Construction Of Transcriptional Signal Amplification Devices In Pichia Pastoris And Their Application For Heterologous Protein Expression

Pichia pastoris is one of the most widely used heterologous expression host for many advantages both in laboratory research and industrial production.Currently,the induction fermentation production of exogenous protein is carried out mainly by using the naturally methanol-inducible AOX1 promoter（PAOX1）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.Many studies have been devoted to the improvement of PAOX1 transcriptional regulation system,or find other naturally methanol-independent promoters in Pichia pastoris,but the effects are not ideal.The expression intensity and regulation sensitivity are not as good as PAOX1.With the development of synthetic biology,based on the identified PAOX1 transcriptional regulation mechanism,the directional modification of PAOX1 provides new ideas and methods for solving this problem.The transcriptional activators and inhibitory factors related to methanol metabolism in Pichia pastoris have been identified in our previous work.In addition,we have clarified the methanol-signaling pathway and deduced the transcriptional regulation model of PAOX1.On the basis of previous studies,we designed and verified two sets of artificial transcriptional activation devices in Pichia pastoris based on CRISPR-dCas9 and lactose operon elements,combined with PAOX1-related transcriptional regulatory elements.The CSAD₅ device based on the design of lactose operon components showed good transcriptional activation under various carbon sources such as glucose,glycerol and methanol,and reached about 5 times of the intensity of PAOXI and about 13 times of the intensity of PGAP under the same conditions,far exceeding the highest reported level to present.In order to further verify the ability of CSAD 5 device to drive heterologous protein expression,three different exogenous proteins,i.e.,human insulin precursor,amylase and human hepatitis B surface antigen,were selected.We expressed the three proteins in shaker level under glucose and methanol conditions,and compared the differences between the new device and traditional PAOX1 methanol-induced system.The results showed that CSAD₅ device could efficiently drive the expression of heterologous proteins whether methanol or glucose as a carbon sources.In the condition of glucose as a carbon source,the intensity of CSAD 5 device driving human insulin precursor,amylase and human hepatitis B surface antigen was 1.3 times,1.7 times and 8.4 times higher than that of PAOX1 system,respectively.In the condition of methanol as a carbon source,the intensity of the CSAD₅ device to drive human insulin precursor and amylase expression was comparable to that of the PAOX1 methanol-induced system.The intensity of the CSAD 5 device to drive human hepatitis B surface antigen expression was about 8 tirmes higher than that of PAOX1 system.In addition,the fermentation process of CSAD₅-driven human insulin precursor expression was explored in a 5-L bioreactor in the low-flow methanol feed process and the glucose-carbon source process.After optimization,a perfect fermentation process using glucose as a carbon source was established.The whole fermentation process used glucose as a carbon source,and glucose was added at a flow rate of 15.5 ml/（h·L broth）at the end of the batch.Compared with the methanol-induced process,the new process is simpler in control,lower in oxygen consumption,and less in heat generation when maintaining comparable yield.Through increasing module-specific antibiotic stress to optimize the biosynthetic gene dosages,a recombinant Pichia pastoris strain producing 1.85 g/L insulin precursor was screened in 108 h 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.