Excavation And Application Of Aspergillus Niger Biological Elements

The filamentous fungus Aspergillus niger was widely used in production of organic acid and enzyme because of its excellent protein expression and secretion ability.With the postgenomic era,the rapid development of transcriptomics can not only play an important theoretical guidance for revealing the regulatory mechanism of A.niger when expressing proteins,but also explore more usable and efficient biological elements.Meanwhile diverse biological elements can provide technical support for the production of more valuable products in the application of A.niger cell factories.The model strain A.niger ATCC1015 was used as the research object in this study.The biological parts were mined by the combined application of transcriptome sequencing and reporting system,and the elements library of the promoter,signal peptide and genome locus were definitively established.In addition engineered living materials and gluconic acid highyielding strains were also established by biological parts combining with each other.The specific content of the research and the main results are as follows:Firstly,the databases of gene differential expression level of pH-stressed and glucosestressed were firmly established by transcriptome sequencing,respectively.And 558 stably expressed genes whose promoters match with potential constitutive promoters were broadcast,18 stressor response expression genes whose promoters correspond with inducible promoters were also screened.Besides,five pH-related promoters and five glucose-related promoters were excavated by monitoring the driving performance through fixed-point integrated reporting system.Secondly,496 coding genes were carefully analyzed through official Signal P-5.0website and 35 key genes were found to have signal peptide.Next these genes were also analyzed via Prot Comp 9.0 websites,of which 18% encoded proteins were located outside the cells,43% encoded proteins were located outside membrane-bound cells and 26%encoded proteins were located on the plasma membrane.18 signal peptide were successfully excavated by examining the secretion promotion performance through fixed-point integrated reporting system and one of which was significantly better than gla A signal peptide.Thirdly,a series of recombinant strains were gained through integrating the random insertion reporter system into A.niger,and the strains with gradient production capacity were selected for gene expression cassette integration site analysis.11 insertion sites were successfully excavated,named IS1～IS1,respectively.Fourthly,two recombinant strains with excellent gluconic acid production performance were gained by properly introducing the expression elements which obtained by screening to modified the chassis cells.When incubated in 2 L fermenter,the attractive yield of gluconic acid of two recombinant strains at 87 h was 153.72 g/L and 155.72 g/L,and the production efficiency was 1.77 g/L/h and 1.79 g/L/h,respectively,which was 1.34 times compared with the current commonly used expression element construction strains.Finally,the chassis cell was modified by properly introducing the inducing expression element and the functional part controlling pigment synthesis,and an environmentally responsive pigment synthesis reporting system was constructed.Additionally,engineered living materials,which has the distinguishing characteristics of strong flexibility,environmental response and self-healing regeneration,was constructed according to colour synthesis reporting system.