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					     Metabolic and engineering integrated approach for the optimisation of
                    recombinant fermentation processes
                               Gerald Striedner, Franz Clementschitsch, Monika Cserjan-Puschmann, Reingard Grabherr and Karl Bayer
                                            Institute of Applied Microbiology, University of Agricultural Sciences Vienna
                                                    Muthgasse 18B, A-1190 Vienna, Austria, bayer@mail.boku.ac.at


Introduction
The main objectives in industrial biopharmaceutical production are to attain high yield in combination                                                                                                     300
                                                                                                                                                                                                                                                                                       PCN
with required product quality. E.coli is a widely used host for the production of recombinant proteins.
                                                                                                                                                                                                           250




                                                                                                                                                                                   BDM, rhSOD, qP, PCN
A common strategy to increase yield is the use of strong expression vectors, such as the T7 system. In                                                                                                                                                    INDUCTION
reality these systems are too strong and host cell metabolism is heavily overstrained after induction.                                                                                                     200
                                                                                                                                                                                                                                                                                                      BDM(g)
Hence, maximum yield cannot be attained, because recombinant protein production can only be                                                                                                                150
maintained for a short period due to too high production rate and heavy increase of plasmid copy                                                                                                                                                                                             10*total rhSOD(g)
                                                                                                                                                                                                           100
number under these conditions (Figure 1). To cope with this situation an integrated systems approach
aiming at maximal exploitation of the cell factory’s potential by adjusting optimal ratios between                                                                                                             50
                                                                                                                                                                                                                                                                                                  5*qP (mg/g,h)


biosynthesis of (1) host cell proteins and (2) recombinant proteins, is applied.                                                                                                                                   0
                                                                                                                                                                                                                       22         24          26        28    30     32      34              36        38         40

Methodology:                                                                                                                                                                                                                                              fermentation time (h)


• Monitoring of host cell metabolic load due to overexpression of recombinant protein                                                                                                                                                                        Figure 1
• Monitoring the increase of plasmid replication occuring at high recombinant protein
  expression rates                                                                                                                                                                      300                                                                                                                            1,0

• Control of plasmid replication                                                                                                                                                        250
                                                                                                                                                                                                                                                                                                                       0,9
                                                                                                                                                                                                                                                                                                                       0,8




                                                                                                                                                             BDM, qP, PCN, rhSOD
• Tuning the expression rate to the load limits of the cell factory                                                                                                                                                                                INDUCTION                              ppGpp(μmol/gBDM)
                                                                                                                                                                                                                                                                                                                       0,7
                                                                                                                                                                                        200
                                                                                                                                                                                                                                                                                                     BDM(g)            0,6




                                                                                                                                                                                                                                                                                                                                              ppGpp
                                                                                                                                                                                        150                                                                                                                            0,5
Monitoring metabolic load                                                                                                                                                                                                                                                                    10*total rhSOD(g)         0,4
                                                                                                                                                                                        100
Metabolic load is a very unspecific state variable. However, significant information can be gained                                                                                                                                                                                                                     0,3

from the hierarchically organised regulatory networks, highly involved in stress response                                                                                                         50
                                                                                                                                                                                                                                                                                                  5*qP(mg/g,h)         0,2
                                                                                                                                                                                                                                                                                                                       0,1
mechanisms (Lengeler et al., 1999). These complex regulatory entities, acting on the highest level of
                                                                                                                                                                                                           0                                                                                                           0,0
regulation, co-ordinate the activity of widely distributed genes by formation of highly specific                                                                                                               22              24            26        28       30      32      34          36        38          40
signal molecules, such as guanosinetetraphosphate (ppGpp), the key molecule of the stringent                                                                                                                                                                fermentation time (h)
regulatory network. The aptitude of ppGpp to monitor metabolic load is shown in Figure 2.
                                                                                                                                                                                                                                                             Figure 2


Monitoring plasmid replication                                                                                                                                                                                 120


Monitoring of plasmid copy number (PCN) during recombinant protein production is important,                                                                                                                    100
                                                                                                                                                                                                                                              START FEED                             INDUCTION
because PCN determines the transcription of foreign DNA. Furthermore, PCN is increased drama-
                                                                                                                                                                                    PCN (real and model)




                                                                                                                                                                                                                   80
tically at high expression rates, because enhanced levels of uncharged tRNAs resulting from amino
                                                                                                                                                                                                                   60
acid starvation interact with key molecules of plasmid replication control of ColE1 plasmids and                                                                                                                                    PCN model

thereby increase metabolic burden. To circumvent this problem a two step strategy was developed:                                                                                                                   40
• online monitoring of plasmid copy number (PCN) by neural network based modelling using easily                                                                                                                    20                                                PCN real
  available online data sets (Figure 3) and
• a molecular biology based solution to make plasmid replication independent from expression rate                                                                                                                      0

  by ori modification (Grabherr, et al., 2000).                                                                                                                                                                             12          14        16        18       20    22     24         26       28         30                          32
                                                                                                                                                                                                                                                                 fermentation time (h)

Tuning of expression rate
                                                                                                                                                                                                                                                             Figure 3
To achieve optimal exploitation of the cell factory the rate of recombinant protein production has to
be tuned in relation to metabolic load and plasmid copy number. An effective approach is the
downregulation of transcription of a strong expression vector, such as the widely used T7 system,                                                                                                                                                                                  total IPTG(μmol)         2,5
by titration of inducer in relation to the repressor. To determine the optimal amount of inducer a
                                                                                                                                                                                                                                                                                                                       IPTG related to BDM


                                                                                                                                                                                                    300                                                      IPTG related to BDM
                                                                                                                                                                                                                                                               ( μmol/gBDM)                                 2,0
time shifted exponential feed regime of substrate and inducer was used in fed batch fermentation.
                                                                                                                                                              BDM , total IPTG




As shown in the simulation (Figure 4) the effect of increasing ratios of inducer to biomass in a range                                                                                              200                                 inducer dosage                                                      1,5
                                                                                                                                                                                                                                         into the media
from zero to maximum can be gained in a single experiment. Overdose of inducer is derived from
                                                                                                                                                                                                                           feed start
                                                                                                                                                                                                                                                                                                            1,0
significant deviations of biomass vs. the theoretical exponential curve due to the metabolic overload                                                                                               100                                                                               BDM(g)
of recombinant protein production. By the application of this experimental setup it was found that                                                                                                                                                                                                          0,5

the maximum amount of IPTG per g BDM must not exceed 0,9 μmol. In fed batch fermentations of                                                                                                                   0                                                                                            0,0
E.coli HMS174(DE3)pET11ahSOD (Figure 5) using exponential feed in combination with the                                                                                                                             14                   19              24              29           34               39
developed induction regime the expression rate of recombinant model protein (rec. human                                                                                                                                                                fermentation time (h)
superoxidedismutase) can be tuned in a way that the recombinant protein production could be
                                                                                                                                                                                                                                                             Figure 4
maintained during the whole fermentation process. Thereby the capacity of host cell metabolism
was almost fully exploited, ppGpp and PCN do not exceed the required limits. By the application of
this regime the total yield was 2,5 times increased compared to the standard fermentation process.                                                                                           45
                                                                                                                                                                                                                               2,5times increase of recombinant protein                                                   300
                                                                                                                                                                                             40
Conclusions:                                                                                                                                                                                 35
                                                                                                                                                                                                                                                                             200*ppGpp(μmol/g BDM)
                                                                                                                                                                                                                                                                                                                          250
                                                                                                                                                                                                                                                                                                                                                  ppGpp, PCN,BDM




                                                                                                                                                                                             30
Computer application made a significant contribution to the efficiency of process
                                                                                                                                                             totaL rhSOD




                                                                                                                                                                                                                                                                                BDM (g)                                   200
                                                                                                                                                                                             25
development by :                                                                                                                                                                             20
                                                                                                                                                                                                                                                                                                                          150

• Enabling monitoring of complex variables by a neural network based modelling approach                                                                                                      15
                                                                                                                                                                                                                                                   PCN
                                                                                                                                                                                                                                                                                                                          100
• Determination of optimal amounts of inducer                                                                                                                                                10
                                                                                                                                                                                                                                                                                                                          50
• Application of exponential substrate feed in combination with control of induction                                                                                                                       5                                                                        total rhSOD (g)
                                                                                                                                                                                                           0                                                                                                              0
                                                                                                                                                                                                               20                         25                30              35                       40
References:                                                                                                                                                                                                                                            fermentation time (h)
Lengeler J.W., Drews G. and Schlegel H.G., Biology of the Prokaryotes, (1999) Georg Thieme Verlag Stuttgart
Grabherr R. , Nilsson E. and Bayer K., Expression vectors with modified ColE1 origin of replication for control of plasmid copy number (EP 00121709.01222)                                                                                                   Figure 5

Acknowledgements
This work was supported in part by a grant from the Jubilaeumsfonds der Oesterreichischen Nationalbank and by Boehringer Ingelheim Austria with support from Austrian Industrial Research Promotion Fund .

				
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