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

Microbial Cell Factories

Research

Caryn J. Fenner¹  Susan T. L. Harrison¹  Bronwyn E. White¹  Martha S. Smit² 

[1] Centre for Bioprocess Engineering Research (CeBER), Department of Chemical Engineering, University of Cape Town, Private Bag X3, Rondebosch, 7701, Cape Town, South Africa;South African DST-NRF Centre of Excellence in Catalysis, c*change, University of Cape Town, Private Bag, Rondebosch, 7701, Cape Town, South Africa;Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, Bloemfontein, South Africa;South African DST-NRF Centre of Excellence in Catalysis, c*change, University of Cape Town, Private Bag, Rondebosch, 7701, Cape Town, South Africa;

关键词: Alkane activation;    Octane;    CYP153A6;    Whole cell biocatalysis;    Transport;    Membrane permeabilisation;    Cofactor regeneration;    Glycerol dehydrogenase;   

DOI  :  10.1186/s12934-017-0763-0

 received in 2017-01-28, accepted in 2017-09-07,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundThe 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.ResultsDehydrogenase 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.ConclusionsThis 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.

【 授权许可】

CC BY   
© The Author(s) 2017