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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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mRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask cultivation of P. pastoris. Predetermined quasi-continuous feed of methanol was investigated. The cells were first grown in BMG medium and changed to BMM medium at 0 h. The first sample represents the time when the methanol feeding was started (13 h). Predetermined constant feed (red filled circles); reference culture with pulse Feeding (open blue circles). The data are from experiment 4 in Table 1.
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Figure 6: mRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask cultivation of P. pastoris. Predetermined quasi-continuous feed of methanol was investigated. The cells were first grown in BMG medium and changed to BMM medium at 0 h. The first sample represents the time when the methanol feeding was started (13 h). Predetermined constant feed (red filled circles); reference culture with pulse Feeding (open blue circles). The data are from experiment 4 in Table 1.

Mentions: The effect of the feeding procedure on the formation of the product was investigated in more detail at the mRNA level. A quantitative sandwich hybridization assay which was developed earlier in our laboratory was applied [21,22]. Figure 6 shows the levels of mRNAs encoding the procollagen II chain, AOX1, EF3 (translational factor), and C-P4H. The level of procollagen II mRNA was already high during the first 12 h when the cultures were grown in a batch mode on methanol in both types of cultivations. In the reference culture with pulse feeding, the procollagen II mRNA level decreased to a very low level after the start of the feeding. In contrast, the mRNA level stayed high during the whole cultivation in the culture with quasi-continuous feeding of methanol, being approximately 10 times higher when compared to the pulse feed method.


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)

mRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask cultivation of P. pastoris. Predetermined quasi-continuous feed of methanol was investigated. The cells were first grown in BMG medium and changed to BMM medium at 0 h. The first sample represents the time when the methanol feeding was started (13 h). Predetermined constant feed (red filled circles); reference culture with pulse Feeding (open blue circles). The data are from experiment 4 in Table 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: mRNA levels of a) procollagen II chain, b) AOX1, c) translation factor EF3, and d) C-P4H α(I) subunit during shake flask cultivation of P. pastoris. Predetermined quasi-continuous feed of methanol was investigated. The cells were first grown in BMG medium and changed to BMM medium at 0 h. The first sample represents the time when the methanol feeding was started (13 h). Predetermined constant feed (red filled circles); reference culture with pulse Feeding (open blue circles). The data are from experiment 4 in Table 1.
Mentions: The effect of the feeding procedure on the formation of the product was investigated in more detail at the mRNA level. A quantitative sandwich hybridization assay which was developed earlier in our laboratory was applied [21,22]. Figure 6 shows the levels of mRNAs encoding the procollagen II chain, AOX1, EF3 (translational factor), and C-P4H. The level of procollagen II mRNA was already high during the first 12 h when the cultures were grown in a batch mode on methanol in both types of cultivations. In the reference culture with pulse feeding, the procollagen II mRNA level decreased to a very low level after the start of the feeding. In contrast, the mRNA level stayed high during the whole cultivation in the culture with quasi-continuous feeding of methanol, being approximately 10 times higher when compared to the pulse feed method.

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