The influence of microbial physiology on biocatalyst activity and efficiency in the terminal hydroxylation of n-octane using Escherichia coli expressing the alkane hydroxylase, CYP153A6.
BACKGROUND
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 (0.20 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.


CONCLUSIONS
This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.
Centre for Bioprocess Engineering Research (CeBER), Department of Chemical Engineering, University of Cape Town, Private Bag X3, Rondebosch 7701, Cape Town, South Africa.
Oluwafemi A
OA
Olaofe
Caryn J
CJ
Fenner
Rama Krishna
RK
Gudiminchi
Martha S
MS
Smit
Susan T L
ST
Harrison
D061249:Batch Cell Culture Techniques; D055162:Biocatalysis; D019149:Bioreactors; D042926:Cytochrome P-450 CYP4A / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000502:physiology; D005947:Glucose / Q000378:metabolism; D006900:Hydroxylation; D009795:Octanes / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013696:Temperature
D009795:Octanes; D011994:Recombinant Proteins; D042926:Cytochrome P-450 CYP4A; D005947:Glucose; C026728:octane