Analysis
Culture samples were appropriately diluted with 0.15 M saline solution
for measuring cell density in OD600 using a
spectrophotometer (DU520, Beckman Coulter, Fullerton, CA). Cell-free
medium was prepared by centrifugation of the culture sample at 9,000×g
for 5 minutes, followed by filter sterilization using a 0.2 µM syringe
filter. Extracellular metabolites and glycerol were quantified using
high-performance liquid chromatography (HPLC) (LC-10AT, Shimadzu, Kyoto,
Japan) with a refractive index detector (RID; RID-10A, Shimadzu, Kyoto,
Japan) and a chromatographic column (Aminex HPX-87H, Bio-Rad
Laboratories, CA, USA). The HPLC column temperature was maintained at
35°C and the mobile phase was 5 mM H2SO4(pH 2) running at 0.6 mL min-1. The RID signal was
acquired and processed by a data processing unit (Clarity Lite,
DataApex, Prague, Czech Republic).
The 5-ALA titer in the cell-free medium was measured using a modified
Ehrlich’s reagent (Mauzerall and Granick
1956). The percentage yield of 5-ALA was defined as the mole (or mass)
ratio of the produced 5-ALA to the theoretically maximal 5-ALA produced
based on the consumed glycerol with a molar ratio of one-to-three. Note
that one-mole succinyl-CoA (derived from two-mole glycerol) and one-mole
glycine (derived from one-mole glycerol) are required to generate
one-mole 5-ALA. The bulk level of secreted porphyrin compounds in the
cell-free medium was estimated using a spectrophotometer at two specific
wavelengths, i.e., OD405 (measuring Soret band) and
OD495 (measuring Q-band).