Cytochrome P450 mediated hydroxylation of ibuprofen using Pichia pastoris as biocatalyst

Rinnofner, Claudia; Kerschbaumer, Bianca; Weber, Hansjörg; Glieder, Anton; Winkler, Margit

doi:10.1016/j.bcab.2018.12.022

Cited by 9 publications

(12 citation statements)

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“…Isolated products (M1–M3) were analyzed via 1D and 2D NMR spectroscopy and were confirmed to be 2‐hydroxy‐ibuprofen (M1), 3‐hydroxy‐ibuprofen (M2) and 1‐hydroxy‐ibuprofen (M3) (Figures , S4–9). These results are similar to biooxidations of ibuprofen with CYP505X from Aspergillus fumigatus, another homologue of P450‐BM3 . In line with its activity on lauric acid, CYP505A30 preferentially oxidizes aliphatic side chains on (sub‐)terminal positions; the API screen demonstrates, however, that oxidation is not restricted to such a structural element.…”

Section: Resultssupporting

confidence: 59%

Exploring the Biocatalytic Potential of a Self‐Sufficient Cytochrome P450 from Thermothelomyces thermophila

et al. 2019

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Among nature's arsenal of oxidative enzymes, cytochrome P450s (CYPs) catalyze the most challenging reactions, the hydroxylations of non-activated CÀ H bonds. Human CYPs are studied in drug development due to their physiological role at the forefront of metabolic detoxification, but their challenging handling makes them unsuitable for application. CYPs have a great potential for biocatalysis, but often lack appropriate features such as high and soluble expression, self-sufficient internal electron transport, high stability, and an engineerable substrate scope. We have probed these characteristics for a recently described CYP that originates from the thermophilic fungus Thermothelomyces thermophila (CYP505A30), a homolog of the well-known P450-BM3 from Bacillus megaterium. CYP505A30 is a natural monooxygenase-reductase fusion, is well expressed, and moderately tolerant towards temperature and solvent exposure. Although overall comparable, we found the stability of the enzyme's domains to be inverse to P450-BM3, with a more stable reductase compared to the heme domain. After analysis of a homology model, we created mutants of the enzyme based on literature data for P450-BM3. We then probed the enzyme variants in bioconversions using a panel of active pharmaceutical ingredients, and activities were detected for a number of structurally diverse compounds. Ibuprofen was biooxidized in a preparative scale whole cell bioconversion to 1-, 2-and 3hydroxyibuprofen.

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Section: Resultssupporting

confidence: 59%

Exploring the Biocatalytic Potential of a Self‐Sufficient Cytochrome P450 from Thermothelomyces thermophila

et al. 2019

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“…Several studies have focused on the oxidation of capsaicin by P450s from mammals, fungi, and insects [15–18,30] . In humans, chemical modifications of the alkyl terminus by P450s greatly reduce the biological responses, likely due to decreased interactions between the metabolites and TRPV1 [33] .…”

Section: Discussionmentioning

confidence: 99%

“…The in vitro conversion of capsaicin by recombinant P450s (CYP1A1, 1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) can be used to investigate whether metabolites of capsaicin cause toxicity. Recently, self‐sufficient CYP505X from Aspergillus fumigatus and self‐sufficient CYP505A30 from Thermothelomyces thermophila were heterologously expressed in E. coli and found to catalyze the hydroxylation of capsaicin at the C8 position as well as the epoxidation of the double bond in the aliphatic chain [15–17] . Both ω‐1 hydroxylation and alkyl dehydrogenation have been suggested to be involved in the production of metabolites of capsaicin by insect P450s such as CYP6B6 and CYP9As [18] .…”

Section: Discussionmentioning

confidence: 99%

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Hydroxylation, Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7

Wang

Han

et al. 2021

Chemistry & Biodiversity

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Cytochrome P450 enzymes (P450s) are versatile biocatalysts, which insert a molecular oxygen into inactivated C−H bonds under mild conditions. CYP105D7 from Streptomyces avermitilis has been reported as a bacterial substrate‐promiscuous P450 which catalyzes the hydroxylation of 1‐deoxypentalenic acid, diclofenac, naringenin, compactin and steroids. In this study, CYP105D7 catalyzes hydroxylation, epoxidation and dehydrogenation of capsaicin, a pharmaceutical agent, revealing its functional diversity. The kinetic parameters of the CYP105D7 oxidation of capsaicin were determined as Km=311.60±87.30 μM and kcat=2.01±0.33 min−1. In addition, we conducted molecular docking, mutagenesis and substrate binding analysis, indicating that Arg81 plays crucial role in the capsaicin binding and catalysis. To our best knowledge, this study presents the first report to illustrate that capsaicin can be catalyzed by prokaryotic P450s.

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“…Both are not scalable for preparative synthesis on multi mg scale. Microbial P450 enzymes provide an interesting alternative but frequently do not produce the desired specific products, or undesired mixtures which are different from the product spectrum made by human P450s are obtained [ 6 , 8 – 10 ]. Adaptation by protein engineering is possible but in cases of unexpected arising needs for new drugs such as arising epidemic outbreaks, but such approach is usually too slow.…”

Section: Introductionmentioning

confidence: 99%

Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

et al. 2021

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Background Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. Results A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3′hydroxyibuprofen. Conclusions For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

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Cytochrome P450 mediated hydroxylation of ibuprofen using Pichia pastoris as biocatalyst

Cited by 9 publications

References 20 publications

Exploring the Biocatalytic Potential of a Self‐Sufficient Cytochrome P450 from Thermothelomyces thermophila

Exploring the Biocatalytic Potential of a Self‐Sufficient Cytochrome P450 from Thermothelomyces thermophila

Hydroxylation, Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7

Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9

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