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

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...

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 control of methanol feeding during high-density fermentation of recombinant Pichia pastoris, "Journal of Wuxi University of Light Industry", 22(1):12-15; Zhou Xiangshan, Fan Weimin, Zhang Yuanxing, 2002, different methanol feeding strategies The impact on hirudin produced by high-density fermentation of recombinant Pichia pastoris, "Journal of Bioengineering" 18(3):348-351], generally controlled within 3g / L [same as [Looser V, etc.], but methanol online detector is easy Affected by other volatile components, it is very easy to drift and fail [Gao Minjie, Research on the fermentation process control of Pichia pastoris based on intelligent engineering and metabolic regulation, doctoral dissertation, Wuxi: Jiangnan University, 2012], which often leads to the metabolism of Pichia pastoris Activity is inhibited by high concentrations of methanol, which greatly affects the yield of heterologous proteins

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