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Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system.

Ruottinen M, Bollok M, Kögler M, Neubauer A, Krause M, Hämäläinen ER, Myllyharju J, Vasala A, Neubauer P - BMC Biotechnol. (2008)

Bottom Line: The problem can be solved by applying the fed-batch technology.The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel.By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioprocess Engineering Laboratory, Dept, of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, FIN-90014 University of Oulu, Finland. maria.ruottinen@aurorabiotec.com

ABSTRACT

Background: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.

Results: By applying on-line pO2 monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.

Conclusion: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

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Related in: MedlinePlus

Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a P. pastoris for production of recombinant human collagen II in shake flasks. Cultivation procedures with two methanol pulses per day (control, blue open circles, interrupted blue line) and pO2-dependent manual feeding of methanol (red filled circles, red continuous line) were compared.
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Figure 1: Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a P. pastoris for production of recombinant human collagen II in shake flasks. Cultivation procedures with two methanol pulses per day (control, blue open circles, interrupted blue line) and pO2-dependent manual feeding of methanol (red filled circles, red continuous line) were compared.

Mentions: We have shown previously by applying on-line monitoring sensors in shake flasks that the commonly used methanol feeding protocol for shake flask cultures of P. pastoris (two manual pulses of methanol per day) leads to long starvation phases between feeding pulses [15]. Starvation is obvious by a pO2 peak after exhaustion of methanol, related to declining respiratory activity (Figure 1A). We concluded from this data that the methanol feeding in this shake flask process is not optimal. In the first step of optimization, the rapidly increasing pO2 level after consumption of methanol was used as a signal to manually add a new methanol pulse. Each single methanol dose was the same as in the standard protocol and consequently much more methanol was added during the course of the cultivation. Although the procedure was laborious and difficult to reproduce manually, a 30% higher cell density was obtained and the product related mRNAs showed higher expression, such as the mRNAs encoding alcohol oxidase 1 (AOX), formaldehyde dehydrogenase, yeast PDI, type II procollagen chain and the C-P4H α(I) and PDI/β subunits (Figure 1B). Unexpectedly, the amount of collagen II was not increased, however (not shown).


Improved production of human type II procollagen in the yeast Pichia pastoris in shake flasks by a wireless-controlled fed-batch system.

Ruottinen M, Bollok M, Kögler M, Neubauer A, Krause M, Hämäläinen ER, Myllyharju J, Vasala A, Neubauer P - BMC Biotechnol. (2008)

Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a P. pastoris for production of recombinant human collagen II in shake flasks. Cultivation procedures with two methanol pulses per day (control, blue open circles, interrupted blue line) and pO2-dependent manual feeding of methanol (red filled circles, red continuous line) were compared.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2315644&req=5

Figure 1: Growth parameters (A) and concentrations of product-related mRNA species (B) during cultivation of a P. pastoris for production of recombinant human collagen II in shake flasks. Cultivation procedures with two methanol pulses per day (control, blue open circles, interrupted blue line) and pO2-dependent manual feeding of methanol (red filled circles, red continuous line) were compared.
Mentions: We have shown previously by applying on-line monitoring sensors in shake flasks that the commonly used methanol feeding protocol for shake flask cultures of P. pastoris (two manual pulses of methanol per day) leads to long starvation phases between feeding pulses [15]. Starvation is obvious by a pO2 peak after exhaustion of methanol, related to declining respiratory activity (Figure 1A). We concluded from this data that the methanol feeding in this shake flask process is not optimal. In the first step of optimization, the rapidly increasing pO2 level after consumption of methanol was used as a signal to manually add a new methanol pulse. Each single methanol dose was the same as in the standard protocol and consequently much more methanol was added during the course of the cultivation. Although the procedure was laborious and difficult to reproduce manually, a 30% higher cell density was obtained and the product related mRNAs showed higher expression, such as the mRNAs encoding alcohol oxidase 1 (AOX), formaldehyde dehydrogenase, yeast PDI, type II procollagen chain and the C-P4H α(I) and PDI/β subunits (Figure 1B). Unexpectedly, the amount of collagen II was not increased, however (not shown).

Bottom Line: The problem can be solved by applying the fed-batch technology.The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel.By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

View Article: PubMed Central - HTML - PubMed

Affiliation: Bioprocess Engineering Laboratory, Dept, of Process and Environmental Engineering, University of Oulu, P.O. Box 4300, FIN-90014 University of Oulu, Finland. maria.ruottinen@aurorabiotec.com

ABSTRACT

Background: Here we describe a new technical solution for optimization of Pichia pastoris shake flask cultures with the example of production of stable human type II collagen. Production of recombinant proteins in P. pastoris is usually performed by controlling gene expression with the strong AOX1 promoter, which is induced by addition of methanol. Optimization of processes using the AOX1 promoter in P. pastoris is generally done in bioreactors by fed-batch fermentation with a controlled continuous addition of methanol for avoiding methanol toxification and carbon/energy starvation. The development of feeding protocols and the study of AOX1-controlled recombinant protein production have been largely made in shake flasks, although shake flasks have very limited possibilities for measurement and control.

Results: By applying on-line pO2 monitoring we demonstrate that the widely used pulse feeding of methanol results in long phases of methanol exhaustion and consequently low expression of AOX1 controlled genes. Furthermore, we provide a solution to apply the fed-batch strategy in shake flasks. The presented solution applies a wireless feeding unit which can be flexibly positioned and allows the use of computer-controlled feeding profiles. By using the human collagen II as an example we show that a quasi-continuous feeding profile, being the simplest way of a fed-batch fermentation, results in a higher production level of human collagen II. Moreover, the product has a higher proteolytic stability compared to control cultures due to the increased expression of human collagen prolyl 4-hydroxylase as monitored by mRNA and protein levels.

Conclusion: The recommended standard protocol for methanol addition in shake flasks using pulse feeding is non-optimal and leads to repeated long phases of methanol starvation. The problem can be solved by applying the fed-batch technology. The presented wireless feeding unit, together with an on-line monitoring system offers a flexible, simple, and low-cost solution for initial optimization of the production in shake flasks which can be performed in parallel. By this way the fed-batch strategy can be applied from the early screening steps also in laboratories which do not have access to high-cost and complicated bioreactor systems.

Show MeSH
Related in: MedlinePlus