Produktion humaner Proteine in Bakterien

1
INTEGRATED SYSTEMS APPROACH for the OPTIMIZATION
of MICROBIAL RECOMBINANT FERMENTATION PROCESSES
Karl Bayer University of Agricultural
Sciences,Vienna
221st ACS Nat. Meeting April 1 - 5, 2001 San
Diego Institute of Applied
Microbiology
2

• OUTLINE
• Targets of recombinant fermentation
  process optimisation
• Monitoring of metabolic load
• Control of plasmid copy number
• Tuning of expression rate
• Substitution of IPTG by lactose for induction

3

• KEY ASPECTS for the OPTIMIZATION of RECOMBINANT
  PROTEIN PRODUCTION on INDUSTRIAL SCALE
• Efficiency of process development

221st ACS Nat. Meeting April 1 - 5, 2001 San
Diego Institute of Applied
Microbiology
4

• KEY ASPECTS for the OPTIMIZATION of RECOMBINANT
  PROTEIN PRODUCTION on INDUSTRIAL SCALE
• Efficiency of process development
• High yield

5

• COMMON APPROACH to INCREASE YIELD
• using strong vector systems

6

• COMMON APPROACH to INCREASE YIELD
• using strong vector systems

• DRAWBACKS of too STRONG VECTOR SYSTEMS
• overwhelming of host cell metabolism
• metabolic potential of expression system
  cannot be maintained throughout the whole
  fermentation process

7
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1h-1
exponential feed
300
theoret. BDM(g)
250
Induction
200
BDM(g)
BDM
150
100
50
0
22
24
26
28
30
32
34
36
38
40
fermentation time h
8
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1h-1
exponential feed
300
theoret. BDM(g)
250
Induction
200
BDM, rhSOD
BDM(g)
150
rhSOD(g)10
100
50
0
22
24
26
28
30
32
34
36
38
40
fermentation time h
9
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1h-1
exponential feed
300
theoret. BDM(g)
250
Induction
BDM, rhSOD, qP
200
BDM(g)
150
rhSOD(g)10
100
qP5(mg/g,h)
50
0
22
24
26
28
30
32
34
36
38
40
fermentation time h
10
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1h -1
exponential feed
300
PCN
250
BDM, rhSOD, qP, PCN
Induction
200
BDM(g)
150
rhSOD(g)10
100
qP5(mg/g,h)
50
0
22
24
26
28
30
32
34
36
38
40
fermentation time h
11

• CONCLUSIONS
• Maximum yield of recombinant protein cannot
  be attained
• ? overexpression of recombinant protein
  leads to metabolic collapse of the cell factory

12

• CONCLUSIONS
• Maximum yield of recombinant protein cannot
  be attained
• ? overexpression of recombinant protein
  leads to metabolic collapse of the cell factory
• ? recombinant protein production can only
  be maintained for a short period

13
GOAL Maximum exploitation of the cell
factory KEY appropriate distribution between
RECOMBINANT and CELLULAR protein production
14

• REQUIREMENTS to ACHIEVE OPTIMAL RECOMBINANT
  PROTEIN PRODUCTION
• 1) Monitoring of the host cells metabolic load

15

• DETERMINATION of METABOLIC LOAD
• Recombinant protein production causes
  metabolic load and triggers stress response
  mechanisms
• Metabolic load can be obtained by the identifi-
  cation of key variables of the hierarchically
  organised regulatory networks and signal
  processing machinery of the E. coli cell

16
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17
METHODOLOGY Determination of the signal molecule
guanosinetetraphosphate (ppGpp) by HPLC
18
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1 exponential
feed
1,0
300
PCN
0,9
250
0,8
ppGpp(µmol/g)
BDM, qP, PCN, rhSOD
Induction
0,7
200
0,6
BDM(g)
150
0,5
ppGpp
0,4
rhSOD(g)10
100
0,3
0,2
qP5(mg/g,h)
50
0,1
0
0,0
22
24
26
28
30
32
34
36
38
40
fermentation time h
19

• REQUIREMENTS to ACHIEVE OPTIMAL RECOMBINANT
  PROTEIN PRODUCTION
• 1) Monitoring of the host cells metabolic
  load
• 2) To cope with the runaway effect of plasmid
  copy number (PCN)
• Monitoring of PCN
• Stabilisation of PCN

20

• RUNAWAY PLASMID REPLICATION at HIGH EXPRESSION
  RATES
• increase of plasmid copy number (PCN) is due
  to the interaction of uncharged tRNA with
  plasmid replication control molecules (RNAI,
  RNA II)
• significant increase of uncharged tRNA at high
  expression rates due to starvation

21
PLASMID REPLICATION CONTROL of ColE1 PLASMIDS
REPLICATION CONTROL
INTERACTION of RNAI/RNAII

22

• MONITORING of PCN by MODELLING
• To circumvent complex off-line analytical
  procedures PCN can be modelled from easily
  obtainable on-line data sets (CO2 production
  rate, alkaline base consumption rate, optical
  density)
• by the application of artificial neural networks

23
MODELLING of PCN in FED- BATCH FERMEN-TATION of
E. coli HMS174(DE3)pET11ahSOD)

• PCN modelled from
• CO2 production rate
• alkal. base consumption
• optical density (OD)

complex data become continuously available
24
PREVENTION of PLASMID OVER-REPLICATION
Objective restore plasmid replication control by
deletion of sequence homology between ColE1 RNA
I/RNA II and tRNAs
25
RESTORING PLASMID REPLICATION CONTROL of ColE1
PLASMIDS
Change of sequence homology of loop 2

26
METHODOLOGY Change of nucleotide sequence of loop
2 of RNAI and RNAII RESULT plasmid copy number
(PCN) constant throughout the fermentation
27
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODclone3growth rate 0,1
exponential feed, synthetic media
25
300
BDM theor.
250
20
BDM
200
15
BDM, BDM theoret., IPTG
150
Induction
10
100
IPTG
5
50
0
0
34
36
38
40
42
44
46
48
50
52
54
56
fermentation time h
221st ACS Nat. Meeting April 1 - 5, 2001 San
Diego Institute of Applied
Microbiology
28
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODclone3growth rate 0,1
exponential feed, synthetic media
25
300
total rhSOD
250
20
BDM
200
15
total rhSOD
BDM, ppGpp, PCN
150
Induction
10
PCN
100
ppGpp100
5
50
0
0
34
36
38
40
42
44
46
48
50
52
54
56
fermentation time h
221st ACS Nat. Meeting April 1 - 5, 2001 San
Diego Institute of Applied
Microbiology
29
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODclone3growth rate 0,1
exponential feed, synthetic media
25
300
2,5 times increase of recomb. protein
total rhSOD
250
20
BDM
200
15
total rhSOD
BDM, ppGpp, PCN
150
Induction
10
PCN
100
ppGpp100
5
50
0
0
34
36
38
40
42
44
46
48
50
52
54
56
fermentation time h
221st ACS Nat. Meeting April 1 - 5, 2001 San
Diego Institute of Applied
Microbiology
30

• REQUIREMENTS to ACHIEVE OPTIMAL RECOMBINANT
  PROTEIN PRODUCTION
• 1) Monitoring of the host cells metabolic
  load
• 2) To cope with runaway effect of plasmid copy
  number (PCN)
• Monitoring of PCN
• Stabilisation of PCN
• 3) Tuning of expression rate in relation to
  host cell metabolism

31

• OBJECTIVETuning of transcription rate by
  controlled feed of the inducer in fed batch
  cultivation
• REQUIREMENTS
• promoter induction below maximum
• constant ratio of inducer to biomass
• continuous feed of inducer related to
  biomass

32
EXPERIMENTAL SET-UP to DETERMINE the APPROPRIATE
AMOUNT of INDUCER
total IPTG
IPTG related to BDM
inducer dosageinto medium
feed start
BDM
33
RESULT critical amount of IPTG below 0,9
µmol/gBDM
34
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1 feed of
inducer (0,9 µmolIPTG/gBDM), synthetic media
40
35
200
BDM
cfu108
30
150
25
PCN, BDM
20
SOD total, cfu108
100
15
10
50
PCN
5
total rhSOD
0
0
20
25
30
35
40
fermentation time h
35
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1 feed of
inducer (0,9 µmolIPTG/gBDM) synthetic media
ppGpp
40
35
200
BDM
30
150
25
SOD total
ppGpp200, PCN, BDM
20
100
15
10
50
PCN
5
total rhSOD
0
0
20
25
30
35
40
fermentation time h
36
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1 feed of
inducer (0,9 µmolIPTG/gBDM) synthetic media
2,5 times increase of recomb. protein
ppGpp
40
35
200
BDM
30
150
25
SOD total
ppGpp200, PCN, BDM
20
100
15
10
50
PCN
5
total rhSOD
0
0
20
25
30
35
40
fermentation time h
37

• SUBSTITUTION of IPTG by LACTOSE due to GMP
  REGULATIONS
• use of lactose for induction instead of IPTG
  requires the following additional conditions
• C-limited fed batch fermentation process to
  avoid inducer exclusion
• maintaining a constant ratio of lactose to
  biomass to compensate consumption

38
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1 feed of
inducer (lactose), synthetic media
BDM
40
250
35
200
cfu108
30
25
150
PCN, ppGpp200, BDM
ppGpp
SOD total, cfu108
20
100
15
total hSOD
10
50
5
PCN
0
0
22
27
32
37
42
fermentation time h
39
Fed batch fermentation of E.coli HMS
174(DE3)pET11ahSODgrowth rate 0,1 feed of
inducer (lactose), synthetic media
2,5 times increase of recomb. protein
BDM
40
250
35
200
cfu108
30
25
150
PCN, ppGpp200, BDM
ppGpp200
SOD total, cfu108
20
100
15
total hSOD
10
50
5
PCN
0
0
22
27
32
37
42
fermentation time h
40

• Franz Clementschitsch
• Monika Cserjan - Puschmann
• Otto Doblhoff-Dier
• Eberhard Dürrschmid
• Philipp Fortunat
• Reingard Grabherr
• Walter Kramer
• Diethard Mattanovich
• Franz Steindl
• Gerald Striedner
• Karola Vorauer-Uhl

Michael Hammerschmid (BIA) Erik Nilsson (Univ.
Linköping)
Austrian Industrial Research Promotion Fund
Boehringer Ingelheim Austria, Vienna