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Evonik.

Power to create.

Dr. Walter Ramos-Vera


July 2014

Dr. Walter Ramos-Vera


Dr. Walter Ramos-Vera
Scientist
Biological Systems and Intellectual Property
HN-AN-IM
Evonik Industries AG
Kantstrae 2
33790 Halle
Germany
2007 2010

Dr. rer.nat.

PhD in Microbiology and Enzyme


Biochemistry

University Freiburg

2003 2006

Biologist

Microbiology, Biochemistry, Cell Biology,


Virology

University Freiburg

2001 2002

Certification

German as foreign language

Sprachenkolleg Freiburg

1995 2001

Bachiller

Ing. Agroindustrial

Universidad Nacional del Santa,


Nuevo Chimbote

Dr. Ramos-Vera

Seite 2

Publications
1.

Journal of Bacteriology (2009)

Autotrophic carbon dioxide assimilation in Thermoproteales revisited.


Ramos-Vera W. H., Berg I. A., Fuchs G.

2.

Microbiology (2010)

Study of the distribution of autotrophic CO2 fixation cycles in Crenarchaeota.


Berg, I. A., Ramos-Vera W. H., Petri A., Huber H., und Fuchs G.

3.

Nature Reviews Microbiology (2010)

Autotrophic carbon fixation in Archaea.


Berg, I. A., Kockelkorn D., Ramos-Vera W. H., Say R. F., Zarzycki J., Hgler M., Alber
B. E., Fuchs G.

4.

Journal of Bacteriology (2010)

Regulation of autotrophic CO2 fixation in the archaeon Thermoproteus neutrophilus.


Ramos-Vera, W. H., Labont V., Michael Weiss, Julia Pauly, Fuchs G.

5.

Journal of Bacteriology (2011)

Identification of missing genes and enzymes for autotrophic carbon fixation in


crenarchaeota. Ramos-Vera, W. H., D, Fuchs G.

6.

Journal of Bacteriology (2011)

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula.
Estelmann S, Hgler M, Eisenreich W, Werner K, Berg IA, Ramos-Vera WH, Say RF,
Kockelkorn D, Gad'on N, Fuchs G.

7.

FEMS Microbiol Lett. (2011)

Dr. Ramos-Vera

Carbon dioxide fixation in 'Archaeoglobus lithotrophicus': are there multiple autotrophic


pathways?
Estelmann S, Ramos-Vera WH, Gad'on N, Huber H, Berg IA, Fuchs G.
Seite 3

Our position

Evonik - the creative


industrial group from
Germany - is one of the
world's leading specialty
chemicals companies.

Dr. Ramos-Vera

Page 4

A worldwide presence
Production plants in 24 countries,
active in more than 100 countries

Dr. Ramos-Vera

Page 5

Molecular Biology
and
Industrial Application

Dr. Walter Ramos-Vera


July.2014

Overview
I.

Evolution of life and Basics of


molecular biology

II. Molecular Biology Techniques and


DNA/Genome manipulation
III. Industrial Application

Dr. Ramos-Vera

Seite 7

Part I
Evolution of life
and
Basics of molecular biology

Dr. Ramos-Vera

Seite 8

Phylogenetic tree of life

Dr. Ramos-Vera

Wikipedia

Seite 9

The phylogenetic tree of Archaea


Euryarchaeota
Halobacteriales

Thermoplasmatales

Methanosarcinales
Methanomicrobiales

Thermococcales

Methanobacteriales

Sulfolobales

Methanococcales

Desulfurococcales
Thermoproteales
Dr. Ramos-Vera

Mesophilic group I
Crenarchaeota

Crenarchaeota

Seite 10

Supervolcano
Yellowstone National Park

Dr. Ramos-Vera

Seite 11

Growth temperatures

Highest growth
temperature:
121C

Dr. Ramos-Vera

Seite 12

Hyperthermophilic Archaeon:

Thermoproteus neutrophilus
Carbon source:

CO2

Growth temperature:

85C

Electron acceptor:

elemental sulfur

Dr. Ramos-Vera

Seite 13

CO2 fixation in Crenarchaeota

Order

CO2 fixation cycle

Model organism

Energy

Sulfolobales

3-Hydroxypropionate/
4-hydroxybutyrate

Metallosphaera sedula
75C, pH 2.0

2 H2 + O2 2 H2O

Stygiolobus azoricus
85C, pH 2.5

H2 + S H2S

Ignicoccus hospitalis
90C, pH 5.5

H2 + S H2S

Pyrolobus fumarii
106C, pH 5.5

5 H2 + 2 HNO3 N2 + 6 H2O

Thermoproteus
neutrophilus
85C, pH 6.8

H2 + S H2S

Desulfurococcales

Thermoproteales

Dr. Ramos-Vera

Dicarboxylate/
4-hydroxybutyrate

Dicarboxylate/
4-hydroxybutyrate

Seite 14

The dicarboxylate/4-hydroxybutyrate
cycle
Acetyl-CoA
CO2

Acetyl-CoA

14

Fdred

Acetoacetyl-CoA CoASH
NADH +
NAD+

Fdox+ CoASH

Pyruvate

H+
13

ATP + H2O
2
Pi + AMP

PEP

(S)-3-Hydroxybutyryl-CoA

HCO33
Pi

12

H2O

Crotonyl-CoA

Oxaloacetate
NADH + H+
4

H2O
11

NAD+

Malate

4-Hydroxybutyryl-CoA
AMP + PPi

10

H2O

ATP + CoASH

Fumarate

4-Hydroxybutyrate
NADP+

6 2 MVred
2 MVox

NADPH + H+

Succinate

Succinic semialdehyde
CoASH + NADP+
Dr. Ramos-Vera

Succinyl-CoA

NADPH +

H+

ADP + Pi

ATP + CoASH
Seite 15

Ramos-Vera et al., 2009

Basics
of
Molecular Biology

Understanding the bacterial cell

Sugar

Peptide

Fruc

Gluc

Succ

Treh

Rib
Sugar

G6P

FBP

R5P

GA3P

S7P

E4P

F6P

GA3P

0.5

Corynebacterium
glutamicum
2.5
Genome
1
3.31 Mbp
2

Xu5P

F6P

Phe
Tyr

6PG
Ru5P

Ser
Cys

6PGL

1.5

PEP

Ala
Val
Leu
Asp
Met
Thr
Lys

His

Try
Tyr
vitamines

Pyruvate
Lactate
Oxalacetate

Citrate

Acetate

Malate
Isocitrate

Fumarate
2 Oxoglutarate
Succinate

Glu
Gln
Pro

Succinyl-CoA
AA

AA
Ions Betain

Dr. Ramos-Vera

PO42-

Page 17

Bacterial Genome

Membrane proteins
Soluble proteins
induced
0.00Mbp

repressed

0.50Mbp
3.00Mbp

C. glutamicum
genome
3,282,708 nt

unchanged
2.50Mbp

1.00Mbp

1.50Mbp
2.00Mbp
-3.0
-1.0
0.0
1.0
3.0

Dr. Ramos-Vera

Page 18

Transcription and Translation

Transcription

DNA

Dr. Ramos-Vera

mRNA

Translation

Protein

Seite 19

Nucleotides

DNA:

A C T G

RNA:

A C U G

Dr. Ramos-Vera

WIkipedia

Seite 20

DNA

Gene
ATGTGATGCTCGACTAGCCTAGCTAGCTA
TACACTACGAGCTGATCGGATCGATCGAT

Dr. Ramos-Vera

Seite 21

Promoter

Gene

Plassmeier et al., 2011

Dr. Ramos-Vera

Seite 22

Standard genetic code

Wikipedia
Dr. Ramos-Vera

Seite 23

Protein
Amino acid sequence

Protein/Enzyme

MAGTATYWWWDDEVILPREKKK
(Met-Ala-Gly-Thr-Ala-Thr-Tyr.)

Dr. Ramos-Vera

Page 24

Transcription and Translation:


From the DNA to the protein
Protein/Enzyme
Protein
Folding

Amino Acid
sequence

mRNA

Ribosome
RNA
Polymerase
Gene

ATGTGATGCTCGACTAGCCTAGCTAGCTA
DNA
Dr. Ramos-Vera

Page 25

Base for biotechnological amino acid


production: microbial physiology

Ornithin

Glutamat

Prolin

Dr. Ramos-Vera

Page 26

Part II
Molecular Biology Techniques
and
DNA/Genome manipulation

Dr. Ramos-Vera

Seite 27

Understanding the bacterial cell

Sugar

Peptide

Fruc

Gluc

Succ

Treh

Rib
Sugar

G6P

FBP

R5P

GA3P

S7P

E4P

F6P

GA3P

0.5

Corynebacterium
glutamicum
2.5
Genome
1
3.31 Mbp
2

Xu5P

F6P

Phe
Tyr

6PG
Ru5P

Ser
Cys

6PGL

1.5

PEP

Ala
Val
Leu
Asp
Met
Thr
Lys

His

Try
Tyr
vitamines

Pyruvate
Lactate
Oxalacetate

Citrate

Acetate

Malate
Isocitrate

Fumarate
2 Oxoglutarate
Succinate

Glu
Gln
Pro

Succinyl-CoA
AA

AA
Ions Betain

Dr. Ramos-Vera

PO42-

Page 28

The world of
biological engineering

stress
BDM

co-factors

substrate

Metabolism

product
CO2,
energy

ions,

bacterial cell
Dr. Ramos-Vera

Page 29

Metabolic engineering

The bacterial metabolic network is very complex.


Dr. Ramos-Vera

Page 30

Metabolic engineering

substrate

product

The flux through metabolic networks can be quantified.


Dr. Ramos-Vera

Page 31

Metabolic engineering

substrate

product

Pathways can be inserted from scratch.


Dr. Ramos-Vera

Page 32

Strain performance

Substrate
(S)

Dr. Ramos-Vera

Product
(P)

Biomass
(X)

Page 33

Principle challenges
Y ps: Product/Substrate
Y xs: Biomass/Substrate
WT: Wild Type
PS: Production Strain

PSx

Y P/S
PS3

PS2

PS1

PS0

WT

Y X/S
Dr. Ramos-Vera

Page 34

Principle challenges

Performance
Y P/S

Y ps: Product/Substrate
WT: Wild Type
PS: Production Strain

PS2

PS3

Significance
PS1

PSb

WT

Strain history
Dr. Ramos-Vera

Page 35

Production ofAminosureproduktion
Amino Acids
Fruc

Gluc

Succ

G6P

Serine family
Ser
Cys

6PGL

6PG
Ru5P

F6P
FBP

Xu5P

R5P

GA3P

S7P

E4P

F6P

GA3P

Pyruvate family
Ala
Val
Leu

Aspartate family
Asp
Met
Thr
Lys

His

3PG
PEP

Aromatic AA
Phe
Trp
Tyr

Pyruvate

Oxalacetate

Citrate

Malate

TCA

Isocitrate

Fumarate
2 Oxoglutarate
Succinate
Succinyl-CoA

Dr. Ramos-Vera

Glutamate family
Glu
Gln
Pro

Page 36

DNA/Genome Manipulation
In vivo Mutagenesis

radiation (short-wavelength Ultraviolet, 200-300nm)

chemical agents (e.g. nitrous acid (HNO2), hydroxylamine (NH2OH)

DNA Manipulation

Dr. Ramos-Vera

Deletion of Genes

Modification of Promoters

Modification of Genes (e.g. Point Mutations, Codon Optimization)

Overexpression of Genes

Overexpression of Operons

Page 37

Improvement of strain performance


3 4.5 kb inserted
50

42

45
40

44

35

35
30
3000000
500000

25

2500000
1000000
2000000
1500000

20
15
10

20

21

2xOP

3xOP

16
12
6

5
0
Ref.

Dr. Ramos-Vera

1xOP

Seite 38

Techniques
in
Molecular Biology

Dr. Ramos-Vera

Seite 39

Techniques
 DNA Manipulation
 Restriction
 Plasmids
 Molecular Cloning
 PCR (polymerase chain reaction)
 In vitro Mutagenesis
 Sequencing of DNA
 Reverse Transcription PCR
 Expression Analysis
 In Silico: Genbank and Tools

Dr. Ramos-Vera

Seite 40

DNA Manipulation:
Restriction
Liste aller
Restriktions
-enzyme

Restriction endonucleases
Enzyme for cut DNA

EcoRI

HindIII

MunI

DraI

SmaI

KpnI

Enzyme

GAATTC
CTTAAG

AAGCTT
TTCGAA

CAATTG
GTTAAC

TTTAAA
AAATTT

CCCGGG
GGGCCC

GGTACC
CCATGG

Detected
sequences

sticky
5-

sticky
5-

sticky
5-

blunt

blunt

sticky
3-

Produced
ends

EcoRI

5-...TCCGCACG
GAATTC
AATTCTGACGCTAAGCT...-3
3-...AGGCGTGCTTAA
CTTAAG
GACTGCGATTCGA...-5
Dr. Ramos-Vera

Seite 41

DNA Manipulation:
Restriction and Ligation
Ligase

Ligase:
Enzyme, for binding compatible DNA Fragments,
under ATP-Consumption

Ligase

Ligation of sticky ends

5-...TCCGCACG
AATTCTGACGCTAAGCT...-3
3-...AGGCGTGCTTAA
GACTGCGATTCGA...-5
Ligase

Ligation of blunt ends

No Ligation

Dr. Ramos-Vera

5-...CTGCACGTTT
3-...GACGTGCAAA

GGGTCTGACGCTAAG...-3
CCCAGACTGCGATTC...-5

5-...TCCGCACG
CTGTGACGCTAAGCT...-3
3-...AGGCGTGCTTAA CATGGACACTGCGATTCGA...-5

Seite 42

DNA Manipulation:
Plasmid
EcoRI

HindIII

E. coli

mcs
lacZ

Plasmid:
extra chromosomal
DNA molecule

Plasmid DNA

pK18mobsacB
chromosomal DNA

origin of replication

Resistance gene for Selektion

Region for mobilization of Plasmids (Conjugation)

Gen for selection of plasmide lost

 Gen for blue/white selection (Insert: yes/no present)


with multiple cloning site (mcs) for Cloning

Dr. Ramos-Vera

ori Ec
KanR

ori Cg
TetR

CmR

mob

EcoRI HindIII

lacZ
mcs
Seite 43

Plasmids examples
EcoRI

HindIII

mcs
lacZ

pK18mobsacB
5.700 bp

EcoRI

HindIII

EcoRI

mcs
lacZ

HindIII

mcs
lacZ

pEC-K18mob2

pEC-T18mob2
6.200 bp

5.700 bp

DNA Manipulation by Using of


suicide vector and recombination

pCRII-blunt
3.500 bp

Dr. Ramos-Vera

Seite 44

Molecular Cloning
Steps:
(1) Choice of host organism (e.g. E. coli) and plasmid
(2) Preparation of plasmid
(3) Preparation of DNA to be cloned (insert)
(4) Restriction
(5) Ligation
(6) Introduction of plasmid with insert into host organism (e.g. E. coli)
(7) Plasmid Replication, Isolation, and Purification
(8) Selection of Clones with desired DNA inserts and biological properties

Dr. Ramos-Vera

Seite 45

Molecular Cloning
Insert DNA

EcoRI

HindIII

gen

EcoRI

5-CCG AATTCAGA
3-GGCTTAA GTCT

HindIII

GCTA AGCTTGA-3
CGATTCGA ACT-5

Vector DNA
EcoRI

HindIII

mcs
EcoRI lacZ
mcs

HindIII

TCCGCACG AATTCTGACGCTA AGCTTCGAG


AGGCGTGCTTAA GACTGCGATTCGA AGCTC
pK18mobsacB
5.700 bp

mob
Dr. Ramos-Vera

Seite 46

Molecular Cloning
HindIII

EcoRI

gen

Dr. Ramos-Vera

Seite 47

Transformation / Electroporation
HindIII

EcoRI

gen

Competent Cell
(e.g. E.coli)
chromosomal DNA

Right clones?
Dr. Ramos-Vera

Seite 48

Selection of Clones

Medium (e.g. LB)

Cell can grow

Antibiotics (z.B. Kanamycin)

Only Plasmid containing cells can


grow

X-Gal (dye:5-bromo-4-chloro-3-indolyl--Dgalactopyranoside, transformed by LacZ)

blue/white selection
E.coli (DH5, TOP10, Mach1)
Only with Plasmids with lacZ Gene

Dr. Ramos-Vera

Plasmid Replication,
Isolation, and Purification

Seite 49

Summary
EcoRI

HindIII

gen

EcoRI HindIIII

Insert-DNA e.g. Restriction


PCR-Product
purification
HindIII

EcoRI

gen
ori E
c

R
Kan

Ligase

mo
b

EcoRI

Ligation

HindIII

R
K an

ori
Ec

lacZ
mcs
EcoRI HindIIII

cB
sa

Plasmid
ready

chromosomale DNA

Transfer to
competent
cells

Selection

pK18mobsacB
sa

ob

cB

5.700 bp

Vector- /
Plasmid DNA

Dr. Ramos-Vera

Restriction
purification

Seite 50

Alternative Cloning:
Gibson Assembly

Dr. Ramos-Vera

Seite | 51

Alternative Cloning:
Gibson Assembly

Exonuclease, DNA-Polymerase und Ligase

Incubation 1h at 50C

Exonuclease, at
50C inactive

Dr. Ramos-Vera

Seite | 52

PCR
Reaction Mix:
1. Template: DNA for amplification
2. Primer 1: Oligonucleotide (downstream)
3. Primer 2: Oligonucleotide (upstream)
4. dNTPs: ATP, TTP, CTP, GTP
5. Buffer with MgCl2
6. DNA-free water
7. DNA Polymerase (e.g. Taq, Pfu, Pwo)
8. (DMSO Dimethylsulfoxide)

Dr. Ramos-Vera

Seite | 53

The Primers

- 20 30 DNA Oligonucleotides
- Similar Smelt Temperature between 50 70C
- GC Content
- No dimers or loops

Dr. Ramos-Vera

Seite | 54

The PCR Cycle

C
94

Denaturation

Elongation

72
55
Annealing

Cycle1

Cycle 2
min

Dr. Ramos-Vera

Seite | 55

The PCR Cycle


DNA

cDNA
Denaturation

Annealing of Primer
Cycle
Repetition

Elongation

Amplifikation

Dr. Ramos-Vera

Seite | 56

Agarose gel electrophoresis

Wikipedia

Dr. Ramos-Vera

Seite | 57

In vitro Mutagenesis

Dr. Ramos-Vera

Seite 58

www.thermoscientificbio.com/

DNA Sequencing
Method:

Cell Lysis of E.coli

Isolation Kit

Adjustment of required DNA concentration

Design of primers

Sequencing

Dr. Ramos-Vera

Seite 59

Reverse Transcription PCR

Sample
Sampling

Liquid Nitrogen
Extraction buffer
RNA storage -80C

RNA

cDNA

nucleic acid
Isolation

Total RNA
Liquid-liquid
Columns
RNA integrity
Nanotrop

RT

real time PCR


amplification

Efficiency of RT:

PCR Efficiency:

Dr. Ramos-Vera

PCR

RT enzyme type
RT temperature
Primers:

Random hexamers

Specific primer
One-step qRT-PCR

Primer design

Specificity

Consensus
mRNA abundance
Polymerase types
Polymerase mixtures

Seite 60

Expression analysis

- Quantitative RT-PCR
- Microarrays (lab-on-chip)

Dr. Ramos-Vera

Seite 61

In Silico
Where can you find bacteria information?
Where can you find bacterial genomes?
Where can you find gene sequences?
Where can you find protein sequences?
Where can you find protein structures?
Where can you compare genes/nucleotides?
Where can you compare proteins?

National Center of Biotechnology Information (NCBI)

Dr. Ramos-Vera

Seite 62

NCBI

NCBI

Dr. Ramos-Vera

Seite 64

NCBI

Dr. Ramos-Vera

Seite 65

NCBI

Dr. Ramos-Vera

Seite 66

Part III
Industrial Application

Dr. Ramos-Vera

Seite 67

Production sites worldwide


MetAMINO: Wesseling (D), Antwerp (B), Mobile (USA)
Biolys:

Blair (USA),

ThreAMINO: Slovenska Lupca (SK), Kaba (HUN)


TrypAMINO: Slovenska Lupca (SK)

Antwerp
Slovenska Lupca
Blair

Wesseling

Kaba

Mobile

Dr. Ramos-Vera

Page 68
http://www.youtube.com/watch?v=8y7r1-MfqUY Biolys plant Russia Link

Product and process definition

Business case
Substrate Process

Product

Key parameters
costs
purity

Dr. Ramos-Vera

YP/S
Qp

purity
biomass content
formulation

Page 69

This is Evonik: Global Leader in


Feed Amino Acids and beyond.
We enable customers and farmers to produce
high value and affordable meat, fish, eggs
and milk. To contribute to feeding the worlds
growing population. Globally accessible.
Today and tomorrow.

Essential Amino Acids

Dr. Ramos-Vera

Integrated Services

Page 70

Feed Additives
essential amino acids
in nature derived from crude protein
important crude protein source: soy, fish meal
1 kg DL-methionine can substitute 54 kg fish meal
1 kg L-Lysine can substitute 35 kg soy grain
Liebigs barrel of nutrition

Dr. Ramos-Vera

Page 71

Our Masterpieces

Dr. Ramos-Vera

Page 72

Product: Biolys

Dr. Ramos-Vera

Page 73

Product: Biolys

Dr. Ramos-Vera

Page 74

Production ofAminosureproduktion
Amino Acids
Src

Frc

Glc
G6P

Serine family
Ser
Cys

6PGL

6PG
Ru5P

F6P
FBP

Xu5P

R5P

GA3P

S7P

E4P

F6P

GA3P

Pyruvate family
Ala
Val
Leu

Aspartate family
Asp
Met
Thr
Lys

His

3PG
PEP

Aromatic AA
Phe
Trp
Tyr

Pyruvate

Oxalacetate

Citrate

Malate

TCA

Isocitrate

Fumarate
2 Oxoglutarate
Succinate
Succinyl-CoA

Dr. Ramos-Vera

Glutamate family
Glu
Gln
Pro

Page 75

Performance is already exceptional


Substrate(s)

CO2

BDM
Lys

Product
Dr. Ramos-Vera

Byproducts

Page 76

L-Lysine metabolism
Gluc

PEP
Pyruvate

CM

L-Aspartat
Aspartat
Kinase Fbr

Oxalacetate

Citrate

Malate

TCA

Isocitrate

Fumarate

L-Aspartatsemialdehyd

2 Oxoglutarate
Succinate
Succinyl-CoA

HDH

Homoserin

L-Threonin

L-Methionin

L-Isoleucin

D,L-Diaminopimelat

LysE

Dr. Ramos-Vera

Lysin
Page 77

Lysine Pathway

Dr. Ramos-Vera

Page 78

L-Lysine metabolism

Gluc

PEP
Pyruvate

CM

L-Aspartate

Aspartate
Kinase

Oxalacetate

Citrate

Malate

TCA

Isocitrate

Fumarate

L-Aspartatsemialdehyde

Homoserine
Dehydrogenase

Homoserine

2 Oxoglutarate
Succinate
Succinyl-CoA

L-Threonine L-Methionine
L-Isoleucine
D,L-Diaminopimelate

Lysine

LysE

Feed back-Inhibition
Dr. Ramos-Vera

Page 79

Optimierter L-Lysin Metabolismus


L-Lysine metabolism

Gluc

PEP
Pyruvate

CM

L-Aspartate
Aspartate
Kinase Fbr

Oxalacetate

Citrate

Malate

TCA

Isocitrate

Fumarate

L-Aspartatsemialdehyde

2 Oxoglutarate
Succinate
Succinyl-CoA

HDH

Homoserine

L-Threonine

LysE

LysE

LysE

Dr. Ramos-Vera

L-Methionine

L-Isoleucine

D,L-Diaminopimelate

Lysine
Page 80

Kinetic models

Dr. Ramos-Vera

Page 81

Strain and process development

Strain

Strain
Development

Screening
tools in

Strain
Evaluation in

Strain
Evaluation in

ml scale

l scale

m3 scale Production

Strain
Evaluation in

PS*

Scale up

Dr. Ramos-Vera

Page 82

Statistic Tools

- Box Plots
- Histogramms
- Normality Tests
- Variance Analysis
- t- Test
- F-Test

Dr. Ramos-Vera

Page 83

Strain and process development


Target
identification

Target and Strain


evaluation
Genetic engineering

Knowledge

Production strain
and process
(KPI: YP/S, QP,
purity, )

Data

Improved
Production strain
and process
(KPI: YP/S, QP,
purity, )

Genetic characterization
A

Undirected
Physiological characterization
Accident

Functional characterization
A

The measures are potential, time, costs and risk.


Dr. Ramos-Vera

Title of presentation

Page 84

The problem of
biological engineering

bacterial cell

fermenter

The tool box of biology does not allow


predictive optimization for process conditions.
Dr. Ramos-Vera

Page 85

Methods for strain evaluation


Early, reproducible (online) characterization

Increase throughput even more

Dr. Ramos-Vera

Page 86

Methods for strain evaluation

Characterization under
process conditions
batch
0,03

OTR

0,02

fed batch
0,01

0,00
0

10

15

20

25

30

35

40

45

50

Time (h)

Dr. Ramos-Vera

Page 87

The world of chemical engineering

Dr. Ramos-Vera

Page 88

Strain and process development

Dr. Ramos-Vera

Page 89

The world of chemical engineering


biomass

C-source

product

N-source

Conc.

Mass/
Balances

Rates/
Kinetics
Dr. Ramos-Vera

Page 90

Scale up of fermentation processes


volume: V = 2l x00 m

Dr. Ramos-Vera

Page 91

Combining the two worlds

Chemical engineering
Add biological read outs
to process evaluation tools

Apply biological thinking


for process optimization

Process
set up or

Update and combine biological and engineering knowledge

optimization
Apply process conditions
as additional targets
for strain optimization

Test strain performance


under process relevant
conditions

Biological engineering
Dr. Ramos-Vera

Page 92

Combining the two worlds


Upon a metabolic pathway improvement Qp decreased in a process.
Solution

feed

Problem

time

A decraese in a substrate conversion rate was derived


and addressed by adjusting the feed profile.
Dr. Ramos-Vera

Page 93

Acknowledgements

UNS:

Prof. Dr. Gilbert Rodriguez Paucar

Rectora Dr. Amrica Odar Rosario

Evonik

HN-AN-IM

R. Kelle
M. Rieping

Dr. Ramos-Vera

Seite 94

Dr. Ramos-Vera

Seite 95

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