Two‐step Screening for Identification of Drug‐metabolizing Bacterial Cytochromes P450 with Diversified Selectivity

Hilberath, Thomas; Windeln, Leonie M.; Decembrino, Davide; Le‐Huu, Priska; Bilsing, Florestan L.; Urlacher, Vlada B.

doi:10.1002/cctc.201901967

Cited by 8 publications

(4 citation statements)

References 51 publications

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“…To this end, a general use of polymyxin B for P450 whole cell catalysis is difficult (White et al 2017 ). Additionally, the use of the antibiotic polymyxin B may be especially problematic for the production of pharmaceuticals with regard to antibiotic resistances and complete removing of this compound in downstream processing (Chokshi et al 2019 ; Hapala 1997 ).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

et al. 2021

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Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

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Section: Discussionmentioning

confidence: 99%

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

et al. 2021

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“…In the first step, substrate profiling with three structurally different model drugs, ritonavir, testosterone, amitriptyline, allowed us to select CYP105D and CYP107Z from Streptomyces platensis DSM 40041 that accepted all model substrates and produced human‐like drug metabolites. In the second step, activity tests with an array of 25 structurally‐related molecules and derivatives of the three model compounds revealed a correlation between structural variations in the target drugs and the enzyme chemo‐ and regioselectivity (Hilberath et al, 2020).…”

Section: Application Potential Of Microbial P450s In Drug Production ...mentioning

confidence: 99%

Microbial P450 repertoire (P450ome) and its application feasibility in pharmaceutical industry, chemical industry, and environmental protection

Wang,

Pan,

Wang

et al. 2023

Biotech & Bioengineering

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Cytochrome P450s (CYPs) are heme‐thiolated enzymes that catalyze the oxidation of C‐H bonds in a regio‐ and stereo‐selective manner. CYPs are widely present in the biological world. With the completion of more biological genome sequencing, the number and types of P450 enzymes have increased rapidly. P450 in microorganisms is easy to clone and express, rich in catalytic types, and strong in substrate adaptability, which has good application potential. Although the number of P450 enzymes found in microorganisms is huge, the function of most of the microorganism P450s has not been studied, and it contains a large number of excellent biocatalysts to be developed. This review is based on the P450 groups in microorganisms. First, it reviews the distribution of P450 groups in different microbial species, and then studies the application of microbial P450 enzymes in the pharmaceutical industry, chemical industry and environmental pollutant treatment in recent years. And focused on the application fields of P450 enzymes of different families to guide the selection of suitable P450s from the huge P450 library. In view of the current shortcomings of microbial P450 in the application process, the final solution is the most likely to assist the application of P450 enzymes in large‐scale, that is, whole cell transformation combined with engineering, fusion P450 combined with immobilization technology.

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“…While they may not serve a physiological role in xenobiotic-metabolism, many bacterial P450s have shown useful activities towards drugs and drug-like molecules, especially those from the CYP102 (Cusack et al, 2013), CYP105 (McLean et al, 2015), CYP106 (Virus et al, 2006;Schmitz et al, 2012;Lee et al, 2015;Bakkes et al, 2017;Schmitz et al, 2018), CYP107 (Schmitz et al, 2018), CYP109 (Bakkes et al, 2017), CYP116 (Klenk et al, 2017), CYP154 (Bracco et al, 2013;Bakkes et al, 2017) and CYP264 (Ringle et al, 2013) families. Recent studies made possible by genome mining have illustrated the catalytic potential present in microbial CYPomes (Agematu et al, 2006;Palmer-Brown et al, 2019;Schmitz et al, 2019;Hilberath et al, 2020;Schmitz et al, 2021).…”

Section: Microbial Systems As An Alternative For Ddd Applicationsmentioning

confidence: 99%

“…For example, a set of 213 mostly Actinomycete P450s in 12 different P450 families were expressed in E. coli and shown to generate testosterone metabolites in yields sufficient for structure elucidation by NMR (Agematu et al, 2006). Two P450s, CYP105D and CYP107Z from Streptomyces platensis, were found to metabolize a broad range of drugs to metabolites seen in humans (Hilberath et al, 2020).…”

Section: Microbial Systems As An Alternative For Ddd Applicationsmentioning

confidence: 99%

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes

Gillam

Kramlinger

2022

Drug Metab Dispos

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The study of drug metabolism is fundamental to drug discovery and development (DDD) since by mediating the clearance of most drugs, metabolic enzymes influence their bioavailability and duration of action. Biotransformation can also produce pharmacologically active or toxic products, which complicates the evaluation of the therapeutic benefit vs. liability of potential drugs, but also provides opportunities to explore the chemical space around a lead. The structures and relative abundance of metabolites are determined by the substrate and reaction specificity of biotransformation enzymes and their catalytic efficiency.Preclinical drug biotransformation studies are done to quantify in vitro intrinsic clearance to estimate likely in vivo pharmacokinetic parameters, to predict an appropriate dose, and to anticipate interindividual variability in response, including from drug-drug-interactions. Such studies need to be done rapidly and cheaply, but native enzymes, especially in microsomes or hepatocytes, do not always produce the full complement of metabolites seen in extrahepatic tissues or preclinical test species. Furthermore, yields of metabolites are usually limiting.Engineered recombinant enzymes can make DDD more comprehensive and systematic.Additionally, as renewable, sustainable and scalable resources, they can also be used for elegant chemoenzymatic, synthetic approaches to optimize or synthesize candidates as well as metabolites. Here we will explore how these new tools can be used to enhance the speed and efficiency of DDD pipelines, and provide a perspective on what will be possible in the future. The focus will be on cytochrome P450 enzymes to illustrate paradigms that can be extended in due course to other drug-metabolizing enzymes. Significance statementProtein engineering can generate enhanced versions of drug-metabolizing enzymes that are more stable, better suited to industrial conditions and have altered catalytic activities,

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Two‐step Screening for Identification of Drug‐metabolizing Bacterial Cytochromes P450 with Diversified Selectivity

Cited by 8 publications

References 51 publications

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

Evaluation of P450 monooxygenase activity in lyophilized recombinant E. coli cells compared to resting cells

Microbial P450 repertoire (P450ome) and its application feasibility in pharmaceutical industry, chemical industry, and environmental protection

Opportunities for Accelerating Drug Discovery and Development by Using Engineered Drug-Metabolizing Enzymes

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