Abstract
BackgroundBiocatalyst 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).ResultsSpecific 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.ConclusionThis study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.
Highlights
Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics
Expression of CYP153A6 in a batch process Temperature For the growth temperature studies, a flask culture was allowed to proceed at the same temperature for both the growth and expression phases while a second flask was incubated at the test temperature for growth and a reduced temperature of 20°C after induction for enzyme expression
Expression of CYP153A6 in recombinant E. coli Studies were carried out to inform the bioprocess factors that govern the biocatalytic activity of CYP153A6 in recombinant E. coli and its effect on the accumulation of 1-octanol in the biotransformation of n-octane and manipulation of these factors to improve biocatalyst activity and efficiency
Summary
Biocatalyst improvement through molecular and recombinant means should be complemented with efficient process design to facilitate process feasibility and improve process economics. Cytochrome P450 monooxygenases (CYPs) have the ability to hydroxylate a wide range of substrates by the regio-and/or stereo-specific insertion of an oxygen atom into a carbon-hydrogen bond at physiological pressure and temperature (Figure 1) This leads to reduced byproduct and waste generation, and thereby potentially reduced environmental burden. The CYP153s have the advantage of terminal alkane hydroxylation with selectivity of over 95%, a notable advantage over biocatalysts performing similar functions including the well-studied alkane mono-oxygenase (alkB) from Pseudomonas. They catalyse the hydroxylation of lower to medium chain linear alkanes, C4-C11, with octane as preferred substrate [4,5,6,7,8]
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