Engineered Pathway for Maltodextrin-to-PHB Conversion

Maltodextrin debranched by isoamylase (IA) was used as the substrate for the production of PHB. Methods for IA treatment and quantification of maltodextrin were described previously^{32 (link)}. 1 mM maltodextrin refers to 1 mM glucose equivalent of maltodextrin. Proof-of-concept experiment was carried out at 37 °C in a 4-mL reaction mixture containing 200 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.5 mM MnCl₂, 10 μg/mL ampicillin, 5 μg/mL kanamycin, 10 mM sodium phosphate (pH 7.4), 0.5 mM TPP, 0.5 mM CoA, 2 mM NADP⁺, 10 g/L (around 55.6 mM glucose equivalent) IA-debranched maltodextrin, and enzymes. PhaA was loaded at a final concentration of 1 mg/mL (0.78 mU/mL), while each of the rest of the enzymes was loaded at a final concentration of 1 U/mL. The absorbance of the reaction mixture at 600 nm (OD₆₀₀) was measured in real-time using a Cary 100 UV-Vis spectrophotometer (Agilent Technologies, USA). Unless specified, one-pot, one-step production of PHB from 100 mM maltodextrin was conducted at 37 °C in a 1-mL reaction mixture containing 200 mM Tris-HCl (pH 7.4), 10 mM MgCl₂, 0.5 mM MnCl₂, 10 μg/mL ampicillin, 5 μg/mL kanamycin, 10 mM sodium phosphate (pH 7.4), 0.5 mM TPP, 0.5 mM CoA, 2 mM NADP⁺, 100 mM IA-debranched maltodextrin, and enzymes. At different time points, a 0.1-mL aliquot was collected, and centrifuged at 8,000×g to harvest the pellet for PHB quantification.
For the complete utilization of maltodextrin, the reaction was performed in a one-pot, two-step mode. When 100 mM IA-debranched maltodextrin was used as the substrate, the first step was initiated under the abovementioned conditions at optimized enzyme concentrations predicted by Model 2 (displayed in Supplementary Table 6) and coenzyme concentrations (2 mM TPP, 0.5 mM CoA, 2 mM NADP⁺), except that the concentration of PhaC was raised to 20 U/mL. After 8 h, 4GT and PPGK were added to the reaction mixture at either 1-fold loading concentrations (0.1 U/mL 4GT, 1.0 U/mL PPGK) or 2-fold loading concentrations (0.2 U/mL 4GT, 2.0 U/mL PPGK) together with 20 mM sodium hexametaphosphate for further PHB production. For the complete utilization of 200 mM maltodextrin using a fed-batch substrate addition strategy, the initial reaction conditions were the same as those using 100 mM maltodextrin. At 4 h, another 100 mM maltodextrin and another 20 U/mL PhaC was added. At 12 h, 4GT, PPGK, and sodium hexametaphosphate were added to the system at 0.2 U/mL (approximately 0.67 mg/mL), 2 U/mL (approximately 0.02 mg/mL), and 20 mM, respectively. For the complete utilization of 200 mM maltodextrin upon a single addition of substrate at 0 h, the concentrations of substrate, MgCl₂, MnCl₂, sodium phosphate, TPP, CoA, NADP⁺, and enzymes were all two times those in the trial using 100 mM substrate. After 8 h, 40 mM sodium hexametaphosphate, 0.4 U/mL (approximately 1.33 mg/mL) 4GT, and 4.0 U/mL (approximately 0.04 mg/mL) PPGK were added to the reaction mixture for further PHB production. At different time points, samples were collected for quantification of maltodextrin and PHB. Residual maltodextrin in the sample supernatant was quantified by the total starch assay kit (Megazyme, Ireland) as instructed.