Optimizations to achieve high-level expression of cytochrome P450 proteins using Escherichia coli expression systems

Zelasko, Susan; Palaria, Amrita; Das, Aditi

doi:10.1016/j.pep.2013.07.017

Cited by 79 publications

(81 citation statements)

References 79 publications

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“…All three modifications were incorporated into the previously truncated, codon optimized versions of these enzymes (26). The first modification was simply the addition of the two amino acids methionine and alanine (MA), because carrying an alanine at the second amino acid position has consistently shown benefit for P450 expression (36). The second was a hydrophilic leader sequence that had been demonstrated useful for the E. coli expression of an Arabidopsis P450 (2B1) (37).…”

Section: Resultsmentioning

confidence: 99%

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

Biggs

Lim

Sagliani

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

192

143

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Recent advances in metabolic engineering have demonstrated the potential to exploit biological chemistry for the synthesis of complex molecules. Much of the progress to date has leveraged increasingly precise genetic tools to control the transcription and translation of enzymes for superior biosynthetic pathway performance. However, applying these approaches and principles to the synthesis of more complex natural products will require a new set of tools for enabling various classes of metabolic chemistries (i.e., cyclization, oxygenation, glycosylation, and halogenation) in vivo. Of these diverse chemistries, oxygenation is one of the most challenging and pivotal for the synthesis of complex natural products. Here, using Taxol as a model system, we use nature's favored oxygenase, the cytochrome P450, to perform high-level oxygenation chemistry in Escherichia coli. An unexpected coupling of P450 expression and the expression of upstream pathway enzymes was discovered and identified as a key obstacle for functional oxidative chemistry. By optimizing P450 expression, reductase partner interactions, and N-terminal modifications, we achieved the highest reported titer of oxygenated taxanes (∼570 ± 45 mg/L) in E. coli. Altogether, this study establishes E. coli as a tractable host for P450 chemistry, highlights the potential magnitude of protein interdependency in the context of synthetic biology and metabolic engineering, and points to a promising future for the microbial synthesis of complex chemical entities.Taxol | P450 | metabolic engineering | natural products | oxygenated taxanes

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Section: Resultsmentioning

confidence: 99%

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

Biggs

Lim

Sagliani

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

192

143

View full text Add to dashboard Cite

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“…These latter efforts required optimal expression in bacteria and purification of the different CYP4B1 isoforms. It is widely recognized, inter alia for eukaryotic P450s and CPRs, that membrane-bound regions can severely reduce the yield of heterologous protein in E. coli (Purnapatre et al, 2008;Zelasko et al, 2013). Here, the idea is that the hydrophobicity of the N-terminal anchor of these enzymes and the absence of an inner membrane system impede efficient expression.…”

Section: Discussionmentioning

confidence: 99%

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Roellecke

Jäger

Gyurov

et al. 2017

Protein Engineering, Design and Selection

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Human CYP4B1, a cytochrome P450 monooxygenase predominantly expressed in the lung, inefficiently metabolizes classical CYP4B1 substrates, such as the naturally occurring furan pro-toxin 4-ipomeanol (4-IPO). Highly active animal forms of the enzyme convert 4-IPO to reactive alkylating metabolite(s) that bind(s) to cellular macromolecules. By substitution of 13 amino acids, we restored the enzymatic activity of human CYP4B1 toward 4-IPO and this modified cDNA is potentially valuable as a suicide gene for adoptive T-cell therapies. In order to find novel pro-toxins, we tested numerous furan analogs in in vitro cell culture cytotoxicity assays by expressing the wildtype rabbit and variants of human CYP4B1 in human liver-derived HepG2 cells. To evaluate the CYP4B1 substrate specificities and furan analog catalysis, we optimized the N-terminal sequence of the CYP4B1 variants by modification/truncation and established their heterologous expression in Escherichia coli (yielding 70 and 800 nmol·l −1 of recombinant human and rabbit enzyme, respectively). Finally, spectral binding affinities and oxidative metabolism of the furan analogs by the purified recombinant CYP4B1 variants were analyzed: the naturally occurring perilla ketone was found to be the tightest binder to CYP4B1, but also the analog that was most extensively metabolized by oxidative processes to numerous non-reactive reaction products.

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“…The recombinant CYP2J2 (construct D34G) was expressed and purified, as published previously (McDougle et al, 2013;Zelasko et al, 2013). Briefly, DH5a cells containing the His-tagged and N-terminally truncated CYP2J2 plasmid and pTGro7 chaperonin plasmid were cultured in 30 ml Luria Bertani media with chloroamphenicol (20 mg/ml) and ampicillin (100 mg/ml) at 37°C and 250 rpm overnight.…”

Section: Methodsmentioning

confidence: 99%

Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

McDougle

Kambalyal

Meling

et al. 2014

J Pharmacol Exp Ther

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The endocannabinoids, anandamide (AEA) and 2-arachidonoylglycerol (2-AG), are arachidonic acid (AA) derivatives that are known to regulate human cardiovascular functions. CYP2J2 is the primary cytochrome P450 in the human heart and is most well known for the metabolism of AA to the biologically active epoxyeicosatrienoic acids. In this study, we demonstrate that both 2-AG and AEA are substrates for metabolism by CYP2J2 epoxygenase in the model membrane bilayers of nanodiscs. Reactions of CYP2J2 with AEA formed four AEAepoxyeicosatrienoic acids, whereas incubations with 2-AG yielded detectable levels of only two 2-AG epoxides. Notably, 2-AG was shown to undergo enzymatic oxidative cleavage to form AA through a NADPH-dependent reaction with CYP2J2 and cytochrome P450 reductase. The formation of the predominant AEA and 2-AG epoxides was confirmed using microsomes prepared from the left myocardium of porcine and bovine heart tissues. The nuances of the ligand-protein interactions were further characterized using spectral titrations, stoppedflow small-molecule ligand egress, and molecular modeling. The experimental and theoretical data were in agreement, which showed that substitution of the AA carboxylic acid with the 2-AG ester-glycerol changes the binding interaction of these lipids within the CYP2J2 active site, leading to different product distributions. In summary, we present data for the functional metabolomics of AEA and 2-AG by a membrane-bound cardiovascular epoxygenase.

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Optimizations to achieve high-level expression of cytochrome P450 proteins using Escherichia coli expression systems

Cited by 79 publications

References 79 publications

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

Overcoming heterologous protein interdependency to optimize P450-mediated Taxol precursor synthesis in Escherichia coli

Ligand characterization of CYP4B1 isoforms modified for high-level expression inEscherichia coliand HepG2 cells

Endocannabinoids Anandamide and 2-Arachidonoylglycerol Are Substrates for Human CYP2J2 Epoxygenase

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