Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts

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dc.contributor.authorHarrison, Susan T L
dc.date.accessioned2021-10-08T07:08:21Z
dc.date.available2021-10-08T07:08:21Z
dc.date.issued2017
dc.description.abstractBACKGROUND: 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.
dc.identifier.apacitationHarrison, S. T. L. (2017). Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts. <i>Microbial Cell Factories</i>, 16(1), 174 - 177. http://hdl.handle.net/11427/34564en_ZA
dc.identifier.chicagocitationHarrison, Susan T L "Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts." <i>Microbial Cell Factories</i> 16, 1. (2017): 174 - 177. http://hdl.handle.net/11427/34564en_ZA
dc.identifier.citationHarrison, S.T.L. 2017. Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts. <i>Microbial Cell Factories.</i> 16(1):174 - 177. http://hdl.handle.net/11427/34564en_ZA
dc.identifier.issn1475-2859
dc.identifier.ris TY - Journal Article AU - Harrison, Susan T L AB - BACKGROUND: 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. DA - 2017 DB - OpenUCT DP - University of Cape Town IS - 1 J1 - Microbial Cell Factories LK - https://open.uct.ac.za PY - 2017 SM - 1475-2859 T1 - Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts TI - Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts UR - http://hdl.handle.net/11427/34564 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/34564
dc.identifier.vancouvercitationHarrison STL. Effect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts. Microbial Cell Factories. 2017;16(1):174 - 177. http://hdl.handle.net/11427/34564.en_ZA
dc.language.isoeng
dc.publisher.departmentDepartment of Chemical Engineering
dc.publisher.facultyFaculty of Engineering and the Built Environment
dc.sourceMicrobial Cell Factories
dc.source.journalissue1
dc.source.journalvolume16
dc.source.pagination174 - 177
dc.source.urihttps://dx.doi.org/10.1186/s12934-017-0763-0
dc.subject.otherAlkane activation
dc.subject.otherOctane
dc.subject.otherCYP153A6
dc.subject.otherWhole cell biocatalysis
dc.subject.otherTransport
dc.subject.otherMembrane permeabilisation
dc.subject.otherCofactor regeneration
dc.subject.otherGlycerol dehydrogenase
dc.titleEffect of cell permeability and dehydrogenase expression on octane activation by CYP153A6-based whole cell Escherichia coli catalysts
dc.typeJournal Article
uct.type.publicationResearch
uct.type.resourceJournal Article
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