Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Cornelissen, Sjef; Julsing, Mattijs K.; Schmid, Andreas; Bühler, Bruno

doi:10.1007/s10295-011-1026-4

Cited by 12 publications

(13 citation statements)

References 79 publications

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“…Active amount of Cyp in whole cells was quantified by means of CO difference spectra previously described (Cornelissen et al, 2011(Cornelissen et al, , 2012. Cells were harvested and resuspended in 100 mM potassium phosphate buffer (pH = 7.4) containing 1% (w/v) glucose to obtain an OD 450 of 15 in a volume of 0.9 mL.…”

Section: Co Difference Spectramentioning

confidence: 99%

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120

Schäfer

Karande

Bühler

2020

Front. Bioeng. Biotechnol.

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Cytochrome P450 monooxygenases (Cyps) effectively catalyze the regiospecific oxyfunctionalization of inert C-H bonds under mild conditions. Due to their cofactor dependency and instability in isolated form, oxygenases are preferably applied in living microbial cells with Pseudomonas strains constituting potent host organisms for Cyps. This study presents a holistic genetic engineering approach, considering gene dosage, transcriptional, and translational levels, to engineer an effective Cyp-based whole-cell biocatalyst, building on recombinant Pseudomonas taiwanensis VLB120 for cyclohexane hydroxylation. A lac-based regulation system turned out to be favorable in terms of orthogonality to the host regulatory network and enabled a remarkable specific whole-cell activity of 34 U g CDW −1. The evaluation of different ribosomal binding sites (RBSs) revealed that a moderate translation rate was favorable in terms of the specific activity. An increase in gene dosage did only slightly elevate the hydroxylation activity, but severely impaired growth and resulted in a large fraction of inactive Cyp. Finally, the introduction of a terminator reduced leakiness. The optimized strain P. taiwanensis VLB120 pSEVA_Cyp allowed for a hydroxylation activity of 55 U g CDW −1. Applying 5 mM cyclohexane, molar conversion and biomass-specific yields of 82.5% and 2.46 mmol cyclohexanol g biomass −1 were achieved, respectively. The strain now serves as a platform to design in vivo cascades and bioprocesses for the production of polymer building blocks such as ε-caprolactone.

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Section: Co Difference Spectramentioning

confidence: 99%

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120

Schäfer

Karande

Bühler

2020

Front. Bioeng. Biotechnol.

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“…To test this hypothesis, we utilized our established model system based on a Synechococcus PCC 7002 (henceforth Synechococcus ) strain expressing the P450 CYP1A1, which has been engineered so that its activity is light-dependent, scaling as a saturating function of irradiance ( 3 ). CYP1A1 is a well-studied mono-oxygenase that plays an important role in the biotransformation of many drugs and toxins, and can degrade the herbicide and environmental pollutant atrazine ( 27 ). Importantly, the in vivo activity of CYP1A1 can be readily evaluated using a fluorescent assay ( 28 ).…”

Section: Introductionmentioning

confidence: 99%

Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity

et al. 2018

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In this study, we exploited a modified photosynthetic electron transfer chain (PET) in the model cyanobacterium Synechococcus PCC 7002, where electrons derived from water-splitting are used to power reactions catalyzed by a heterologous cytochrome P450 (CYP1A1). A simple in vivo fluorescent assay for CYP1A1 activity was employed to determine the impact of rationally engineering of photosynthetic electron flow. This showed that knocking out a subunit of the type I NADH dehydrogenase complex (NDH-1), suggested to be involved in cyclic photosynthetic electron flow (ΔndhD2), can double the activity of CYP1A1, with a concomitant increase in the flux of electrons from photosynthesis. This also resulted in an increase in cellular adenosine triphosphate (ATP) and the ATP/nicotinamide adenine dinucleotide phosphate (NADPH) ratio, suggesting that expression of a heterologous electron sink in photosynthetic organisms can be used to modify the bioenergetic landscape of the cell. We therefore demonstrate that CYP1A1 is limited by electron supply and that photosynthesis can be re-engineered to increase heterologous P450 activity for the production of high-value bioproducts. The increase in cellular ATP achieved could be harnessed to support metabolically demanding heterologous processes. Furthermore, this experimental system could provide valuable insights into the mechanisms of photosynthesis.

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“…CYP1A1 plays a key role in the biotransformation of drugs and other chemical compounds in mammals and has been widely studied. The catalytic activity of CYP1A1 is well-defined and easily assayed, its structure has been resolved at 2.6 Å and it has been expressed in other microbial hosts, allowing comparison of expression and activity levels among species . We designed plasmid pSy21 to express CYP1A1 in Synechococcus .…”

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confidence: 99%

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

et al. 2016

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Photosynthesis is the pivotal biochemical reaction on the planet, providing energy for the 30 global ecosystem. The evolution of oxygenic photosynthesis 2.7-3.2 billion years ago led to 31 the oxygenation of the planet. 1 This subsequently hampered the efficiency of 32 photosynthesis as oxygen competed for binding to the active site of the carbon-fixing 33 catalyst ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco). As a result, the 34 potential of photosynthesis is not achieved as the capacity for light capture and electron 35 transport is often greater than the capacity for carbon-fixation. Photosynthetic cells therefore 36 lose excess energy as heat and fluorescence, or through a number of alternative electron 37 dissipation pathways. 2 Improving photosynthetic efficiency is central to efforts to increase 38 food and/or biofuel yield, and also to realize the biotechnological potential of photosynthetic 39 species. 3 These efforts typically focus on modification of the pigments and proteins involved 40 in light capture, improving the efficiency/specificity of Rubisco or metabolic engineering of 41 product formation downstream of carbon fixation. 4 However, rewiring photosynthesis such 42 that excess reducing potential from light capture is diverted to catalyse the formation of high 43 value products has received little attention. 5 Such a strategy can, in theory, increase the 44 overall efficiency of photosynthetic electron usage by enabling the utilisation of electrons that 45 would otherwise be wasted. 46To increase overall photosynthetic efficiency, we installed the P450 CYP1A1 from 47Rattus norvegicus (brown rat) into Synechococcus PCC 7002 (henceforth Synechococcus) 48 as a new electron-sink in the photosynthetic electron transport chain (Figure 1a). 49Cytochrome P450s are a large and diverse class of monooxygenases that split molecular 50 oxygen (O2), inserting one atom into the substrate and reducing the other to water (Figure 51 1a). CYP1A1 plays a key role in the biotransformation of drugs and other chemical 52 compounds in mammals and has been widely studied. The catalytic activity of CYP1A1 is 53 well-defined and easily assayed, 6 its structure has been resolved at 2.6 Å 7 and it has been 54 expressed in other microbial hosts, allowing comparison of expression and activity levels 55 among species. 8 We designed plasmid pSy21 to express CYP1A1 in Synechococcus. The 56 expression cassette consisted of the cyp1a1 gene, a constitutive phycocyanin promoter from 57 4 Synechocystis PCC 6803, a kanamycin resistance cassette, the rrnB terminator from 58Escherichia coli, and targeting flanks to guide integration to the glpK genomic neutral site 59 (Figure 1b). The cyp1a1 gene was codon-optimized for expression in Synechococcus and 60 the FLAG peptide sequence was added in-frame, along with a 4 x glycine-alanine peptide 61 linker, to the 3' of cyp1a1 to simplify detection, quantification and purification; no other 62 modifications were made to the cyp1a1 sequence. This multipart construct was assemb...

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Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Cited by 12 publications

References 79 publications

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120

Maximizing Biocatalytic Cyclohexane Hydroxylation by Modulating Cytochrome P450 Monooxygenase Expression in P. taiwanensis VLB120

Rational engineering of photosynthetic electron flux enhances light-powered cytochrome P450 activity

Tapping the Unused Potential of Photosynthesis with a Heterologous Electron Sink

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