpression (in purple). Metabolite abbreviations: 5,10-MTH, 5,10-methenyltetrahydrofolic acid; 5-ALA, 5-aminolevulinic acid; 3-PG, 3-phosphoglycerate; 3-PP, 3-phosphooxypyruvate; O-P-Serine, O-phospho-L-serine; PBG, porphobilinogen; PEP, phosphoenolpyruvate. The number of carbon atoms for each metabolite is specified in orange. Protein abbreviations: AceA, isocitrate lyase; AceB, malate synthase A; AceK, isocitrate dehydrogenase kinase/phosphatase; AckA, acetate kinase; AdhE, aldehyde-alcohol dehydrogenase; FHL, formate hydrogenlyase; HemA, 5-aminolevulinate synthase; HemB, 5-aminolevulinate dehydratase; IclR, AceBAK operon repressor; IDH, isocitrate dehydrogenase; IDH-P, isocitrate dehydrogenase-phosphate; LdhA, lactate dehydrogenase A; PC, pyruvate carboxylase; PckA, phosphoenolpyruvate carboxykinase; PDH, pyruvate dehydrogenase; PFL, pyruvate formate-lyase; PK, pyruvate kinase; PPC, phosphoenolpyruvate carboxylase; Pta, phosphotransacetylase; SdhA, succinate dehydrogenase complex (subunit A).
  2. Design strategy for CRISPRi-mediated hemB repression .The three plasmids with their major genetic features, such as promoters, selection markers, key genes, are shown. The design ofhemB -targeting sequences and their associated interacting spots in the hemB gene (i.e., L1, L2, L3, and L4) and the predicted repression efficiencies (numbers in parenthesis) are shown. The resulting hemB -repressed strains, i.e., DMH-CT (control), DMH-L1, DMH-L2, DMH-L3, and DMH-L4, were characterized for quantification of the relative hemB expression using qRT-PCR. All qRT-PCR values are reported as means ± SD (n = 2).
  3. Shake-flask cultivation of hemB-repressed strains for 5-ALA accumulation. Strains compared include BW∆ldhA , DMH, DMH-CT, DMH-L1, DMH-L2, DMH-L3, and DMH-L4. Results of the 48h shake-flask cultivation in (a ) cell density (OD600), (b ) glycerol consumption, (c ) 5-ALA titer and percentage yield, and (d ) porphyrin biosynthesis (represented by the absorbance readings of the Soret peak (OD405) and Q-band (OD495) and the images of cell-free media) are shown. All values are reported as means ± SD (n = 3).
  4. Bioreactor cultivation of DMH for 5-ALA biosynthesis under different oxygenic conditions. Time profiles of cell density (OD600), glycerol consumption and metabolite production profiles, acetate and 5-APA percentage yields, and porphyrin biosynthesis (represented by the absorbance readings of the Soret peak in OD405 and Q-band in OD495 and the images of cell-free media) are shown. The percentage yields of acetate and 5-ALA are calculated based on the consumed glycerol at the end of cultivation. (I ) AL-I: microaerobic, (II ) AL-II: semi-aerobic, and (III ) AL-III: aerobic. All values are reported as means ± SD (n = 2).
  5. Bioreactor cultivation of engineered E. coli for 5-ALA biosynthesis under microaerobic (AL-I) conditions. Strains compared include DMH∆sdhA , DMH-L4, and DMH-L4∆sdhA . Time profiles of cell density (OD600), glycerol consumption and metabolite production profiles, acetate and 5-APA percentage yields, and porphyrin biosynthesis (represented by the absorbance readings of the Soret peak in OD405 and Q-band in OD495 and the images of cell-free media) are shown. The percentage yields of acetate and 5-ALA are calculated based on the consumed glycerol at the end of cultivation. (I ) DMH∆sdhA , (II ) DMH-L4, and (III ) DMH-L4∆sdhA . All values are reported as means ± SD (n = 2).
  6. Bioreactor cultivation of engineered E. coli for 5-ALA biosynthesis under aerobic (AL-III) conditions. Strains compared include DMH-L4, DMH-L4∆sdhA , DMH-L4∆iclR , DMH∆sdhAiclR , and DMH-L4∆sdhAiclR . Time profiles of cell density (OD600), glycerol consumption and metabolite production profiles, acetate and 5-APA percentage yields, and porphyrin biosynthesis (represented by the absorbance readings of the Soret peak in OD405 and Q-band in OD495 and the images of cell-free media) are shown. The percentage yields of acetate and 5-ALA are calculated based on the consumed glycerol at the end of cultivation. (I ) DMH-L4 (II ) DMH-L4∆sdhA , (III ) DMH-L4∆iclR , (IV ) DMH∆sdhAiclR , and (V ) DMH-L4∆sdhAiclR . All values are reported as means ± SD (n = 2).