Figure captions
- 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).
- 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).
- 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).
- 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).
- 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).
- 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∆sdhA ∆iclR , and DMH-L4∆sdhA ∆iclR . 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∆sdhA ∆iclR , and (V )
DMH-L4∆sdhA ∆iclR . All values are reported as means ± SD
(n = 2).