Figure 1.
Mevalonate pathway for amorpha-4,11-diene production.
The abbreviations are as follows. Erg12: mevalonate kinase, Erg8: phosphomevalonate kinase, Erg19: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: farnesyl pyrophosphate synthase, Ads: amorpha-4,11-diene synthase, Pi: phosphate, Ppi: pyrophosphate.
Table 1.
Purification and characterizations of individual pathway enzymes from bacterial culture.
Figure 2.
The Taguchi orthogonal array design results.
To examine the influence of each enzyme on amopha-4,11-diene (AD) yield, the average effects analysis was determined. The five enzymes can be classified into two main groups. A: Average values of each level of factors Erg12, Erg8 and Idi on AD yield. The group of enzymes has a positive correlation with AD yield. B: Average values of each level of factors Erg19 and IspA on AD yield. The group of enzymes has little or no effect on AD yield. C: The half-normal plot indicates the significant factors on AD yield. Factor A, B, D represent Erg12, Erg8 and Idi respectively. The abbreviations are as follows. Erg12: mevalonate kinase, Erg8: phosphomevalonate kinase, Erg19: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: farnesyl pyrophosphate synthase.
Table 2.
Taguchi L16 (45) orthogonal arraray design and results.
Figure 3.
Inhibitory effect of IspA and analysis of the precipitates.
A set of separate experiments was conducted to validate the inhibitory effect of IspA. This was attributed to the precipitation of FPP. A: Fold change in amorpha-4,11-diene (AD) yield when increasing IspA and Idi concentrations while keeping other enzymes at reference level. Fold change in AD yield was calculated by normalizing against AD yield obtained by reference enzyme levels, as indicated by the arrows. Presented data were average of triplicates and standard errors were drawn on the plot. B: UPLC-(TOF)MS analysis of the intermediates in the precipitates. Presented data were average of triplicates and standard errors were drawn on the plot. C: SDS-PAGE analysis of enzymes in the precipitates. The molecular weight of the each band present in the protein marker is indicated. The abbreviations are as follows. Erg19: diphosphomevalonate decarboxylase, Idi: isopentenyl pyrophosphate isomerase, IspA: farnesyl pyrophosphate synthase, MVA: mevalonic acid, FPP: farnesyl pyrophosphate.
Table 3.
Coded level combinations for a five-level, two factor response surface methodology with central composite design.
Figure 4.
Monovalent ions were used to increase the specific activity of amorpha-4,11-diene synthase (Ads) and hence the specific amorpha-4,11-diene (AD) yield of the multienzyme synthesis reaction. A: Titration of potassium chloride concentrations, and their effects on Ads specific activity. Presented data were average of triplicates and standard errors were drawn on the plots. Student’s t-Test with paired two samples for means was used to calculate the p-value in the statistical analysis. B: Titration of different monovalent ions concentrations and their effects on AD yield by reference enzymatic levels. Fold change in AD yield was calculated by normalizing against AD yield obtained by reaction without addition of monovalent ions, as indicated by the arrow. Presented data were average of triplicates and standard errors were drawn on the plots.
Figure 5.
Optimization of buffer pH and magnesium concentration.
Varying buffer pH and magnesium concentrations was found to be helpful to enhance the specific amorpha-4,11-diene (AD) yield. Fold change in AD yield was calculated by normalizing against AD yield obtained with buffer pH 7.4 and 10 mM Mg2+, as indicated by the arrow. Presented data were average of triplicates and standard errors were drawn on the plots.