Towards new biocatalytic activity of ATIM by structure based directed evolution Projects,...

Towards new biocatalytic activity of ATIM by structure based directed

evolution

Projects, Bottlenecks and Where to Go Next?

Biocatalysts - trends

Davenport, R. VOL. 4 NO. 1 March 2008 INDUSTRIAL BIOTECHNOLOGY

Biocatalysts - Bottlenecks

Metagenomics”finding Enzymes”

Protein Engineering”making enzymes”

Bioprocess Development”using enzymes”

• Finding the right markers (M)

• Vast amount of genetic data (M, PE) amount of DNA, processing many clones and sequences, library strategies

• Vast amount of gene products (M, PE, BD) purity, activity, selectivity

• Effective screening of activity (M, PE, BD)data mining, gene isolation, product isolation

• Adjustable product-gene expression, inteference-free operation (BD)

Biocatalysts - Solutions

• Miniaturization

• Parallelization

• High Throughput approaches

• Modelling

• Bioinformatics

Metagenomics”finding Enzymes”

Protein Engineering”making enzymes”

Bioprocess Development”using enzymes”

Biocatalysis at our FacilitiesWhere three key components meet...

Biocatalysts

Thermostabilityexample moleculesphosphorylases

TIM barrelsversatile platform for isomerisation

A

A

B

B

Ligands

SubstratesUsed for validation and process optimization

InhibitorsUsed to find ideal starting biomolecules for directed evolution

ProcessDevelopment

0.100 ml

10 000 mlSmall scale High Throughput is scaleable to Production

ModellingIn solico design of future experiments

Prof. Peter NeubauerDirected evolution Molecular biologyEnzymologyProf. Rik Wierenga Structural studies

Ph.D Mari YlianttilaPh.D.Markus AlahuhtaMarco Casteleijn / Mikko SalinMirja Krause/ Kathleen Szeker

Prof. Marja LajunenOrganic chemistryPh.D. Sampo Mattila NMR

Matti VaismaaNanna Alho

Prof. Peter NeubauerProcess Development

Ph.D Tomi HillukkalaJaakko SoiniJohanna Panula-PeräläNarendar Kumar Khatri


Biocatalysts



A

A

B

B

ProcessDevelopment

0.100 ml





Prof. Marja Lajunen Organic chemistryPh.D. Sampo Mattila NMR




Ligands



Biocatalysis at our FacilitiesEnzymes...

Biocatalysts



A

A

B

B

BIOCAT-HT: Production of active thermostable phosphorylases based on High Throughput strategies

Parallel transformations and expressions of phosphorylases isolated from thermophilic organisms by using a fusion-partner plasmid library.

• High quantity approach: automated, fed-batch small scalecultivations, on-line evaluation of proper folding

• Starting points

•Novel thermostable phosphorylases

•Development of High Throughput methods

45

gene cultivation product High Throughput parallel

optimization

Thermostable Phosphorylases

Simple and cheap Purification

Higher general Stability

Suitability for specific industrial

Processes

Strategies for improved Protein stability

Structure-stability Relationships

EvolutionarySignificance of

Thermophilic MO

A. Thermostabilityexample moleculesphosphorylases

Basic research Industrial application


Biocatalysts



A

A

B

B

BIOCAT: New enzymes for the chiral synthesis ofnew chemical compounds by structure based directed evolution

Structure based directed evolution towards new tailormade active enzymes

• Interdisciplinary approach: Structural biochemistry, chemical synthesis, molecular biology, enzymology.

• Starting points

•a superior structural framework

•a highly interesting chemical reaction: chiral hydroxy compounds

Wild Type Kealases

α-hydroxy keton α-hydroxy keton

R R

α-hydroxy aldehyde α-hydroxy aldehyde

Novel enzymes Kealases

Chirally pureα-hydroxy aldehydes

Xylose isomerase without cofactors

Ribose sugers for modified nucleosides

Strategies with altered Substrate specificity

Structure-function Relationships

Directed evolution Potential enzyme libraries

B. TIM barrelsversatile platform for isomerisation

Basic research Industrial application

Wild TypeDimer

4000 s-1 (!!!)

ml8b TIMml8b TIM

monoTIMmonoTIM

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

A-TIMvariants

A-TIMvariants

Wild Type

Loop 3 deletion

Dimer

4000 s-1 (!!!)

ml8b TIMml8b TIM

monoTIMmonoTIM

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

A-TIMvariants

A-TIMvariants

monoTIMMonomer

Loop 1 rigdify

5 s-1 (!)

ml8b TIMml8b TIM

monoTIMmonoTIM

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

A-TIMvariants

A-TIMvariants

Ml1 TIMMonomer

Loop 8 deletion

5 s-1 (!)

ml8b TIMml8b TIM

monoTIMmonoTIM

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

A-TIMvariants

A-TIMvariants

Ml8b TIMMonomer

Point mutationV233A

Not Active

ml8b TIMml8b TIM

monoTIMmonoTIM

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

A-TIMvariants

A-TIMvariants

ATIMMonomer

Active site = ok

Perfect startfor Directed Evolution

ml8b TIMml8b TIM

monoTIMmonoTIM

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

A-TIMvariant

s

A-TIMvariant

s


Biocatalysts



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B

• WT-TIM is very active and very well studied

• Small size: easy to crystallize, suitable for NMR, suitable for biocomputational studies

• Easily actively expressed in high amounts in E. coli

• Stable

• Monomeric protein

• No cofactors needed

Monomeric TIM is a very suitable protein for biocatalysis:

Mutant Libraries

Random mutagenesis

DNA SequenceStructural

changes

Screening NMRChemistryGrowthAutomated

A-TIMA-TIM-A178LA-TIM-S96PA-TIM-I245A

Characterization of monomeric TIMs

Binding studiesNMR/Mass Spectrometry

Chemical synthesisX-ray/docking

Start

Proof-Of-Principle studies

A-TIM-X*

*RpiA/B activity **new activity

A-TIM-Y**DirectedEvolution

Screening

NMR

Enzyme based

Chemical based

Growth based

Automated

*AraA activity

*XylA activity

Active enzymesActive enzymes


Biocatalysis at our FacilitiesDirected evolution...

Biocatalysts



A

A

B

B

Lead Enzyme

ATIM

Improved

Variants

Mutagenesis

A) fully random

B) targeted random

Screening

in vivo

Mutant Libraries

Random mutagenesis

DNA SequenceStructural

changes

Screening NMRChemistryGrowthAutomated

Rational Design:

Site-directed mutagenesis creates four starting points

for the directed evolution approach

Starting points (4)

ATIM (A)

ATIM-S96P (ASP)

ATIM-A178L (AAL)

ATIM-I245A (AIA)

The libraries – selection of good targets

A178L

I245A

S96PLead

enzyme

ATIM

4 Starting points

- ATIM (A)

- ATIM-S96P (ASP)

- ATIM-A178L (AAL)

- ATIM-I245A (AIA)

Loop 6

Loop 8

Loop 4

error prone PCR:

GeneMorph II Random Mutagenesis Kit

ATIM

MutagenesisI) fully random

MutagenesisII) targeted random

Megaprimer PCR(WuWu et al. 2005)

Rational Design:

Megaprimer PCR creates different libraries

of ATIM mutants

Regions (3)

W100 (W)

V214/N215 (VN)

A233/G234/K239/E241

(AGKE)

V214/ N215

A233/G234/

K239/E241

W100Mutagenesis

II) targeted random

The libraries – selection of good targets

Targeted mutagenesis(megaprimer

method )3 Regions

- W100 (W)

- V214/N215 (VN)

- A233/G234/K239/E241 (AGKE)

Loop 7

Loop 8

Loop 4

Fully randomizedmutagenesis

Targeted mutagenesis

(megaprimer method )

Starting points (4)

- ATIM (A)

- ATIM-S96P (ASP)

- ATIM-A178L (AAL)

- ATIM-I245A (AIA)

Regions (3)

- W100 (W)

- V214/N215 (VN)

- A233/G234/K239/E241 (AGKE)

Error rate 0.3-0.6 %

amino acid change

(Fu)

Results

Libraries (16)

- A (Fu,W,VN,AGKE)

- ASP (Fu,W,VN,AGKE)

- AAL(Fu,W,VN,AGKE)

- AIA (Fu,W,VN,AGKE)

16 libraries of A-TIM variants

The libraries – creating the experimental space

• All methods are verified and introduced mutations into the A-TIM sequence.

• Screening based on Growth of Knock-out strains on selective media is ongoing.

• Screening methods for High Throughput approaches are under development

A-TIM-X*

*RpiA/B activity*AraA activity

*XylA activity


Knock-out strains

Fully randomizedmutagenesis

Targeted mutagenesis

(megaprimer method )

Starting points (4)

- ATIM (A)

- ATIM-S96P (ASP)

- ATIM-A178L (AAL)

- ATIM-I245A (AIA)

Regions (3)

- W100 (W)

- V214/N215 (VN)

- A233/G234/K239/E241 (AGKE)

Error rate 0.3-0.6 %

amino acid change

(Fu)

Results

Libraries (16)

- A (Fu,W,VN,AGKE)

- ASP (Fu,W,VN,AGKE)

- AAL(Fu,W,VN,AGKE)

- AIA (Fu,W,VN,AGKE)

16 libraries of A-TIM variants

The libraries – creating the experimental space

93750 to 9.4x1010 days of screening required

Every screening 4 plates à

2000 colonies = 8000

4 screenings every day

3x 109 to

3x1015

Pool III

epPCR

525 days of screening required


2000 colonies = 8000

4 screenings every day

46

412

Pool II

V214/N215

A233/G234…

1 screening required


2000 colonies = 800043

Pool I

W100

Biocatalysts - Solutions

• Miniaturization

• Parallelization

• High Throughput approaches

• Modelling

• Bioinformatics

Metagenomics

Protein Engineering

Bioprocess Development

Biocatalysis at our FacilitiesThe right Tools for the Right Methods...

Tools

High Throughput* Hamilton pipetting station

Parallelization* Small scale cultivation technology (EnBase)* Parallel cloning library

Miniaturization * Cultivations* Parallel cloning library

Methods

High Throughput transformation

High Throughput optimization of protein expression

From Small Scale to Large Scale without further optimization

High Throughput production of crystals for Crystallography

A

A

B

B

Examples

Thermostabilityexpression of themophilicphosphorylases(diploma work – end 2008)

TIM barrelsParallel optimization of expression of a known active, instable monomer (project work – end 2008)

High Throughput production of monomeric TIM crystals for Crystallography(diploma work – feb 2009)

Kathleen Zseker


Biocatalysts



Metagenomics

Protein Engineering

Bioprocess Development

A A

B B

C C

Ligands



ProcessDevelopment

0.100 ml









Ligands



Biocatalysis at our FacilitiesThe chemistry of interactions...

O

OHS

R

OO

OH

O

SR

OO Cl

HO

O

O

HO

Detailed understanding

Of interactions

Matti Weismaa

Nano Alho

(NMR)


Biocatalysts



A

A

B

B

ProcessDevelopment

0.100 ml







Ligands





ProcessDevelopment

0.100 ml



Biocatalysis at our FacilitiesMore is less...

Cultivation time

Cell density

Fed batch

EnBase

Traditional

d (Vy)dt = Fi yi + Qiyg,i - F0 δy – Q0yg,0 + Vry (formula for mass balance [kg h-1]

Kathleen

Szeker

Biocatalysis at our FacilitiesPresentations...

BIOCAT: New enzymes for the chiral synthesis of new chemical compounds by structure based directed evolution

Towards new biocatalytic activity of ATIM by structure based directed evolution

Marco Casteleijn

High throughput methods for the production of thermostable enzymes

Kathleen Szeker

The design, synthesis and evaluation of new substrate candidates based on Triosephosphate

isomerase.

Matti Vaismaa

Utilization of NMR and MS techniques in biocatalysis research

Nanna Alho

Protein crystallographic characterization of the A-TIM binding properties

Mikko Salin

BIOCAT - Network summary

Analytical tools

ml8b TIMml8b TIM

monoTIMmonoTIM

Kealases

iterative directed evolution

Pool of enzymes

Random mutage-

nesis/shuffling

Selectionof best

mutants

Screen for activity

Chemical compounds

Input Output

Process development

ICM docking Technology

Applications

Wild type TIM

Wild type TIM

ml1 TIMml1 TIM

Input

Wild type studies

X-Ray Crystallograph

y

NMRMass Spec.

High Throughput

methods

BindingStudies

A-TIMvariants

A-TIMvariants

The search continues...

Methods

What was done:

error prone PCR:

GeneMorph II Random Mutagenesis Kit

A) Why? 1. Combination of two polymerases

lowers bias of single bases

2. includes a cloning kit

A) Usually done, when structure of enzyme is

not well or not at all known

Mutagenesis

A) fully random

B) targeted random

B) Why?

1.Simple primers with Wobbles (Ns)

within the targeted areas

2.one-step PCR, no interference

necessary

B) Structure of TIM is well known:

Rational DesignMegaprimer PCR:

according to Wu et al. 2005

Towards new biocatalytic activity of ATIM by structure based directed evolution Projects,...

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