dr. grzegorz kubik february 5th 2020...identify best enzyme project idea enzyme fermentation...

Dr. Grzegorz Kubik

February 5th 2020

The Information Presented here is the Property of Enzymaster (Ningbo) Bio-Engineering Co., Ltd.

Broad Spectrum of Chemical

Transformations Available

e.g.C-C-bond coupling,

RedOx chemistry

Problem:Natural enzymes are

often industrially not applicable

Shorter Synthetic Routes

No need for protective group chemistry

No need for prior substrate activation

Mild Environment Friendly Reaction

Conditions

e.g.Ambient temperature

No heavy metals

Extraordinary Selectivity

Stereo SelectivityRegio Selectivity

Chemo Selectivity

By specific substrate binding

Solution:Enzyme engineeringby directed evolution

2


Commercialization

Process Development

Enzyme Fermentation Development

Down Stream Process Development

Optimization of Process Set-up & Up-scaling

3

Screening of Available Enzyme Panels

Computational Activity Prediction

Enzyme Identification

Enzyme and Reaction Engineering

Optimizing Enzyme by Directed Evolution

Optimization of Reaction Conditions

The Information Presented here is the Property of Enzymaster (Ningbo) Bio-Engineering Co., Ltd. 4

High throughput screening

Selection of most improved

enzyme

DNA library creation

Recombinant enzyme library

expression

wt Enzyme

DNA preparation

Nobel Prize in Chemistry 2018

Industrial enzyme

Library Creation Strategies

Random MutagenesisSingle Point Mutations at Random Positions

at a low Frequency

Site Saturation MutagenesisFull Randomization of Chosen Positions

Based on Rational Reasoning

DNA Shuffling/RecombinationShuffling of sequence Motifs or Mutations

Between Similar Enzymes

All these Methods suffer from different drawbacks, creating the need for more

efficient strategies.


Computational enrichment of in house enzyme

libraries

Panel Screening

Enzyme screening to identify best enzyme

Project idea

Enzyme fermentation development

Industrial biocatalytic process

Commercialization

5

In silico pre-screening

In silico recombination

High throughput screening using HPLC, GC

Selection of most improved

enzyme

In silicoidentification of

hot spots

Smart library creation by- Gene synthesis

- Mutagenesis

Recombinant enzyme library

expression

Use all collected data to improve computational

models

Directed Evolution


random epPCR MutagenesisSite Saturation Mutagenesis

Computer Aided Evolution

Global Maximum

TargetFitness

®

6


Amrein et al., IUCrJ. 2017 Jan 1; 4 (Pt 1): 50–64.

In Silico Enzyme Activity

Screening

10,000 variants/day

In SilicoEnzyme Stability

Screening

1,000 variants/day

Genome Mining

NR, Uniref90, Uniprot: 164 Billion Proteins

Quantum Mechanics

Activity determination by 3D Protein - Ligand Interactions analysis

Molecular Dynamics

Simulation of protein/environment interactions

Molecular Docking

High Throughput Inhibition/Substrate binding studies

Bioinformatics and big-data driven CADEE

Computer Aided Directed Enzyme

Evolution


Directed Evolution of Enzymes

Bioinformaticsand Data

Management

Automation

High-Throughput Biochemistryand Analytics

Molecular Biology

Strain-Engineering

Enzymatic Process Dev. and Production

Commercialization

Process Dev. and Downstream

Microbiology and Fermentation

Quality Management

Manufacturing


3 Examples from Real Applications Showing The Benefits of BioEngine®

In silicostudies/simulations

provide high sequence space coverage

All screenings and studies under real process conditions

Only 4-5 weeks per round of evolution

Continuous online improvement of in

silico methods

HPLC/GC analytics provide comprehensive

reaction insights

Capacity for up to 6 evolution projects in

parallel


(R)-Phenylethylamine

Wildtype Enzyme:

• Inhibited at 5 g/L product • Inactive at 2M iso-propylamine (IPM) • Space-time-yield: 50 g/L/day

Industrial Enzyme:• Tolerates > 300 g/L product• Stable at 2M IPM for 180 h• Space-time-yield: 160 g/L/day 10 MT/a

10 rounds of evolution

Transaminase

IPMcontacts

in 126-loopregion

solvent contact analysis

2M IPM 554 Residues0.8M PEA 188 Residues

1M IPM 227 Residues0.4M PEA 94 Residues


(R)-1,3-butanediol

Wildtype Enzyme:

• Not thermally stable enough for heat treatment • ~ 50% conv. • 60 g/L substrate loading• 30% (v/v) loading of enzyme

Industrial Enzyme:• Stable at 85℃ for 2h • 99% conv.• 400 g/L substrate loading• 8% (v/v) loading of enzyme

Ketoreductase

50 MT/a


Library Design Strategy Library Size Instrument Time*

Fully combinatorial 6 x NNK 1,000,000,000 >10,000 years

Fully combinatorial 6 x 20 amino acids 64,000,000 732 years

Library of 2x3x20 amino acids 2x 8000 2 months

Enzymaster combinatorial library 2000 8 days

*Calculated on 2 min/sample and 95% coverage


Enzymatic Synthesis of Chloramphenicol IM

+

Chloramphenicol

Aldolase

R =

Bromination

Ac2O

Aldol Addition Reduction

HCl

Resolution Amidation

Br2 Hexamine

Traditional Chemical Synthesis of Chloramphenicol

H2CO Al(i-PrO)3

Amination Protection Deprotection

Synthetic step which introduces atoms being part of target molecule

Synthetic steps not directly contributing to building the target molecule

• 5 steps (60%) Shorter than Chemical Synthesis

• Avoiding Protecting Group Chemistry

• Avoiding Activation Chemistry


Chloramphenicol IM

Wildtype Enzyme:

• de 50%• 10% conversion in 8 h • 40 g/L substrate loading

Industrial Enzyme:• de > 90%• >80% conversion in 8 h• 200 g/L substrate loading Piloting


+

Chloramphenicol

Aldolase

A

Benzaldehyde points towards C-terminal domain → desired 2S-3R -diastereomer

Benzaldehyde points towards N-terminal domain → wrong diastereomer

Y40

W322

BG318

Y40

G318

W322

• 5 steps (60%) Shorter Than chemical Synthesis

• Avoiding Protecting Group Chemistry

• Avoiding Activation Chemistry


1

2

1

2

Enoate Reduction

Imine Reduction

1 2 1 2

Transamination Hydroxylation

1 2

1 2

Alcohol Oxidation

Nitrile Hydrolysis

Aldehyde Cyanation

Aldehyde OxidationKetone Reduction1 2 1 2

Aldol Reaction

Decarboxylation

CO2

Key Enzyme Classes

Aminotransferases

Ketoreductases/ADHs

Aldolases

Esterases/Lipases

Decarboxylases

Oxynitrilases

Nitrilases

TPP-dep. Lyases

Carboligation

14


Enzyme- Crude extract

- Whole cell- Immobilized

Application as “normal” Catalyst Application in Microbial Fermentation

Substrates

Solvent

Product

Standard Reactor

+ No new equipment needed

+ High substrate concentrations possible

+ Reaction in aqueous or organic media

Enzyme

Strain Engineering

GlucoseOr other biobased

feed stock

Culture media Product

Fermenter

Oxygen

+ Cheap and biobased starting material

+ Cofactor regeneration by production host

+ Increased stability of enzymes


Chiral buildingblock:Methyl(R)-3-hydroxybutyrateR-MHB

Production Scale: Up to 100 mt

Application: Pharma and Food

Target Industries Pharma

Chiral building block:(R)-1,3-ButanediolR-BDO


Application: Pharma and Cosmetics

Building block and food supplement:β-Alanine

Production Scale: 100 – 150 mt/month

Application: Pharma and Food

Chiral auxiliary:(R)-α-PhenylethylamineR-PEA


Application: Pharma

Chiral building block:L-tert-Leucine


Application: Pharma

16

Fine Chemicals Cosmetics

Food and Nutrition Agri Business Household Care


Enzyme panel screening

Enzyme catalytic process development

Custom biocatalytic manufacturing

Enzyme preparation by fermentation

„Smart“ enzyme directed evolution

Idea

Industrial process

R&D

17


Dr. Thomas Daußmann

EVP

Yong KoyBong, PhDChairman

ZhenlinLv

President

Haibin Chen, PhD

VP

Shanghai:SJTU π-Supercomputer account

Computational enzyme engineering500,000 CPU hours/year

International consortium of founders

Enzymaster (Ningbo) Bio-Engineering Co., Ltd.in Ningbo (China): Lab space: ca. 3000 m2

Fermentation pilot plant: up to 1000 LEmployees: ca. 100 (60% in R&D and Tech)

Enzymaster Deutschland GmbH in Düsseldorf (Germany): Employees: 4

18

We look forward to discuss your needs!

Contact: Enzymaster Deutschland [email protected]+49 211 1582 1610Neusser Str. 3940219 DüsseldorfGermany

dr. grzegorz kubik february 5th 2020...identify best enzyme project idea enzyme fermentation...

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