Browsing by Subject "Whole cell biocatalysis"
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- ItemOpen AccessEffect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts(2017) Harrison, Susan T LBACKGROUND: The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. RESULTS: Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. CONCLUSIONS: This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.
- ItemOpen AccessEffect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts(BioMed Central, 2017-09-20) White, Bronwyn E; Fenner, Caryn J; Smit, Martha S; Harrison, Susan T LBackground: The regeneration of cofactors and the supply of alkane substrate are key considerations for the biocatalytic activation of hydrocarbons by cytochrome P450s. This study focused on the biotransformation of n-octane to 1-octanol using resting Escherichia coli cells expressing the CYP153A6 operon, which includes the electron transport proteins ferredoxin and ferredoxin reductase. Glycerol dehydrogenase was co-expressed with the CYP153A6 operon to investigate the effects of boosting cofactor regeneration. In order to overcome the alkane supply bottleneck, various chemical and physical approaches to membrane permeabilisation were tested in strains with or without additional dehydrogenase expression. Results: Dehydrogenase co-expression in whole cells did not improve product formation and reduced the stability of the system at high cell densities. Chemical permeabilisation resulted in initial hydroxylation rates that were up to two times higher than the whole cell system, but severely impacted biocatalyst stability. Mechanical cell breakage led to improved enzyme stability, but additional dehydrogenase expression was necessary to improve product formation. The best-performing system (in terms of final titres) consisted of mechanically ruptured cells expressing additional dehydrogenase. This system had an initial activity of 1.67 ± 0.12 U/gDCW (32% improvement on whole cells) and attained a product concentration of 34.8 ± 1.6 mM after 24 h (22% improvement on whole cells). Furthermore, the system was able to maintain activity when biotransformation was extended to 72 h, resulting in a final product titre of 60.9 ± 1.1 mM. Conclusions: This study suggests that CYP153A6 in whole cells is limited by coupling efficiencies rather than cofactor supply. However, the most significant limitation in the current system is hydrocarbon transport, with substrate import being the main determinant of hydroxylation rates, and product export playing a key role in system stability.
- ItemOpen AccessThe influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6(BioMed Central Ltd, 2013) Olaofe, Oluwafemi; Fenner, Caryn; Gudiminchi, Rama Krishna; Smit, Martha; Harrison, SusanBACKGROUND: Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n-octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR). RESULTS: Specific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20mumol gDCW-1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature. CONCLUSION: This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.
- ItemOpen AccessThe influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6(BioMed Central, 2013) Olaofe, Oluwafemi A; Fenner, Caryn J; Gudiminchi, Rama Krishna; Smit, Martha S; Harrison, Susan T LBiocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. This study focused on understanding the bioprocess limitations to identify factors that impact the expression of the terminal hydroxylase CYP153A6 and also influence the biocatalytic transformation of n–octane to 1-octanol using resting whole cells of recombinant E. coli expressing the CYP153A6 operon which includes the ferredoxin (Fdx) and the ferredoxin reductase (FdR). Results: Specific hydroxylation activity decreased with increasing protein expression showing that the concentration of active biocatalyst is not the sole determinant of optimum process efficiency. Process physiological conditions including the medium composition, temperature, glucose metabolism and product toxicity were investigated. A fed-batch system with intermittent glucose feeding was necessary to ease overflow metabolism and improve process efficiency while the introduction of a product sink (BEHP) was required to alleviate octanol toxicity. Resting cells cultivated on complex LB and glucose-based defined medium with similar CYP level (0.20 μmol gDCW -1) showed different biocatalyst activity and efficiency in the hydroxylation of octane over a period of 120 h. This was influenced by differing glucose uptake rate which is directly coupled to cofactor regeneration and cell energy in whole cell biocatalysis. The maximum activity and biocatalyst efficiency achieved presents a significant improvement in the use of CYP153A6 for alkane activation. This biocatalyst system shows potential to improve productivity if substrate transfer limitation across the cell membrane and enzyme stability can be addressed especially at higher temperature. Conclusion: This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.