One-Stop Metabolite Shop

Recombinant Enzymes

Recombinant Enzymes for Metabolite Synthesis

To access and identify phase 1 drug metabolites, Hypha have developed several key enzymes including a set of proprietary diverse microbial CYPs called PolyCYPs®, as well as human recombinant aldehyde oxidase (AOX1) and all five human isoforms of flavin monooxygenase enzymes (FMOs). Each enzyme is cloned into E.coli and validated against a panel of relevant drug substrates to demonstrate catalytic activity.

A set of 18 PolyCYPs enzymes, AOX1 and FMO3 are available in a screening kit form called PolyCYPs+,which clients can use in their own labs. The kit is fully supported through the availability of scale-up kits and the option of outsourcing the screening and preparation of larger amounts of metabolites to Hypha. We can also undertake purification and structure elucidation of metabolites.

PolyCYPs+ Kits

PolyCYPs+ kits are effective for synthesis of phase 1 metabolites. The kits contain all co-factors needed to screen a drug substrate for metabolites, as well as a control compound and formulant for poorly water-soluble test compounds.  All reagents are provided as lyophilised powders in kit form together with a 24-well reaction plate and air-permeable seal.

Once a target metabolite has been synthesised by one or more of the enzymes in the screening kit, a scale-up reaction with the best performing enzyme can be performed in order to access material for definitive identification of the metabolite and biological testing. Higher amounts can be generated at Hypha, using either a bulk enzyme extract or through fermentation of a recombinant Streptomyces clone expressing the enzyme responsible for the biotransformation.

PolyCYPs screening kit opened

About PolyCYPs

PolyCYPs are promiscuous class 1 microbial CYPs mined from talented actinomycete bacteria in Hypha’s biotransformation panel, providing a wide diversity of CYPs that oxidise a variety of drug and agrochemical compounds. The enzymes are expressed in E.coli along with the redox partners ferredoxin and ferredoxin reductase. Although 18 PolyCYP isoforms have been selected to form a screening kit, over 100 isoforms form the PolyCYPs platform, access to which is possible through outsourcing to Hypha.

Both human and other mammalian CYP-mediated metabolites can be produced. The use of multiple PolyCYPs enzymes broadens coverage of reactions, which have been proven to metabolise some low turnover drugs and produce multi-step oxidised metabolites.

meloxicam metabolites case study image

Different PolyCYPs isoforms are capable of oxidising a broad range of substrate compounds, including aliphatic/benzylic, -CH, -CH2, -CH3, –tert-butyl, and isopropyl moieties, and aromatic systems for phenols and epoxides. De-alkylation of N– and O-alkyl moieties is also possible.

Case Study

Rapid production of hydroxylated drug metabolites using PolyCYPs

A US client urgently needed at least 2 mg of a monohydroxylated metabolite (M4), originally observed in rat liver microsomes. Screening of the parent compound against 22 PolyCYPs enzymes and 23 microbes revealed the production of two main monohydroxylated products, one of which was M4.  M4 was best produced by PolyCYPs 152 and 359, and by microbial biotransformation. The other monohydroxylated metabolite was produced by a different PolyCYPs isoform, (PolyCYP 350) and a wild-type bacterial strain.

Rapid OH metabolite PC scale-up case study

Scale-up of PolyCYP 152 was selected as the quickest route for producing mg quantities of M4, prior to which a follow-up dose escalation study performed in 96-well plates showed that the substrate loading could be increased from 100 mg/L to 300 mg/L to provide higher volumetric yield of M4.

The scale-up reaction provided a higher conversion than the 11% conversion seen at the screening stage, increasing to 35% total conversion. A total of 20.1 mg of M4 was purified from the scale-up material using two rounds of HPLC purification. The metabolite was determined to be >97% pure by LC-UV-ELSD, and this was confirmed by 1H NMR. Lyophilised M4 was supplied to the client together with a Certificate of Analysis within 22 days from receipt of order, exemplifying the short timelines achievable using PolyCYPs to access CYP-derived metabolites.


Explore our library of resources comprising brochures, case studies, posters and publications about the work we do.

This poster illustrates the application of a new biocatalysis kit, PolyCYPs®, to enable scalable synthesis of CYP-derived metabolites of drugs. The PolyCYPs platform is comprised of a set of recombinant cytochrome P450 enzymes and redox partners cloned from some of the talented actinomycetes in Hypha’s biotransformation panel. Enzymes in the kit catalyze the oxidation of a wide variety of substrate types to generate multiple mammalian and microbial-derived CYP metabolites.

Access a brochure on Hypha’s PolyCYPs+ kits. The kits contain 20 enzymes effective for producing a wide range of phase 1 metabolites. As well as 18 PolyCYPs enzymes, the kit also contains human aldehyde oxidase (AOX1) and the main human hepatic flavin-containing monooxygenase (FMO3), with other human FMO isoforms also available from Hypha.

In this case study at least 2 mg of a monohydroxylated metabolite (M4), originally observed in rat liver microsomes, was required by a US pharma company. Using PolyCYPs, the target metabolite was supplied to the client together with a Certificate of Analysis within 22 days from receipt of order, exemplifying the short timelines achievable using PolyCYPs to access CYP-derived metabolites.

Our other one-stop shop solutions for accessing and characterising metabolites

Hypha’s One-Stop Metabolite Shop enables synthesis, purification and characterization of all the main types of mammalian phase 1 and 2 metabolites.

Chemical Synthesis

Late-stage chemical glucuronidation

Mammalian biotransformation

Mammalian biotransformation

Purification & structure elucidation

COAs, acquisition / interpretation of NMR data

Test tubes with dripper
251A3171-DE01-48EB-AA27-BD82BC5AC10DCreated with sketchtool.

Hypha Discovery was a huge help to our drug development timeline, when an ADME study revealed significant metabolites that were challenging to synthesize chemically. Hypha was able to rapidly reproduce the metabolites to confirm chemical structure, and then scale up to support nonclinical testing and bioanalytical method development, with far greater speed than chemical synthesis could achieve. The Hypha people were very pleasant to work with and the material they produced was of very high quality, which rounded out an overall great experience. I would recommend them without reservation.

Senior VP, Pharmaceutical Sciences

US Pharma company

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