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).