Abstract
BackgroundHere 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.ResultsBy 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.ConclusionThe 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.
Highlights
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
It has been demonstrated that P. pastoris is an efficient production system for very large and complex proteins, such as collagens, which besides the recombinant gene(s) needed for the collagen polypeptide chain(s) needs the parallel expression of two different genes coding for collagen prolyl 4-hydroxylase (C-P4H), an enzyme required for the thermal stability of collagens [7,8,9]
Results pO2 level-dependent manual feeding of methanol improves expression 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 leads to long starvation phases between feeding pulses [15]
Summary
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. Different promoter systems exist for a controlled or continuous expression of heterologous proteins in P. pastoris, frequently the strong and tightly controlled promoter of alcohol oxidase 1 (AOX1) is applied. Aside from being the inducer of the promoter is a carbon/ energy substrate In such P. pastoris processes, methanol is the only carbon substrate during the production phase of the AOX1-promoter controlled protein. Methanol concentrations above 3.6% inhibit the yeast growth and lead to death [2]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
