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

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.

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.

Figure 2

Schematic presentation of the wireless data collection and control system for shake flask cultivations.

Figure 3

P. pastoris cultivation in shake flasks with methanol feeding without (A, B) or with (C, D) manual pH adjustment. Cultivations were performed in two phases: initial batch phase in BMG-medium and fed-batch in BMM-medium. Dissolved oxygen (pO₂) and pH were measured with a wireless measuring system, cell growth was followed by measurement of the OD₆₀₀. Vertical lines represent methanol feeding points in A and C or start of methanol feed in B and D.

Figure 4

SDS-PAGE analysis of expression of human collagen II in P. pastoris shake flask cultivation (experiment 3, Table 1) after 21, 46 and 72 hours cultivation. R represents a reference culture with pulse feeding of methanol and F predetermined quasi-continuous feed. Collagen chains were derived from correctly folded collagen II molecules by HCl-extraction and pepsin digestion. The collagen II chains are marked with an arrow.

Figure 5

C-P4H activity in three different experiments, each with a quasi-continuous feeding culture (black bars) and a reference culture (manual feeding twice a day, white bars). Additionally, in experiment no. 4 double concentrated methanol was fed (grey bar).

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.