Regulation Research Of Highly Secreted Protein Gene From Aspergillus Niger CICC2462

Since the genetic engineering technology being established,construction of a new-type recombinant protein expression system has been a hotspot in the field of biological research and development.Escherichia coli expression system and Pichia expression system have been widely applied and created a huge commercial value.But so far,the deficient of protein expression system with intellectual property rights is the significant bottleneck of domestic industrial protein development.In order to solve this problem,we need to study the molecular basis of the protein expression system and establish the accurate regulatory technique,which is also the hotspot of international competition in this field.Excellent expression and secretion ability is one of the most striking characteristics of filamentous fungi which has attracted many people to research.Filamentous fungi is labeled as "cellular factory of eukaryotic protein" for some unique advantages in production of eukaryotic protein.A.niger is one of filamentous fungus and an important industrial organic acids and enzymes producing strain,which is widely used in basic genetic research.The glucoamylase high-producing strain A.niger CICC2462 has been used as a host strain to construct a food-grade A.niger secreting expression system by our research team,which is based on gene replacement technology by the insertion of target genes to the high-level expressing glucoamylase gene site.This system could highly express xylanase,mannanase,feruloyl esterase and so on,which have a clear industrial value.But there still some highly secreted background proteins,the proportion of target proteins to total proteins is very low,which not only limits the purity of target proteins and increasing the cost of target proteins purification but also leads the continuous yield upgrading of target proteins.Thus,it is very important that the total secreted proteins of the overall expression system will be reduced to get a host strain which has a low background of secreted proteins.Meanwhile,strengthen the expression of amylolytic enzyme,to further improve the ability and the purity of A.niger CICC2462 produce glucoamylase,and it have an important research value through obtain high-level expression promoter to increase the production of target protein.The purpose of this study is to identify the major constituents of secretome and expression regulation of this expression system based on analysis of A.niger CICC2462 and recombinant strain PglaA-xynB secretomes under the shake flask fermentation condition.The secretome will be analyzed under the condition of some key regulatory genes(such as creA and amyR)overexpressed or knocked out.And combined with construction and screening high-level expression promoters to regulate the expression of target gene.In order to achieve the regulation of the major secreted proteins from A.niger CICC2462,and to meet the demand of construct different engineering strains.The main research results are shown as follows:1.Secretome analysis of A.niger CICC2462Identification of secretomes from A.niger CICC2462 and recombinant strain PglaA-xynB under the shake flask fermentation condition through two-dimensional electophoresis and shotgun LC-MS/MS proteome technology,respectively.The results showed that the main secretory protein components of A.niger CICC2462 is amylolytic enzyme,including glucoamylase(P69328)and amylase(P56271).The secreted proteins of recombinant strain PglaA-xynB lacked of glucoamylase,increased xylanase,other amylolytic enzymes proteins have no change.The distribution of specific cis elements in control region sequences were analyzed,such as SYGGRG and CGGN8(C/A)GG,and it was initially identified that those genes mainly regulated by carbon metabolism inhibitor CreA and amylolytic enzymes transcriptional activator AmyR.2.Deletion research of amylolytic enzyme positive regulation factor amyRConstruction of positive regulatory factor amyR gene deletion mutant.Shaking fermentation of the wild-type strain A.niger CICC2462 and amyR gene deletion strain with 10%glucose as the sole carbon source was performed.By measuring enzyme activity,SDS-PAGE and shotgun LC-MS/MS proteome measurement of amyR gene deletion strain,it is easy to learn that,compared to wild-type strain A.niger CICC2462,total secreted protein level of amyR gene deletion strain was reduced and the glucoamylase and amylase were significantly reduced.In addition,using RNA-sequencing technology,the transcriptional levels of wild-type strain A.niger CICC2462 and amyR gene deletion strain were compared to each other by transcriptome sequencing analysis.According to the transcriptome data,transcript levels of glucoamylase(An03g06550),a-amylase(Anl2g06930),acid stable a-amylase(Anllg03340)and glucosidase(An04g06920)were significantly reduced,which was further verified by real-time PCR.In addition,with the significant reduction of highly secreted protein genes,some protease-related genes were significantly down-regulated,which was fully confirmed by that activity of the protease in the fermentation supernatant was lower 50%-90%than wild type strain.There are many glucose transport-related down-regulated genes,but their specific regulation mechanisms is unclear.There were a lot of genes related to the oxidative phosphorylation and the citric acid cycle were found in up-regulated genes.The up-regulation of these genes related to the energy metabolism of organisms is a nice indication of well mycelium growth.Through the growth measurement of the wild-type and amyR gene deletion strains cultured in PDA media and shake flask fermentation media,it can be seen that the growth of amyR gene deletion strain was not inhibited and better than wild type.Comprehensive the above results,whether the background knockout or the growth of the mycelium,amyR deletion strain is more suitable for host strain with low secretion background protein.3.Regulation research of carbon metabolism inhibitor factor CreAConstruction of negative regulatory factor creA gene overexpression and deletion mutant.Shaking fermentation of the wild-type strain A.niger CICC2462 and creA gene overexpression strain with 10%glucose as the sole carbon source was performed.By measuring enzyme activity and SDS-PAGE measurement of creA gene overexpression strain,it is easy to learn that,compared to the wild-type strain A.niger CICC2462,total secreted protein level of creA gene overexpression strain was reduced and the glucoamylase and amylase were significantly reduced.In addition,using RNA-sequencing technology,the transcriptional levels of the wild-type strain A.niger CICC2462 and creA gene overexpression strain were compared to each other by transcriptome sequencing analysis.According to the transcriptome data,transcript level of creA gene overexpression strain is apparently higher than the wild-type strain A.niger CICC2462.Transcript levels of glucoamylase(An03g06550),a-amylase(An12g06930),acid stable a-amylase(An11g03340)and glucosidase(An04g06920)were significantly reduced,but significantly higher than amyR deletion strain,which was further verified by real-time PCR.There were a lot of genes related to the glycolysis and the citric acid cycle were found in up-regulated genes,including glyceraldehyde-3-phosphate dehydrogenase(An15g07390),triosephosphate isomerase(An14g04920),citrate synthase(Anl5g01920),acetyl coenzyme A(An04g03290)and isocitric acid acid dehydrogenase(Anl5g02490).etc.But there are many difference from amyR deletion strain that a lot of protease-associated genes were up-regulated in creA overexpression strain.The genes related to apoptosis(An 12g00600)and cell stress response(An 16g04420)were also significantly up-regulation.Many unnamed and putative genes have significant changes in creA overexpression strain,which may play an important role in the regulation of this strain and need to be further,studied.Through the growth measurement of the wild-type and creA overexpression strain cultured in PDA media,it can be seen that the growth of creA gene overexpression strain was apparently inhibited,mycelium growth not only smaller but also slower than the wild-type strain,and the colour was darker.The results prove that creA overexpression strain is unfit for host strain with low secretion background protein compared with amyR deletion strain.Shaking fermentation of the wild-type strain A.niger CICC2462 and creA gene deletion strain with 15%liquefied starch and 15%glucose was performed,respectively.The glucoamylase enzyme activity reached the peak when cultured 7 d.The glucoamylase enzyme activity were 48966.09 U/mL(A.niger CICC2462)and 49121.05 U/mL(creA4)in fermentation supernatant with 15%glucose as the carbon source,the glucoamylase enzyme activity were 56016.59 U/mL(A.niger CICC2462)and 42654.30 U/mL(creA4)in fermentation supernatant with 15%starch as the carbon source,combined with enzymatic activity measurement and SDS-PAGE analysis,it can be seen that,compared with the wild type strain A.niger CICC2462,the glucoamylase enzyme activity of creA gene deletion strain changed small compared with the wild-type strain when with 15%glucose as the carbon source.The results prove that the function of creA gene is weaker in the wild-type A.niger CICC2462 strain,and regulation effect is smaller for amylolytic enzyme after deletion creA gene.4.Research of improving transcription efficiency for glaA promoterThe transcriptome sequencing analysis shows that the transcription level(FPKM=46008.13)of glucoamylase gene glaA was highest in A.niger CICC2462.It have a significant research value to further improving transcription efficiency for glaA promoter.Repetition of AmyR and CCAAT binding protein combination domains and deletion of CreA binding sites from glucoamylase gene glaA promoter,so construct a repetitive-type promoter PglaAR and a repetition and derepression-type promoter PglaARD.With the A.niger endo-xylanase gene xynB as report gene to confirm these promoter's function by transgenic.Detection the xylanase enzyme activities of fermentation supernatant from the wild-type strain A.niger CICC2462,recombinant strain PglaA-xynB,recombinant strain PglaAR-xynB and recombinant strain PglaARD-xynB in the shake flask fermentation conditions,respectively.The xylanase enzyme activity reached the peak when cultured 8 d,the xylanase enzyme activities of fermentation supernatant were 32.57 U/mL,5534.16 U/mL,4904.61 U/mL and 6219.83 U/mL,respectively.Through xylanase enzyme activity measurement and SDS-PAGE,it can be seen that,PglaARD promoter more effcient.And then with 12%starch and 2%starch with 10%glucose as carbon sources to shake flask fermentation culture recombinant strain PglaA-xynB and PglaARD-xynB,respectively.The effect of different carbon sources on the promoter of enzyme production by fermentation was measured through enzyme activity,real-time PCR and SDS-PAGE.Comprehensive analysis the detection results of enzyme activities could observed that recombinant strain PglaARD-xynB higher 1-2 fold than recombinant strain PglaA-xynB for xylanase enzyme activity.Comprehensive analysis the detection results of real-time PCR could observed that the transcription level of xynB was higher 6-8 fold in recombinant strain PglaARD-xynB than in recombinant strain PglaA-xynB.The research results showed that in can markedly improving transcription efficiency of glaA promoter through transformation.