Fermentation control process for Mut<s> type recombinant Pichia pastoris

A technology of Pichia pastoris and fermented liquid, which is applied in the direction of microorganism-based methods, measurement/inspection of microorganisms, microorganisms, etc., and can solve the problems of slow methanol utilization rate, easy drift and failure, etc.

Active Publication Date: 2017-07-04
WILMAR SHANGHAI BIOTECH RES & DEV CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The first two host bacteria are the most widely used, and most of the current methanol feeding control processes are aimed at Mut + type, such as DO-stat, q s -stat etc. to control the rate of methanol feeding, while Mut s Due to the slow methanol utilization rate of Pichia type pastoris, DO-stat, q s -stat and other control strategies are invalid, so the reasonable control of methanol feeding is Mut s Difficulties in the fermentation of Pichia pastoris can only be (1) through experience to control the feeding of methanol (conventional empirical control process), such as pulse feeding to observe the change of DO, if DO drops after feeding, then mechanically increase methanol flow Acceleration rate, or (2) Feedback control of methanol concentration in a constant state through methanol online detector [Xie Jingli, Zhang Li, Ye Qin, Zhou Qingwei, Xin Li, Du Peng, Gan Renbao, 2003, Mut s The

Method used

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  • Fermentation control process for Mut&lt;s&gt; type recombinant Pichia pastoris
  • Fermentation control process for Mut&lt;s&gt; type recombinant Pichia pastoris
  • Fermentation control process for Mut&lt;s&gt; type recombinant Pichia pastoris

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Embodiment 1 adopts a single DO control strategy.

[0157] Such as figure 1 As shown, the whole fermentation process is divided into three stages, 0-24h is the glycerol batch fermentation stage, DO is reduced from the initial 100% to 14.1%, 24-28h is the glycerol feeding stage, DO% is maintained at 15-20%, After the wet weight of the bacteria reaches 200g / L, pulse 1g / L methanol every 1h for 4 times, so that Pichia pastoris enters the methanol adaptation period. During the adaptation period, the DO is kept at about 70-80%, and the DO drops rapidly. to 61.5%, indicating that Pichia pastoris has been able to adapt to and utilize methanol well, and the methanol feeding stage is started. The feeding control of methanol is controlled by DO feedback, and the DO is maintained at 55-65% by adjusting the methanol feeding rate between until the end of fermentation.

[0158] The fermentation results of methanol flow rate controlled by DO feedback are as follows figure 1 As shown...

Embodiment 2

[0160] Embodiment 2 adopts the strategy of separate OUR control.

[0161] Such as figure 2 As shown, the whole fermentation process is divided into three stages, 0-24h is the glycerol batch fermentation stage, OUR rises from the initial 0mmol / L / h to 131.71mmol / L / h, 24-28h is the glycerol feeding stage, and OUR maintains At 117-131mmol / L / h, after the wet weight of the bacteria reaches 200g / L, pulse 1g / L of methanol every 1h for 4 times, so that Pichia pastoris enters the methanol adaptation period, and the OUR keeps decreasing during the adaptation period. The lowest dropped to 21.3mmol / L / h, and then began to rebound to 50mmol / L / h, and then OUR entered a plateau, indicating that Pichia pastoris has been able to adapt and utilize methanol well, and began to carry out the methanol feeding stage, methanol The feeding control of OUR is controlled by OUR feedback, and OUR is maintained between 45-50mmol / L / h by adjusting the methanol feeding rate until the end of fermentation.

[...

Embodiment 3

[0164] Embodiment 3 adopts the strategy of methanol concentration feedback control.

[0165] Such as image 3 As shown, the whole fermentation process is divided into three stages, 0-24h is glycerol batch fermentation stage, Pichia pastoris uses glycerol as carbon source, 24-28h is glycerol feed stage, Pichia pastoris still uses glycerol as carbon source, bacteria After the wet weight of the body reaches 200g / L, the Pichia pastoris enters the methanol adaptation period by pulse-filling 1g / L methanol every 1h for 4 times. Concentration feedback controls the concentration of methanol in the fermentation broth at 3-5g / L until the end of fermentation.

[0166] The final fermentation result of methanol flow rate controlled by methanol concentration feedback is as follows: image 3 As shown, it can be seen that the final yield is 2238U / ml.

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Abstract

The invention relates to a fermentation control process for Mut<s> type recombinant Pichia pastoris. Specifically, the invention relates to a method for determining whether methanol is excess or insufficient in the process of fermentation of Mut<s> type Pichia pastoris. The method comprises the following steps: adding methanol into fermentation broth every 3 to 18 h in the fermentation process when feeding at a basic methanol supplementation rate of 0.8 to 1.8 g/L/h is carried out, wherein 1.0 to 2.0 g of methanol is added for each L of the fermentation broth; and detecting the changes of DO and OUR of the fermentation broth in a designated period of time after addition of the methanol relative to the DO and OUR of the fermentation broth before addition of the methanol so as to determine whether the methanol in the fermentation broth is excess or insufficient. Compared with control with conventional processes, the fermentation control process provided by the invention enables the output of heterologous protein to be increased by 2 to 5 times.

Description

technical field [0001] The invention belongs to the field of microbial fermentation, in particular to Mut s Fermentation control process of recombinant Pichia pastoris. Background technique [0002] Pichia methanolica is the most important platform for expressing heterologous proteins. Currently, more than 600 recombinant proteins can be expressed in Pichia pastoris [Zhang AL, Luo JX, Zhang TY, Pan YW, Tan YH, Fu CY and Tu FZ, 2009, Recent advances on the GAP promoter derived expression system of Pichia pastoris, Mol Biol Rep 36:1611-1619]. The optimal control of methanol feeding rate during fermentation is the key to ensure the maximum expression of heterologous protein activity. [0003] Pichia methanolica is divided into three types according to the destruction of alcohol oxidase genes (AOX1 and AOX2) during recombination [Looser V, Bruhlmann B, Bumbak F, Stenger C, Costa M, Camattari A., Fotiadis D, Kovar K., 2015, Cultivation strategies to enhance productivity of Pic...

Claims

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Application Information

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IPC IPC(8): C12Q3/00C12R1/84
CPCC12Q3/00
Inventor 杨倚铭关惠琴吴清杭杰西卡·弗吉尼亚
Owner WILMAR SHANGHAI BIOTECH RES & DEV CENT
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