Integrative Approach to Promote Hydroxylations with Novel ... · ERA-IB-2 Final conference, Berlin, 16./17.02.2016 NA-ss NA-ss SPL SER AAU SHY SFR RST MVI CCA Annotation CCA MVI RST

Integrative Approach to Promote Hydroxylations with Novel P450 Enzymes for Industrial Processes

Project acronym: HyPerIn

Project no: EIB.12.026

Prof. Dr. Vlada B. Urlacher

ERA-IB-2 Final conference, Berlin, 16./17.02.2016

www.era-ib.net HyPerIn ERA-IB-2 Final conference, Berlin, 16./17.02.2016

Project partners

• Total project budget: 2024 k€

Dr. Inger Reidun Aukrust

Dr. Dominik Gront

Dr. Stephan Luetz

Prof. Gary Lye

Prof. Vlada Urlacher

Dr. Andreas Vogel

Prof. Steffen Rupp

Novartis


Cytochrome P450 monooxygenases

• Heme b containing oxidoreductases

• > 30 types of oxidation reactions

• Hydroxylation of non-activated C-atoms

• Accept a vast variety of substrates

Limitations for application

• Requirement of electron transfer partner(s)

• Limited access to suitable P450 biocatalysts

• Inefficient biotransformation processes

Introduction

NAD(P)H + RH + O2 + H+ R–OH + NAD(P)+ + H2O

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Introduction

• HyPerIn objectives:

To provide a platform of novel cytochrome P450 biocatalysts

To overcome bottlenecks for the application of cytochrome P450 monooxygenases

• General project approach:

Discovery of novel P450s through genome mining

Construction of designer biocatalysts with optimized P450 systems in recombinant hosts

Development of a high-throughput platform for P450 biocatalyst evaluation

Representative process examples at a lab- and a preparative scale

HyPerIn ERA-IB-2 Final conference, Berlin, 16./17.02.2016

www.era-ib.net

Technical overview


WP 6 Overall project management

WP 1

Target

compound

and strain

identification

&

Assay

development

WP 2

New enzyme

identification

through

genome

mining

WP 3

Enzyme

expression

&

Development

of whole-cell

biocatalysts

WP 4

High-

throughput

P450

bioprocess

development

&

Optimization

WP 5

Synthetic

application

&

Process

evaluation

Novartis Novartis

www.era-ib.net

Technical overview


Tamoxifen Amodiaquine Haloperidol Ritonavir

Estrogen receptor

antagonist

Histamine N-methyltransferase

inhibitor

Dopamine receptor inhibitor

HIV-1 protease inhibitor

Breast cancer Malaria Psychosis HIV infection

• Identification of target compounds and synthesis of reference standards

Novartis

www.era-ib.net

Technical overview


Valencene Grundmann’s ketone Fatty acids

Ingredient of orange peel oil

Precursor for vitamin D3 derivatives

ω-hydroxylation

Grapefruit flavor Synthesis of medicines Polymer synthesis

• Identification of target compounds and synthesis of reference standards

Novartis

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Technical overview


• Development of analytical methods

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Technical overview


Strain Number of target compound hits

No. ID VAL AM HAL TAM RIT GK

1 AAU 1 4

2 SHR 1

3 SHY 2 2

4 SPL 2 3 4 3 9

5 SER 3 5

6 TBF 3 1

7 MVI 1 3 1

8 RST 2 3 2 2

• Screening of strains with P450 activity

www.era-ib.net

Technical overview


• DNA-sequencing of strains with P450 activity

www.era-ib.net

Technical overview


RN

A-s

eq

uen

ce d

ata

in p

rogr

ess

RN

A-s

eq

uen

ce d

ata

in p

rogr

ess

SPL SER AAU SHY SFR RST MVI CCA

Annotation

CCA MVI RST

SFR SHY

AAU SER SPL

Genome sizes

108 prokaryotic P450 genes

~ 150 putative redox partners

> 300 eukaryotic P450 genes

• DNA-sequencing of strains with P450 activity • Transcriptomics analysis

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Technical overview

Internet-based platform:

open for partners

~ 53,000 P450-sequences

186 structures

annotation of sequences

structural information

description of domains

available publications

authors and their affiliations

sequential patterns


http://193.239.206.14/menu_login.php

• Establishment of a P450 database

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Technical overview


Concentration of recombinant P450s in E. coli: 20 - 400 mg per L culture

• Recombinant expression of P450s in E. coli

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Redox partners P450 gene of interest

Technical overview


YkuN/FdR Pdx/PdR

+

in vitro & in vivo +

• Identification and (co-)expression of appropriate redox partners for P450s

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Technical overview


• Identification of lead P450 candidates (selected examples)


Technical overview

Optimization of: Cell concentration Substrate concentration Co-solvents Redox partners Cofactor availability

• Designer biocatalyst with optimized P450 system expression in host systems

www.era-ib.net

Automated platform

HPLC


Technical overview

• Creation of automated high throughput methods


Technical overview

• Creation of automated high throughput methods

Thermomixer

Centrifuge

Plate reader

Liquid handling arm

Plate carrier

Disposable tips

Reagents troughs

Robotic mani-pulation arm

Sample and reagent plates

Fermentation plate

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Technical overview


LB

Inoc

1. Plate

preparation:

Addition of LB

and inoculum

2. Growth

Phase: 3h 3. Induction:

Addition of IPTG,

ALA and FeSO4

4. Expression

Phase: 18h,

sample OD

every 6h

OD readings:

Microplate reader, absorption at 595

nm

5. Media

Exchange

and Substrate

Addition

6. Biooxidation:

24h, sample OD

every 6h

Bioconversion samples:

Liquid-liquid extraction and

GC analysis (offline)

P450 content

samples: CO

assay (offline)

IPTG

Add.

Buffer

Add.

GK

• Optimization of P450 biooxidation processes at µl-scale

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Technical overview


0

0,5

1

1,5

2

2,5

3

3,5

0 5 10 15 20 25

Op

tica

l de

nsi

ty

Time (h)

Cell growth during P450 expression

PP TECAN PP SF YK TECAN YK SF

0102030405060708090

15 20 25 30 35 40 45 50 55

Co

nce

ntr

atio

n (

mg

/L)

Time (h)

25-hydroxy-GK concentration

PP SF YK SK PP TECAN YK TECAN

• Optimization of P450 biooxidation processes at µl-scale

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Summary

• Start of HyPerIn was delayed

• Extension of project duration until 03/2017

• Work packages in progress: Process scale-up and verification

Pilot scale production of a fine chemical compound

Industrial evaluation for a pharma compound


Novartis

www.era-ib.net

Project outcome


I. A collection of wild-type strains with known P450 activities

II. Genomes of 8 microbial strains with P450 activities are sequenced and annotated III. More than 450 new P450-genes identified

IV. A new P450 database created

www.era-ib.net

Project outcome


V. A library of novel P450 enzymes expressed in recombinant E. coli

VI. A set of recombinant P450-based whole-cell biocatalysts with activity toward target compounds

VII. A high-throughput platform for P450 biocatalyst evaluation at µl-scale


WP 1

Synthesized

compounds

WP 2

DNA-

Sequences

WP 6 Partner meetings 2014/02 Kick-off 2014/07 6 month 2015/02 12 month 2016/03 24 month

WP 3

Enzyme

WP 5

Strains

Enzyme

DNA-

Sequences

Enzyme

Database information

WP 4

High-

throughput

bioprocess

at µl-scale

Process conditions

General Evaluation

www.era-ib.net EIB.12.026, HyPerIn, Brussels, 29.09.2014

National funding organizations ERA-IB network


Acknowledgements

www.era-ib.net

Contact details

Prof. Dr. Vlada B. Urlacher

Heinrich-Heine University Düsseldorf

Institute of Biochemistry II

Universitätsstraße 1

40225 Düsseldorf


Documents

Integrative Approach to Promote Hydroxylations with Novel ... · ERA-IB-2 Final conference, Berlin, 16./17.02.2016 NA-ss NA-ss SPL SER AAU SHY SFR RST MVI CCA Annotation CCA MVI RST