Академический Документы
Профессиональный Документы
Культура Документы
Power to create.
Dr. rer.nat.
University Freiburg
2003 2006
Biologist
University Freiburg
2001 2002
Certification
Sprachenkolleg Freiburg
1995 2001
Bachiller
Ing. Agroindustrial
Dr. Ramos-Vera
Seite 2
Publications
1.
2.
Microbiology (2010)
3.
4.
5.
6.
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.
Dr. Ramos-Vera
Our position
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
Overview
I.
Dr. Ramos-Vera
Seite 7
Part I
Evolution of life
and
Basics of molecular biology
Dr. Ramos-Vera
Seite 8
Dr. Ramos-Vera
Wikipedia
Seite 9
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
Order
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
Basics
of
Molecular Biology
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
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
Dr. Ramos-Vera
Seite 22
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
Amino Acid
sequence
mRNA
Ribosome
RNA
Polymerase
Gene
ATGTGATGCTCGACTAGCCTAGCTAGCTA
DNA
Dr. Ramos-Vera
Page 25
Ornithin
Glutamat
Prolin
Dr. Ramos-Vera
Page 26
Part II
Molecular Biology Techniques
and
DNA/Genome manipulation
Dr. Ramos-Vera
Seite 27
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
Page 30
Metabolic engineering
substrate
product
Page 31
Metabolic engineering
substrate
product
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
DNA Manipulation
Dr. Ramos-Vera
Deletion of Genes
Modification of Promoters
Overexpression of Genes
Overexpression of Operons
Page 37
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
5-...TCCGCACG
AATTCTGACGCTAAGCT...-3
3-...AGGCGTGCTTAA
GACTGCGATTCGA...-5
Ligase
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
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
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
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
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
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
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
C
94
Denaturation
Elongation
72
55
Annealing
Cycle1
Cycle 2
min
Dr. Ramos-Vera
Seite | 55
cDNA
Denaturation
Annealing of Primer
Cycle
Repetition
Elongation
Amplifikation
Dr. Ramos-Vera
Seite | 56
Wikipedia
Dr. Ramos-Vera
Seite | 57
In vitro Mutagenesis
Dr. Ramos-Vera
Seite 58
www.thermoscientificbio.com/
DNA Sequencing
Method:
Isolation Kit
Design of primers
Sequencing
Dr. Ramos-Vera
Seite 59
Sample
Sampling
Liquid Nitrogen
Extraction buffer
RNA storage -80C
RNA
cDNA
nucleic acid
Isolation
Total RNA
Liquid-liquid
Columns
RNA integrity
Nanotrop
RT
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?
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
Blair (USA),
Antwerp
Slovenska Lupca
Blair
Wesseling
Kaba
Mobile
Dr. Ramos-Vera
Page 68
http://www.youtube.com/watch?v=8y7r1-MfqUY Biolys plant Russia Link
Business case
Substrate Process
Product
Key parameters
costs
purity
Dr. Ramos-Vera
YP/S
Qp
purity
biomass content
formulation
Page 69
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
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
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
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
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
Title of presentation
Page 84
The problem of
biological engineering
bacterial cell
fermenter
Page 85
Dr. Ramos-Vera
Page 86
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
Dr. Ramos-Vera
Page 88
Dr. Ramos-Vera
Page 89
C-source
product
N-source
Conc.
Mass/
Balances
Rates/
Kinetics
Dr. Ramos-Vera
Page 90
Dr. Ramos-Vera
Page 91
Chemical engineering
Add biological read outs
to process evaluation tools
Process
set up or
optimization
Apply process conditions
as additional targets
for strain optimization
Biological engineering
Dr. Ramos-Vera
Page 92
feed
Problem
time
Page 93
Acknowledgements
UNS:
Evonik
HN-AN-IM
R. Kelle
M. Rieping
Dr. Ramos-Vera
Seite 94
Dr. Ramos-Vera
Seite 95