Fig 1.
Time courses of DO, cell and ethanol concentrations during cells growth phase under different strategies.
○: by standard strategy initiating induction at high cells concentration with pure oxygen aeration at 14 h and implementing the modified glycerol feeding strategy at about 22 h (A). ●: initiating induction at low cells concentration with air aeration/standard DO-Stat feeding (C).
Fig 2.
Curves of fermentation data and monellin synthesis/SDS analysis results during induction phase with different strategies.
(A)-(D): initiating methanol induction at high cell concentration; (E)-(H): initiating induction at low cell concentration. ●, ■ and □: induction at 30°C; ○, □ and ┄: at 20 °C; ▲ and —: at 30 °C but aerating pure oxygen.
Fig 3.
A simplified carbon/metabolic map and the carbon/energy distribution ratios for monellin synthesis by P. pastoris.
CY: methanol distribution ratio for cells growth and maintenance; CP: methanol distribution ratio for protein synthesis; CE: methanol distribution ratio for NADH (energy) formation. ηH: the ratio of data categorized into high energy utilization efficiency η (η>0.8). 1, 2, 3 and 6: referred to fermentation runs numbers.
Fig 4.
Methanol metabolism patterns under different induction conditions.
(A): specific cells growth rates (μ); (B): specific methanol consumption rates (v); (C): specific monellin production rates (ρ); (D): specific methanol consumption rates (v) versus specific cells growth rates (μ); (E): specific monellin production rates (ρ) versus specific cells growth rates (μ). ● and □: induction at low cells concentration and 30°C (run #3); ○ and —: at low cells concentration and 20°C (run # 6); ■ and …: at high cells concentration and 30°C (run #1); □ and ┄: at high cells concentration and 20°C (run #2).
Fig 5.
Energy (NADH) metabolism patterns □ under different induction strategies.
(A): Categories of NADH distribution η (rCNADH/rFNADH) under different induction strategies (runs #1–3, #6). █: η≥1.0; ░: 0.8<η<1.0; □: 0.0≤η≤0.8. (B): NADH distribution η (rCNADH/rFNADH) versus induction time under the two extreme induction strategies (run #1 and #3). □: run #1; ●: run #3. (C): specific rFNADH versus specific growth rate (μ) when initiating induction at low cell concentration while maintaining induction temperature at 30°C/20°C. ●: run #3, ○: run #6. (D): NADH distribution η (rCNADH/rFNADH) versus specific growth rate (μ) when initiating induction at low cell concentration while maintaining induction temperature at 30°C/20°C. ●: run #3, ○: run #6.
Fig 6.
Specific activities of AOX, FLD, and FDH in run #1 and #3.
○: run#1; ●: run#3.