Application Research Of The Inducible Promoter Cbh1 In Mortierella Alpina

The promoter of cellobiohydrolase I gene is a strong inducible promoter induced by cellulose,which can realize the expression of target gene regularly and quantificationally.It is considered to be an ideal choice for the construction of eukaryotic expression vector and is also the only inducible promoter reported to be used in the CRISPR system of filamentous fungi.Mortierella alpina(M.alpina),an oleaginous fungus,has strong lipid synthesis ability,which has been used in the production of arachidonic acid(AA)in industry.The fermentation process of M.alpina can be obviously divided into two stages,cell proliferation stage and lipid accumulation stage.However,at present,conventional constitutive promoters cannot artificially and efficiently regulate the transcription of target genes according to the characteristics of lipid-producing fermentation of M.alpina.The tools for conditionally controlling gene expression are urgently needed to be developed.In this study,the omega-3 fatty acid desaturase gene oPpFADS17 was used as the reporter to investigate the feasibility of the application of inducible promoter cbh1 in M.alpina by constructing the recombinant MA-Pcbh1-oPpFADS17 containing the promoter cbh1.The induction conditions of promoter cbh1 were optimized from many aspects.In addition,based on the GC-MS metabolomic research method,the metabolites of recombinant were analyzed to determine the biomarkers that reflect the inducibility changes of promoter cbh1.The main results of this study are as follows:1.Eight genes encoding three kinds of cellulose were screened from the genome data of M.alpina ATCC 32222.Analysis of transcriptome data of M.alpina showed that the eight genes were effectively transcribed.According to the analysis of transcriptome sequencing data,these genes were transcribed to different degrees.Besides,the strain can grow in the medium with avicel as the only carbon source,which indicates that M.alpina has the ability to metabolize avicel,the inducer of cbh1 promoter,and has the application basis of the cbh1 promoter.2.The recombinant MA-Pcbh1-oPpFADS17 with inducible promoter cbh1 was successfully constructed using the omega-3 fatty acid desaturase gene oPpFADS17 as the reporter gene and the insertion of the promoter cbh1 had no significant influence on the lipid production and growth of the recombinant under fermentation condition,which met the requirements of subsequent application.The recombinant can produce EPA under induction condition with avicel,but can not produce EPA under the condition without avicel,indicating that the recombinant strain can respond to the induction effect of avicel,and the cbh1 promoter has inducibility in M.alpina.3.The induction conditions were optimized from the inducer type,the inducer concentration and glucose concentration(additional carbon source concentration).The optimal induction condition of the recombinant MA-Pcbh1-oPpFADS17 was the Broth medium supplemented with 1% avicel and 5% glucose.After being cultivated for 2,4 and 6 days under this induction condition,the recombinant's reporter gene transcriptional level was 2.08,7.53 and 18.15 times higher than that of the control group respectively,indicating that the induction efficiency of the promoter cbh1 could be significantly improved with the extension of induction time4.The key inducers affecting the induction efficiency of promoter cbh1 were analyzed from the metabolite level of M.alpina.The results of principal component analysis of prototrophy M.alpina CCFM 505,recombinant MA-His550-oPpFADS17 constructed by our laboratory in the early stage and recombinant MA-Pcbh1-oPpFADS17 constructed in this study,showed that metabolites of three strains had no significant difference.Both insertion of the constitutive promoter His550 and the inducible promoter cbh1 had no significant effect on the metabolism of M.alpina.By analyzing the metabolites of the recombinant strains and prototrophy M.alpina cultivated in the condition with and without avicel,four different metabolites were identified,which were pinitol,ononitol,glutamic acid and adenosine monophosphate.The correlation analysis of the content of the four metabolites in the recombinant strain cultured for 2,4,and 6 days under induction conditions and the transcriptional level of the reporter gene showed that the content of pinitol and adenosine monophosphate were relevant to changes of the transcriptional level of the reporter gene.Pinitol was positively correlated and adenosine monophosphate was negatively correlated,which indicates that pinitol and adenosine monophosphate were the biomarkers of inducibility of the promoter cbh1,pinitol may be a key inducer of the promoter cbh1.