Final Activity Report Summary - GALBIOPRO (Non-conventional methods for galanthamine bioproduction)
The main objective of the project was the reintegration of the project fellow to the Host Institute. This task was fulfilled through the scientific work performed:
- Development of two-phase cultivation systems for galanthamine production (incl. model studies);
- Elicitation of in vitro shoot cultures of Leucojum aestivum with exogenous elicitors;
- Up-scaling of the galanthamine biosynthetic process in different cultivation systems.
The first stage of the investigations on the development of two-phase cultivation systems for galanthamine bioproduction were the model experiment with galanthamine hyrdobromide to determine the adsorption capacity of Amberlite XAD-2, XAD-4 and XAD-7 resins. It was found the greatest adsorption capacity showed Amberlite XAD-4 with 11.53 mg galanthamine/g resin. Then the elution process was studied using ethanol, methanol, and their water mixtures as well as acidified variants of the mentioned eluents. The most suitable eluents appeared to be the acidified pure methanol and 60 % (v/v) ethanol-water mixture, as well as their acidified variants. Further the obtained dependences were verified in real system using cultural medium from the cultivation of in vitro Leucojum aestivum shoot cultures. The results showed that the Amberlite XAD-4 adsorbed all alkaloids from the L. aestivum cultural media, which further were desorbed with acidified pure methanol at 91.1 % (from total galanthamine content).
The elicitation process was studied using two exogenous elicitors: jasmonic and methyl jasmonate. The possibility to boost the yields of desired compounds was clearly outlined as the outcome of the performed experiments was the enhancement of desired compounds volumetric yields: galanthamine and norgalanthamine were enhanced with about 142 % and 236 % (compared to the non-elicited cells), respectively 7-days after elicitation with 25µM jasmonic acid, while lycorine was enhanced with about 51 % 24 hours after elicitation with 25µM methyl jasmonate. The phenolic compounds biosynthesis was also influenced after elicitation with both elicitors.
The further up-scaling of the galanthamine biosynthetic process was studied in two different cultivation systems: authors' designed column bioreactor and RITA® temporary immersion systems. The first step from the optimization of the column bioreactor internal environment was the investigation of the temperature of cultivation. It was found that the temperature strongly influences the growth of the shoot cultures as the highest biomass amounts were accumulated at temperature of 22 C (20.82 g ADB/L), while at 18 C and 26 C the accumulated dry biomasses were with 32.6 % and 14.3 % lower, respectively. The accumulated biomass was also higher compared to the control shake-flasks cultivation, which imposes the necessity of optimisation of the cultivation conditions. The production of secondary metabolites in the frame of alkaloids determined (galanthamine, norgalanthamine and lycorine) was also influenced by the cultivation temperature. The highest volumetric yields of galanthamine (1.55 mg/L), norgalanthamine (0.979 mg/L) and lycorine (7.69 mg/L) were achieved at 22 C. In the second case when the temporary immersion systems were used for galanthamine bioproduction the influence the immersion frequency was studied. It was observed that the optimal immersion frequency for highest growth of shoot cultures (2.4 g ADB/RITA) appeared to be 15 min flooding period per 480 min stand-by period. The maximum in galanthamine production appeared to be at the same immersion frequency (264.3 µg/RITA), while the peaks in norgalanthamine and lycorine production were at immersion frequencies 15 min flooding period per 360 min stand-by period and 15 min flooding period per 60 min stand-by period, respectively.
The new objectives established during the project were the development of fast HPLC method for simultaneous determination of lycorine and norgalanthamine together with galanthamine. As a conclusion the overall outcomes of the project matched the original expectations.
- Development of two-phase cultivation systems for galanthamine production (incl. model studies);
- Elicitation of in vitro shoot cultures of Leucojum aestivum with exogenous elicitors;
- Up-scaling of the galanthamine biosynthetic process in different cultivation systems.
The first stage of the investigations on the development of two-phase cultivation systems for galanthamine bioproduction were the model experiment with galanthamine hyrdobromide to determine the adsorption capacity of Amberlite XAD-2, XAD-4 and XAD-7 resins. It was found the greatest adsorption capacity showed Amberlite XAD-4 with 11.53 mg galanthamine/g resin. Then the elution process was studied using ethanol, methanol, and their water mixtures as well as acidified variants of the mentioned eluents. The most suitable eluents appeared to be the acidified pure methanol and 60 % (v/v) ethanol-water mixture, as well as their acidified variants. Further the obtained dependences were verified in real system using cultural medium from the cultivation of in vitro Leucojum aestivum shoot cultures. The results showed that the Amberlite XAD-4 adsorbed all alkaloids from the L. aestivum cultural media, which further were desorbed with acidified pure methanol at 91.1 % (from total galanthamine content).
The elicitation process was studied using two exogenous elicitors: jasmonic and methyl jasmonate. The possibility to boost the yields of desired compounds was clearly outlined as the outcome of the performed experiments was the enhancement of desired compounds volumetric yields: galanthamine and norgalanthamine were enhanced with about 142 % and 236 % (compared to the non-elicited cells), respectively 7-days after elicitation with 25µM jasmonic acid, while lycorine was enhanced with about 51 % 24 hours after elicitation with 25µM methyl jasmonate. The phenolic compounds biosynthesis was also influenced after elicitation with both elicitors.
The further up-scaling of the galanthamine biosynthetic process was studied in two different cultivation systems: authors' designed column bioreactor and RITA® temporary immersion systems. The first step from the optimization of the column bioreactor internal environment was the investigation of the temperature of cultivation. It was found that the temperature strongly influences the growth of the shoot cultures as the highest biomass amounts were accumulated at temperature of 22 C (20.82 g ADB/L), while at 18 C and 26 C the accumulated dry biomasses were with 32.6 % and 14.3 % lower, respectively. The accumulated biomass was also higher compared to the control shake-flasks cultivation, which imposes the necessity of optimisation of the cultivation conditions. The production of secondary metabolites in the frame of alkaloids determined (galanthamine, norgalanthamine and lycorine) was also influenced by the cultivation temperature. The highest volumetric yields of galanthamine (1.55 mg/L), norgalanthamine (0.979 mg/L) and lycorine (7.69 mg/L) were achieved at 22 C. In the second case when the temporary immersion systems were used for galanthamine bioproduction the influence the immersion frequency was studied. It was observed that the optimal immersion frequency for highest growth of shoot cultures (2.4 g ADB/RITA) appeared to be 15 min flooding period per 480 min stand-by period. The maximum in galanthamine production appeared to be at the same immersion frequency (264.3 µg/RITA), while the peaks in norgalanthamine and lycorine production were at immersion frequencies 15 min flooding period per 360 min stand-by period and 15 min flooding period per 60 min stand-by period, respectively.
The new objectives established during the project were the development of fast HPLC method for simultaneous determination of lycorine and norgalanthamine together with galanthamine. As a conclusion the overall outcomes of the project matched the original expectations.