A Preliminary Study On The Heterologous Expression Of Insulin Glargine By The Filamentous Fungus Trichoderma Reesei

In 2019,approximately 463 million adults worldwide have diabetes,which is equivalent to 1 out of 11 people.Insulin drugs are an essential life-saving medicine for people with diabetes.Insulin Glargine(IG,long-acting insulin drugs),as an important insulin drug,can effectively prolong the action time of insulin in the human body,and has great commercial value.In 2017 alone,the sales of Thermo Fisher’s IG drugs reached 6.72 billion U.S.dollars.The rapid increase in the number of diabetic patients and the rapid increase in the commercial value of insulin poses new challenges to the high-level production of IG,and the IG production system needs to be optimized.The filamentous fungus Trichoderma reesei is a potential excellent host for heterologous protein production.On the one hand,Trichoderma reesei can secrete a large amount of extracellular protein.According to reports,the maximum output of extracellular protein of Trichoderma reesei can reach 100g/L;On the other hand,Trichoderma reesei can also produce different kinds of proteins,and has a wide range of applications in the production of industrial enzymes,etc.It is particularly noteworthy that in Trichoderma reesei,the catalytic domain of cellobiohydrolase Ⅰ(CBH1,or Cellobiohydrolase Ⅰ)has been used as a carrier protein to successfully produce mammalian small-molecule proteins.In order to improve the production of heterologous proteins from Trichoderma reesei,different carrier proteins such as cellobiohydrolase Ⅱ(CBH2,or Cellobiohydrolase Ⅱ)were used for protein expression experiments.In addition,the use of signal peptides with high secretion ability(such as CBH1 signal peptide,A.niger glucoamylase signal peptide,A.awamori α-amylase signal peptide,etc.)can also increase the production of heterologous proteins to a certain extent.In this paper,the filamentous fungus Trichoderma reesei was used as the IG expression and secretion system,and different expression strategies were used to study the expression and secretion of IG protein in Trichoderma reesei.The specific research content is as follows:(1).An attempt to directly express and secrete IG in Trichoderma reesei using a strong promoter and signal peptide.The IG protein expression strain S1r was constructed using the strong constitutive promoter Pcdna1 of Trichoderma reesei and the high-efficiency CBH1 secretion signal peptide;the IG protein expression strain S1HCr was constructed using Pcdna1,CBH1 signal peptide,modified insulin C peptide and His tag.The detection of the transcription level of the strains showed that the above-mentioned IG protein expression strains all transcribe and expressed the IG gene,but the IG protein could not be detected in the fermentation broth.In order to find the reason why the IG protein cannot be secreted,the transcription levels of the unfolded protein response(UPR response),the endoplasmic reticulum-related protein degradation pathway(ERAD pathway),and protein folding monitoring element of S1r and S1HCr strains were analyzed.The results showed that the UPR response-related genes pdil and bipl of the S 1r strain were 7.3 times and 6.4 times that of the control strain,respectively.The S1r and S1HCr strains activated the ERAD degradation pathway to a certain extent.At the same time,the expression of protein folding monitor element in S1r and S1HCr strains was also up-regulated.The above results indicate that the IG protein may not complete the correct folding during the process of synthesis and secretion,triggering a UPR response and undergoing intracellular degradation,thus making it undetectable in the fermentation broth.(2).Three carrier proteins(CBH1,CBH2 and artificially synthesized protein LA20)were fused with IG to promote the expression of IG in Trichoderma reesei.CBH1 was used as the carrier protein,and CBH1 signal peptide was selected to construct an IG expression strain.The strain was named S1C1r(CBH1 signal peptide-CBH1 carrier protein).Using CBH2 as carrier protein,CBH1 signal peptide and CBH2 signal peptide were selected to construct IG expression strains.The strains were named S1C2r(CBH1 signal peptide-CBH2 carrier protein)and S2C2r(CBH2 signal peptide-CBH2 carrier protein).At the same time,a strain S1CLr(CBH1 signal peptide-LA20)expressing IG with artificially synthesized protein LA20 as a carrier protein combined with CBH1 signal peptide was constructed.After shaking flask fermentation and qPCR detection,it was found that the above-mentioned strains successfully expressed carrier protein and IG protein at the transcriptional level.Identification by SDS-PAGE and mass spectrometry revealed that no IG secretion was detected in the fermentation broth of S1C2r,S2C2r and S1CLr strains.However,S1C1r successfully detected the secretion of the carrier protein CBH1 and IG protein,especially when the S1C1r strain was cultured in a 1L fermentor,the IG protein secretion was significantly increased.Further testing the expression of genes such as UPR response,ERAD pathway,and protein folding monitoring element found that the transcription of the UPR response-related gene bip1 of S2C2r and S1CLr strains was 2.2 times and 3.3 times that of the control strain,and the transcription of pdi1 was 3.4 times and 3.6 times that of the control strain.The transcription levels of the protein folding monitoring element related genes slp1 and emp65 have been increased to varying degrees.At the same time,the transcription levels of genes related to the ERAD degradation pathway have also been up-regulated,indicating that the UPR response in this strain has been activated,and Induced the ERAD pathway.In the S1C2r strain,the UPR response was not activated,but the ERAD pathway was induced.These results indicate that SlC2r,S2C2r and S1CLr did not detect the secretion of IG protein,which may be due to the protein degradation related to the intracellular endoplasmic reticulum of IG,resulting in the inability to detect IG protein in the fermentation broth.It is worth noting that the UPR response-related gene pdi1 transcription in the S1C1r strain increased by 2.9 times,and the ERAD pathway was not activated,indicating to a certain extent that the CBH1 carrier protein promotes the folding and secretion of the IG protein.The above results indicate that the selection of different carrier proteins has an important impact on the expression and secretion of IG.After that,the strains SAC1r and SGC1r were constructed.After shaking flask fermentation and qPCR detection,it was found that the above-mentioned strains successfully expressed the carrier protein CBH1 and IG protein at the transcription level,but the IG protein gene transcription amount in the SAC1r strain was lower than that of SGC1r.SDS-PAGE analysis and mass spectrometry analysis revealed that no IG protein was detected in the fermentation broth of SAC1r strain,but IG protein was successfully detected in the fermentation broth of SGC1r.Further examination of the transcription level of genes such as UPR response,ERAD pathway and protein folding monitoring element found that the UPR response of SGClr strain was strongly activated,the bip1 gene was increased by 6.8 times,and the pdi1 gene was increased by 4.8 times;the transcription of protein folding monitoring element emp65 increased by 11 times.The UPR pathway and protein folding monitoring of SGC1r strain are activated,which may promote the folding of intracellular IG protein.(3)The strategy of using three reporter proteins(Aspergillus niger β-glucosidase BGLA,Trichoderma reesei endoglucanase EG2 and EG3)to fuse with IG protein to improve the screening of Trichoderma reesei IG expression and secretion strains effectiveness.Using BGLA as the reporter protein,select the BGLA-IG protein gene expression sequence to construct the strain and name it BgIr(BGLA-IG protein);use the IG protein-BGLA gene expression sequence to construct the strain and name it IBgr(IG protein-BGLA).Using EG2 as the reporter protein,the strain was constructed in the order of EG2-IG protein gene expression and named E2Ir(EG2-IG protein);the strain was constructed in the order of IG protein-EG2 gene expression and named IE2r(IG protein-EG2).Using EG3 as the reporter protein,the strain was constructed in the order of EG3-IG protein gene expression and named E3Ir(EG3-IG protein);the strain was constructed in the order of IG protein-EG3 gene expression and named IE3r(IG protein-EG3).Using the CMC-esculin glucose color plate,4 strains secreting BGLA reporter protein were successfully screened and named:BgI-2-18,BgI-2-12;IBg-193,IBg-280.Using the CMC glucose plate,9 strains secreting EG2 and 18 strains secreting EG3 reporter protein were successfully screened;the ratio of transparent circle to colony diameter was further measured and calculated,and 6 strains of E2Ir with higher secretion of reporter protein were selected.5 strains of E3Ir strain and 5 strains of IE3r strain.Through shake flask fermentation and qPCR detection of the selected strains,it was found that the reporter protein gene was successfully transcribed in the selected strains,and the IG protein gene was successfully transcribed except for the strain IBg-280.SDS-PAGE analysis of the fermentation broth revealed that all the screened strains except for the IE3r strain successfully secreted the reporter protein,but no obvious IG protein band was detected.The UPR response of some strains and the transcription of ERAD pathway genes were further analyzed,and it was found that using reporter protein to express IG protein may activate the UPR and ERAD pathways of strains,thereby affecting the secretion of IG protein.In this part of the study,the reporter protein and IG protein fusion expression and secretion strains were successfully constructed,which laid a good foundation for subsequent strain fermentation optimization,IG protein identification and in-depth protein secretion mechanism analysis.