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中国生物工程杂志

China Biotechnology
China Biotechnology
China Biotechnology  2019, Vol. 39 Issue (7): 48-55    DOI: 10.13523/j.cb.20190707
    
Heterologous Expression of Insulin Precursor in A Newly Engineered Pichia pastoris
Qiang-qiang PENG,Qi LIU,Ming-qiang XU,Yuan-xing ZHANG,Meng-hao CAI()
State Key Laboratory of Bioreactor Engineering, East China University of Science & Technology, Shanghai 200237, China
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Abstract  

Pichia pastoris is one of the most widely used heterologous expression host in the synthesis of human insulin precursors both in laboratory research and industrial production. Currently, the induction fermentation production of insulin precursors is carried out mainly by using the naturally methanol-inducible AOX1 promoter and utilizing methanol as the sole carbon source. High oxygen consumption, heat release and complicated feeding process control caused by methanol catabolism usually bring difficulties to industrial scale-up and limited application for protein expression by high cell densities fermentation in Pichia pastoris. A synthetic efficient constitutive transcriptional signal amplification device of CSAD_5 was constructed to drive insulin precursor gene expression based on the transcriptional regulation design of the AOX1 promoter in our previous work. In order to reduce the oxygen consumption, heat generation and bioprocess regulation during fermentation process, a glucose supported fermentation process is established. Through increasing module-specific antibiotic stress to optimize the biosynthetic gene dosages, a recombinant Pichia pastoris strain producing 1.85g/L insulin precursor was screened in 108h in a 5-L bioreactor, which is the highest productivity to produce human insulin precursor by utilizing glucose as a carbon source according to reports. Our findings provide a new alternative choice to the high-yield production of insulin precursor in industrial application and reveal the potential of Pichia pastoris to produce a wide range of compounds.

Key words： Pichia pastoris      Insulin precursor      Fermentation process      High-producing strains     
Received: 30 November 2018      Published: 05 August 2019
ZTFLH:  Q815  
Corresponding Authors: Meng-hao CAI     E-mail: cmh022199@ecust.edu.cn
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Qiang-qiang PENG
Qi LIU
Ming-qiang XU
Yuan-xing ZHANG
Meng-hao CAI
Cite this article:

Qiang-qiang PENG,Qi LIU,Ming-qiang XU,Yuan-xing ZHANG,Meng-hao CAI. Heterologous Expression of Insulin Precursor in A Newly Engineered Pichia pastoris. China Biotechnology, 2019, 39(7): 48-55.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20190707     OR     https://manu60.magtech.com.cn/biotech/Y2019/V39/I7/48

Fig.1 Production of insulin precursor of WT-IP and CSAD_5-IP in baffled flask culture Error bars are plotted from 3 individual replicates. The asterisk marks indicate the significance levels between WT-IP and CSAD_5-IP at the same time points (* P<0.05; NS, not significant)
Fig.2 Wet cell weight (WCW) and insulin precursor (IP) expression of CSAD_5-IP and WT-IP at different methanol feeding rates in a 5L bioreactor fermentation Three different methanol feeding rates of 3.33ml/(h·L broth), 6.67ml/(h·L broth), and 12.0ml/(h·L broth) were used for CSAD_5-IP and an optimal methanol feeding rate of 12.0ml/(h·L broth) was used for WT-IP in induction phase. Error bars are plotted from 3 individual replicates
Fig.3 Wet cell weight (WCW) and insulin precursor (IP) expression of CSAD_5-IP at different glucose feeding rates in a 5-L bioreactor fermentation Three different glucose feeding rates is 11.5ml/(h·L broth), 15.5ml/(h·L broth), and 21.5ml/(h·L broth), respectively. Error bars are plotted from 3 individual replicates
Fig.4 Comparison of fermentation process for recombinant protein expression in the WT-IP and the CSAD_5-IP strains (a) WT-IP (b) CSAD_5-IP Time profiles of dissolved oxygen (DO), rotational speed, wet cell weight (WCW), insulin precursor (IP) in a 5L bioreactor fermentation
Fig.5 Comparison of insert air flow for recombinant protein expression in the WT-IP and the CSAD_5-IP strains (a)WT-IP (b) CSAD_5-IP Time profiles of total flow and 99.999% oxygen flow in a 5L bioreactor fermentation
Fig.6 Production of insulin precursor of CSAD_5 -IP1~CSAD_5-IP10 in baffled flask culture Error bars are plotted from 3 individual replicates. The asterisk marks indicate the significance levels between CSAD_5-IP and high-producing strains in the same time (* P<0.05 NS, not significant)
Fig.7 The fermentation process of the CSAD_5-IP10 strains in recombinant protein expression in a 5 L bioreactor Time profiles of dissolved oxygen (DO), rotational speed, wet cell weight (WCW), insulin precursor (IP). Error bars are plotted from 3 individual replicates
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