High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase

Bordeaux, Mélanie; Girval, Diane de; Rullaud, Robin; Subileau, Maeva; Dubreucq, Éric; Drone, Jullien

doi:10.1007/s00253-014-5671-1

Cited by 7 publications

(7 citation statements)

References 45 publications

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“…Compared with CYP153A33 (50 nmol/g cdw ), the expression level of CYP153A13 was only 11 nmol/g cdw (Table S4). Similarly, the expression level of selfsufficient CYP153A13-Red was only 10 % as that of CYP102A1 (i.e., BM3) in E. coli BL21 (DE3), which is well known for its high soluble expression (Bordeaux et al 2014), indicating that in solublization and functional expression of CYP153A13, it would be critical to achieve high product yields, becoming a big challenge for application in industry. Although the CYP153A35 from G. alkanivorans was not yet characterized as ω-hydroxylase, G. alkanivorans strain was identified as indigenous diesel-degrading strain (Young et al 2005), and three alkanal monooxygenase genes related to hydrocarbon degradation were suggested by genome sequencing (Wang et al 2014), suggesting that CYP153A35 is very likely to show ω-hydroxylation activity toward fatty acids.…”

Section: Discussionmentioning

confidence: 98%

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

Jung

Park

Ahsan

et al. 2016

Appl Microbiol Biotechnol

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Bacterial cytochrome P450 enzymes in cytochrome P450 (CYP)153 family were recently reported as fatty acid ω-hydroxylase. Among them, CYP153As from Marinobacter aquaeolei VT8 (CYP153A33), Alcanivorax borkumensis SK2 (CYP153A13), and Gordonia alkanivorans (CYP153A35) were selected, and their specific activities and product yields of ω-hydroxy palmitic acid based on whole cell reactions toward palmitic acid were compared. Using CamAB as redox partner, CYP153A35 and CYP153A13 showed the highest product yields of ω-hydroxy palmitic acid in whole cell and in vitro reactions, respectively. Artificial self-sufficient CYP153A35-BMR was constructed by fusing it to the reductase domain of CYP102A1 (i.e., BM3) from Bacillus megaterium, and its catalytic activity was compared with CYP153A35 and CamAB systems. Unexpectedly, the system with CamAB resulted in a 1.5-fold higher yield of ω-hydroxy palmitic acid than that using A35-BMR in whole cell reactions, whereas the electron coupling efficiency of CYP153A35-BM3 reductase was 4-fold higher than that of CYP153A35 and CamAB system. Furthermore, various CamAB expression systems according to gene arrangements of the three proteins and promoter strength in their gene expression were compared in terms of product yields and productivities. Tricistronic expression of the three proteins in the order of putidaredoxin (CamB), CYP153A35, and putidaredoxin reductase (CamA), i.e., A35-AB2, showed the highest product yield from 5 mM palmitic acid for 9 h in batch reaction owing to the concentration of CamB, which is the rate-limiting factor for the activity of CYP153A35. However, in fed-batch reaction, A35-AB1, which expressed the three proteins individually using three T7 promoters, resulted with the highest product yield of 17.0 mM (4.6 g/L) ω-hydroxy palmitic acid from 20 mM (5.1 g/L) palmitic acid for 30 h.

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Section: Discussionmentioning

confidence: 98%

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

Jung

Park

Ahsan

et al. 2016

Appl Microbiol Biotechnol

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“…To date, most studies have focused on the creation of self-sufficient CYP153s and WC (or permeabilized WC) biotransformations, with very few studies performed using CYP153s in vitro or as purified enzymes. Activity and stability of the self-sufficient CYP153A13RhFRed were shown to be improved through the addition of BSA and catalase [25]. Since we have observed that using CFE instead of purified enzymes led to significantly higher final product concentrations, we revisited the influence of BSA and catalase as well as CFE on the activity and stability of CYP153 biotransformations.…”

Section: Discussionmentioning

confidence: 99%

“…Purified biocatalysts were used in a 1:1:6 (CYP153A6:FdR:Fdx) molar ratio as previous investigation have indicated that Fdx concentration can significantly influence product yields [15,22]. Bovine serum albumin (BSA) and catalase have been shown to improve the stability of reactions using purified CYP153A13RhfRed [25] and were included in the reaction mixture to final concentrations of 10 mg mL −1 and 0.5 U mL −1 , respectively. Glucose dehydrogenase (GDH) was selected as cofactor regeneration system as it can accept both NAD + and NADP + with similar turnover frequencies [26,27].…”

Section: Cofactor Regeneration Using Glucose Dehydrogenasementioning

confidence: 99%

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

Kochius¹,

Marwijk²,

Ebrecht³

et al. 2018

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Some of the most promising results for bacterial alkane hydroxylation to alcohols have been obtained with the cytochrome P450 monooxygenase CYP153A6. CYP153A6 belongs to the class I CYPs and is generally expressed from an operon that also encodes the ferredoxin (Fdx) and ferredoxin reductase (FdR) which transfer electrons to CYP153A6. In this study, purified enzymes (CYP, Fdx, FdR and dehydrogenases for cofactor regeneration) were used to deconstruct the CYP153A6 system into its separate components, to investigate the factors limiting octane hydroxylation in vitro. Proteins in the cytoplasm (cell-free extract) were found to better enhance and stabilize hydroxylase activity compared to bovine serum albumin (BSA) and catalase. Optimization of the CYP:Fdx:FdR ratio also significantly improved both turnover frequencies (TFs) and total turnover numbers (TTNs) with the ratio of 1:1:60 giving the highest values of 3872 h−1 and 45,828 moloctanol molCYP−1, respectively. Choice and concentration of dehydrogenase for cofactor regeneration also significantly influenced the reaction. Glucose dehydrogenase concentrations had to be as low as possible to avoid fast acidification of the reaction medium, which in the extreme caused precipitation of the CYP and other proteins. Cofactor regeneration based on glycerol failed, likely due to accumulation of dihydroxyacetone. Scaling the reactions up from 1 mL in vials to 60 mL in shake flasks and 120 mL in bioreactors showed that mixing and shear forces will be important obstacles to overcome in preparative scale reactions.

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“…Considered over extended periods, the use of Triton X-100 has led to mixed results in the literature, with Grant et al reporting a positive effect on the oxidation of dodecane in microwells and shake flasks when Triton X-100 was present [ 19 ], while Julsing et al found that Triton X-100 was not helpful in the oxidation of fatty acid methyl esters [ 18 ]. Bordeaux et al found that Triton X-100 was moderately helpful in octane bioconversions when the organic phase included a co-solvent, but that the surfactant suppressed turnovers considerably when the organic phase consisted of pure octane [ 34 ]. Julsing et al also used pure substrate as the organic phase, while Grant et al had dimethylsulfoxide (DMSO) present in the organic phase, and found that the beneficial effects of Triton X-100 were enhanced when more DMSO was present.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2017

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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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0763-0) contains supplementary material, which is available to authorized users.

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High-cell-density cultivation of recombinant Escherichia coli, purification and characterization of a self-sufficient biosynthetic octane ω-hydroxylase

Cited by 7 publications

References 45 publications

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

Production of ω-hydroxy palmitic acid using CYP153A35 and comparison of cytochrome P450 electron transfer system in vivo

Deconstruction of the CYP153A6 Alkane Hydroxylase System: Limitations and Optimization of In Vitro Alkane Hydroxylation

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

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