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
© Olaofe et al.; licensee BioMed Central Ltd. 2013
Received: 24 September 2012
Accepted: 17 January 2013
Published: 25 January 2013
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).
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.
This study emphasises that the overall process efficiency is primarily dependent on the interaction between the whole cell biocatalyst and bioprocess conditions.
In biocatalysis, productivity is a function of biocatalyst activity. This can be maximised by protein engineering using molecular techniques or by adopting a bioprocess-based approach. To date, most studies on CYPs as biocatalysts have focused on the use of molecular methods to manipulate and improve enzyme properties, with the process and reaction engineering given less attention. Molecular methods for enzyme engineering such as rational design, directed evolution and site directed mutagenesis have been reported to yield up to 8-fold improvement for CYP whole-cell processes while increments of over 25-fold have been achieved by considering factors such as microbial host selection, reaction and process optimisation [3, 9–15]. For the hydroxylation of alkanes using whole cell biocatalysts expressing CYP153s, significant studies have centred on the choice of host organism, vector systems and other molecular tools to improve protein expression and whole cell activity [4, 5, 8, 16–18]. Although this has led to meaningful progress in biocatalyst design, productivity in most in vivo alkane biotransformation studies fall outside the operating window described as essential for fine chemical synthesis, a minimum of 0.1 g-1 l-1 h-1. The system is often characterised by poor activity and low stabilities, resulting in short reaction time and unacceptable productivity. In a recent study to improve the recombinant expression of CYP153A6 using a pET vector, Gudiminchi et al.  reported notable improvements in biocatalyst activity and productivity. In this study, the major influence resulted from biocatalyst loading, increase in oxygen content and choice of carbon source to supply energy and reducing equivalents for biotransformation. Although the biocatalyst efficiency achieved was over 12 fold better than literature values, the factors constraining further process improvement were not well understood. From the studies, it was apparent that the overall process efficiency is dictated by the physiological state of the cell, determined by the interaction between whole cell biocatalyst, bioprocess conditions and their history. The physiology of whole cell biocatalyst during long term biotransformation is largely unknown and may also be responsible for dynamic biocatalyst activities and stabilities, decreasing predictability of the bioprocess. These physiological factors include overexpression of the cloned gene(s), supply of nutrients and growth factors through the growth medium and mass transfer, product toxicity, and cofactor regeneration [20, 21]. These environmental process conditions determine the extent to which the maximum active enzyme concentration achieved in cell cultivation is exploited in the alkane bioconversion phase. Therefore, in conjunction with strain development and metabolic engineering, it is important that parameters that constrain physiological state and subsequent process performance be considered to improve the specific biocatalyst activity and productivity. These include cell environmental factors that impact the expression level of the cloned gene, biocatalyst activity and stability.
In this study, the expression of the CYP153A6 gene product in E. coli, its activity, stability and octane biotransformation to 1-octanol were investigated as a function of induction conditions, FeCl3 concentration, temperature, glucose availability to facilitate co-factor regeneration and product toxicity. The influence of overflow metabolism and product toxicity were assessed to inform further process development.
Expression of CYP153A6 in a batch process
Effect of temperature conditions on growth rate, CYP153A6 expression and subsequent bioconversion of octane to 1-octanol in a 72 h reaction
Maximum growth rate
Induction conditions in a batch process
The results from LB complex medium did not show a major difference in CYP expression (0.17 to 0.19 μmol gDCW-1) with FeCl3 concentration in the range of 5 to 1000 μM relative to the control without FeCl3 added (0.15 μmol gDCW-1). The rate of octanol formation varied between 4 and 5 μmol (gDCW min)-1 with FeCl3 in the range of 0 to 100 μM. A further increase in iron concentration resulted in reduced biocatalyst activity (Figure 3).
Whole cell hydroxylation of n-octane
Further experiments to understand limiting factors in octane bio-oxidation were conducted using resting cells grown in LB and glucose-based chemically defined medium. The conditions that favour the maximum expression of CYP153A6 were also adopted for cell cultivation and protein expression prior to the use of biomass in bioconversion.
Temperature - rate of reaction and biocatalyst stability
Comparison of key bioprocess parameters obtained in this study with previous reports on 1-octanol production using E. coli and cytochrome P450, especially the CYP153 operon
Fujii et al., 2006
Bordeaux et al., 2011
Koch, et al., 2009
E. coli biocatalyst
CYP, Fdx& Fdr
23 ± 1
Max octanol formed
Time max octanol (h)
Max volumetric rate
(g LBRM-1 h-1)
Biocat efficiency at max octanoll
Max specific octanol formation rate per biomass
(μmol min-1 gDCW-1)
Periodmax rate (h)
0 – 24
2 – 6
0 – 2
0 – 1
0 – 24
0 – 24
0 – 4
24 – 48
Glucose fed-batch process
Product toxicity using cells grown on LB and glucose based defined medium
Cell permeabilisation and biocatalyst activity
Hydroxylation of n-octane using permeabilised cell in 60 ml amber vials
Max specific octanol formation rate per biomass
(μmol min -1 g DCW -1 )
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. The role of cultivation parameters such as temperature, induction conditions and supply of nutrients were investigated with respect to its effect on CYP153A6 expression driven by the T7 promoter in a recombinant E. coli BL21(DE3) strain carrying the pET28b-PFR1500 plasmid. Performance of the resultant whole cell biocatalyst under various biotransformation conditions was determined.
Reduced temperature usually helps to control metabolic fluctuations and protein synthesis rate thereby avoiding the formation of insoluble protein aggregates typical of cells at higher growth rate . In the case of CYP153A6 expression, post-induction temperatures of 25°C and above yielded very little or no properly folded active P450. To further improve protein concentration and possibly improve gene expression, a glucose-based defined medium was investigated due to an expected increase in biomass concentration . This necessitated varying inducer concentration (IPTG), as previous investigations have suggested that inducer concentration should be determined on a basis of biomass concentration . The concentration of δ-ALA which is required for heme synthesis was varied simultaneously across the same range. Again, increased concentration of active biocatalyst does not necessarily guarantee the higher hydroxylation rate . Previous studies involving the use of E. coli as whole cell biocatalyst in alkane hydroxylation have also shown that specific activity was independent of expression levels of alkane mono-oxygenase in different E. coli hosts . These observations thus support findings from a previous study that microbial physiology may indeed be the overall limiting factor in CYP catalysed bioprocesses [20, 21]. Hence, further investigations were aimed at understanding the bioprocess limitations that contribute to cell physiology in octane bio-oxidation.
Hydroxylation of n-octane in a batch and fed-batch reaction using cells cultured on complex LB and glucose-based defined medium
The key bioprocess parameters and results presented in Table 2 shows a relatively improved process for 1-octanol production using resting whole E. coli cells expressing cytochrome P450 in this present study. The highest biocatalyst activity(16 μmol gDCW-1 min-1) in this study at 37°C presents a minimum of 4-fold increase over data from previous studies using the CYP153 protein in alkane hydrolysis. CYP153A6 is unfortunately not stable at this temperature and the activity was maintained for only 6 h. The addition of glycerol to the biotransformation mixture did not alleviate biocatalyst instability, even though it is often regarded to have a stabilising role in recombinant E. coli - CYP450 biocatalysis systems . Therefore, further experiments were conducted at 20°C to understand other factors influencing octane hydroxylation activity.
A fed-batch mode of glucose addition was adopted to ease overflow metabolism as by-products such as acetic acid reduce reaction pH and also inhibit metabolism [27, 28]. This fed-batch system led to reduced acetic acid formation (which was prevalent in the batch system at glucose concentration above 1 g gDCW-1), thus improving the coupling of glucose usage to product formation which is essential for enhanced process efficiency . In addition, this reduces the potential formation of octylacetate (condensation of octanol with acetate) which has been observed at very high glucose concentration in a batch system (Smit, personal communication). Further process intensification identified product toxicity as a limiting factor. Using cells grown on LB medium, it was observed that 1-octanol concentrations as low as 0.25 g gDCW-1 inhibited biocatalyst activity when added at the start of the reaction. However, the cells were able to tolerate increasing 1-octanol produced during the reaction up to 0.8 g gDCW-1 (Figure 6A.) It suggests the ability of the whole cells to adapt the cell membrane to the toxic nature of the increasing concentration of product secreted into the medium. Solvent toxicity is usually described using the logarithm of the partition coefficient P of a particular solvent between a standardised 1:1 mixture of octanol and water, log P OW . Low log P OW solvents (log P OW < 4) are regarded as highly toxic due to their relative water solubility and ease partitioning within the cell membrane. This causes membrane fluidity and permeability leading to leakages, disruption in energy metabolism and cell death. Octanol has a logPOW of 2.9, hence the need to alleviate its impact on the cell cytoplasmic membrane where it accumulates preferentially. Toxicity of octanol was relieved by the addition of BEHP which acted as a co-solvent and product sink. This prolonged the time that the cells remained catalytically active and increased the final octanol levels obtained with LB grown cells from 0.80 to 1.25 g gDCW-1 after 60 h. It had an even more pronounced effect on the conversions obtained with cells grown in chemically defined medium. In this case the cells without BEHP remained active for only 48 h, while the cells with BEHP added maintained activity for 120 h, so that a final octanol production of 1.45 g gDCW-1 was obtained, compared with only 0.65 g gDCW-1 in the absence of BEHP. The addition of the BEHP did not affect the reaction rate but extended the time over which the cells remained active. Notable was the lower reaction rates obtained with cell grown in chemically defined medium (2.40 μmoloctanol gDCW-1 min-1), compared with LB grown cells (4.2 μmoloctanol gDCW-1 min-1), even though the CYP153A6 expression was similar (0.20 μmol gDCW-1). This may be explained by the difference in glucose uptake rate, 0.48 and 0.30 mmol (gDCW h)-1 for LB and defined medium cells respectively. It is supported by the similar octanol yield on glucose of 0.49 ± 0.06 mmol-1 mmol-1 in both systems. Glucose metabolism is directly coupled to cofactor regeneration and cell energy which are required for effective substrate conversion . Other effects might also include the presence of essential metabolites in complex medium which may have aided the cofactor pool or improved flux of NAD(P)H to the enzyme system thus influencing the product formation rate . The physiology conferred on the cells grown in defined medium may also explain the different metabolic pattern shown during octane biotransformation. Although rate of octane hydroxylation was slower to reach its peak, the decrease was also slower, with the cells actively producing octanol up to 120 h, a 2.5-fold increase over duration achieved with LB grown cells. It is likely that this pattern shown in octane hydroxylation may be due to cell metabolism as depicted by glucose uptake rate. The cessation of octanol accumulation using the complex LB and defined medium cultured biomass is apparently due to the decrease in the pH of the reaction mixture from pH 7.2 to 5.4 ± 0.2.
Attempts to improve biocatalyst activity by pre-treating with acetone to permeabilise the cells yielded up to 2-fold increase except where LB grown cells were used at the higher temperature of 37°C. The latter could be due to toxicity arising from high metabolic rate afforded by the medium and temperature, aggravating the cell stress caused by the compromised cell membrane and structure. It has been shown that cells with higher metabolism are more prone to chemical inactivation . This result suggests an apparent limitation of octane mass transfer across the cell membrane. A similar effect of uptake limitation has been reported for biotransformation of octane  and n-dodecane  using E. coli expressing alkane monooxygenase. Octane hydroxylation rate was improved by 4 fold through cloning an additional protein, AlkL, in the outer membrane of the E. coli cells to facilitate efficient uptake of the substrate . The authors reported a better result using this system over cell permeabilisation via chemical agents. This shows that further strain design may be necessary for enhanced activity.
This study provides the first detailed basis for process intensification involving the use of CYP153A6 for the hydroxylation of linear alkanes which is regarded as the physiological substrate of CYP153s. The maximum rate of 1-octanol formation of 16 μmol gDCW-1 min-1 in this system compares favourably with studies using alkane hydroxylase (AlkB) expressed in E. coli. With the latter, the product is usually subjected to further product oxidation and degradation, negatively affecting downstream processing cost . Hence the CYP153A6 system is favoured. The present study shows that the success of this CYP process is not solely dependent on the concentration of the active monooxygenase, but also on other process conditions with cell physiology playing an important role. Composition of growth medium and supply of nutrients (affecting cofactor pool and regeneration), optimum process conditions (induction and temperature studies) and process engineering (fed-batch supply of glucose and in situ product removal) were necessary to enhance biocatalyst efficiency.
Blank et al.  estimated a maximum specific activity of 370 μmol gDCW-1 min-1 for resting E. coli cells in oxygenase biotransformation, based on an equimolar NADH/product stoichiometry at a glucose uptake rate of 2.4 mmol (gDCW h)-1 allowing maximal NADH yields on glucose. From this prediction, it is apparent that the maximum hydroxylation activity achieved in this study was not limited by the metabolic capacity of the whole cell biocatalyst. This illustrates the ongoing scope for process improvement through biocatalyst and bioprocess design to favour increased CYP153A6 activity and stability.
Materials and Methods
Chemicals, bacterial strains and plasmids
Chemicals and antibiotics were obtained from Sigma-Aldrich. E. coli BL21(DE3) pET28b-PFR1500 containing the CYP153A6 operon from Mycobacterium sp. HXN-1500 cloned into plasmid pET28b(+) was used as biocatalyst .
Pre-cultures, medium preparation and expression of CYP153A6
Luria Bertani (LB) broth (tryptone 10 g l-1, yeast extract 5 g l-1 and NaCl 10 g l-1) supplemented with 30 μg ml-1 kanamycin was inoculated with E. coli BL21(DE3) pET28b-PFR1500 glycerol stock (previously maintained at -60°C). The pre-inoculum (50 ml in a 500 ml shake flask) was incubated at 30°C and 160 rpm for 12 h. This was used to inoculate LB medium or defined medium adapted from Pflug et al.  containing 10 g l-1 glucose at 2% v/v (using a working volume of 200 ml in a 2 L flask). This was supplemented with 30 μg ml-1 kanamycin and incubated at 37°C with constant shaking at 160 rpm. At A578nm of 0.6 – 0.8, the medium was supplemented with, unless stated otherwise, 0.5 mM δ-aminolevulinic acid (δ-ALA), 50 μM FeCl3.6H2O and 0.5 μM isopropyl-β-d-thiogalactopyranoside (IPTG). Cultures were further incubated at 20°C, 160 rpm for a total of 24–26 h after inoculation.
Studies on CYP153A6 expression and whole cell activity
The effect of inducer concentration (0 – 1.0 mM IPTG) and concentration of heme precursors (FeCl3.6H2O and δ-ALA, 0 – 1.0 mM) on the expression of CYP153A6 was investigated. These compounds were added at A578nm of 0.6 – 0.8 and cultures were incubated at 20°C. In another set of experiments, the cultivation temperature preceding and following induction was varied from 20 to 37°C.
Whole cell biotransformation
Following protein expression, the E. coli BL21(DE3) cells were harvested through centrifugation at 7000 g and 4°C for 10 min. The cell pellet was resuspended in 200 mM sodium phosphate buffer (pH 7.2) containing glucose and 100 μg ml-1 FeSO4.7H2O. This biotransformation reaction mixture (BRM) was used for octane biotransformation under non-growing, but metabolically active conditions. One millilitre aliquots of this mixture was transferred into 40 or 60 ml sterile amber screw cap vials and 300 μl of n-octane added to each vial before capping and placing on an orbital shaker at 200 rpm and 20°C (unless otherwise stated). Vials were removed at specified time intervals for product extraction. The biotransformation reactions were stopped by adding 100 μl of 5 M HCl to each vial. The octanol, residual glucose and acetic acid concentrations were determined.
Influence of bioprocess conditions on biocatalyst efficiency
Non-growing, but metabolically active cells were used in studying the influence of various bioprocess parameters such as temperature, overflow metabolism, product toxicity and biocatalyst stability on octane biotransformation. The glucose concentration at the start of reaction was varied from 0.5 to 2.5 gglucose gDCW-1. The preferred concentration was used in another set of experiments to study the influence of reaction temperature (20 - 37°C) on product accumulation and CYP stability. For the toxicity studies, a carrier solvent into which the product was selectively drawn was added using hexadecene or bis(2-ethylhexyl) phthalate (BEHP), due to properties such as recovery efficiency, non-toxicity to E. coli cells and no interference with reaction mixture . To 1 ml of re-suspended biomass, 100 μl of the carrier solvent and 200 μl of octane was identified as the preferred ratio and added to the reaction mixture.
Effect of cell permeabilisation on whole cell biocatalyst activity
To determine the extent of mass transfer in the reaction vials, studies were carried out by pre-treating (permeabilising) the whole cell biocatalyst with acetone and toluene in the range of 5 to 20% (v/v). The permeabilising agents were added to the re-suspended cells (in 200 mM sodium phosphate buffer, pH 7.2) and vortexed in an Eppendorf tube for 2 – 3 mins. A 1 ml aliquot of the pre-treated cells was transferred into the 60 ml reaction vial containing 300 μl of octane and 40 mM glucose (final concentration). This was mixed on an orbital shaker at 200 rpm while temperature was maintained at either 23 or 37°C.
Quantification of cell growth and substrate utilisation
Biomass concentration was measured gravimetrically as cell dry weight and by absorbance at A578nm. Glucose utilisation was analysed by determining the generation of H2O2 spectrophotometrically, following reaction catalysed by glucose oxidase using Roche glucose oxidase kit.
Quantification of whole-cell CYP153A6
The functional CYP153A6 protein in whole cells was quantified using CO-difference spectra . Cell suspension samples of 1000 μl were placed in 10 ml test tubes and saturated with carbon monoxide. The samples were reduced immediately by adding sodium dithionite powder. The absorbance spectra between 400 and 500 nm of reduced samples with and without CO were recorded using a UV-Vis spectrophotometer (UNICAM Helios α). The A450-490 difference was determined and the active concentration of the CYP153A6 protein was calculated using an extinction coefficient of 91 mM-1 cm-1.
Analysis of overflow metabolism through acetic acid accumulation
The concentration of acetic acid was quantified by high pressure liquid chromatography (HPLC) using Aminex HPX-87H column (Bio-Rad). The operating temperature was 60°C. Separation was achieved using 0.01 M H2SO4 at a flow-rate of 0.6 ml min-1 and analysed by UV absorbance at 210 nm.
Analysis of octanol formation
The contents of each vial were extracted using 1000 μl of ethyl acetate containing 0.3% 1-decanol (v/v) as the internal standard. GC analysis was carried out using a Varian 3900 series gas chromatograph equipped with a flame ionisation detector and a non-polar Varian factor four column (VF–1 ms) composing of dimethylpolysiloxane, measuring 15 m × 0.25 mm. The inlet temperature was 280°C, the flow of nitrogen (carrier gas) through the column was 15 ml min-1 and the sample volume was 1 μl. The temperature program used was adapted from Gudiminchi et al. : isotherm maintained at 120°C for 5 min, temperature ramped at 20°C min-1 to 280°C, followed by a 7 min isotherm. Product concentrations were calculated using a standard curve of 1-octanol in the range 5 to 100 mM.
Financial support of the University of Cape Town, the South African National Research Foundation and the South African Department of Science and Technology through the national Centre of Excellence in Catalysis (c*change) are gratefully acknowledged.
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