Screening Of Key Factors For Protein Secretion Pathway In Aspergillus Niger And Constrcution Of Industrial Glucoamylase-hyperproducing Strains

Filamentous fungi are important microorganisms in the fermentation industry and have been widely used as cell factories due to their outstanding capacity in expression and secretion of heterologous proteins with posttranslational processing.They have been used in the production of a wide variety of products for human beings,such as organic acids and proteins.As an important member of filamentous fungi,Aspergillus niger has the advantages of high protein secretion capacity,high production economy and high food safety.A.niger is a typical species used for producing enzymes such as glucoamylase,proteases,phytases,glucose oxidase,and organic acids including citric acid and gluconic acid.Glucoamylase(Gla A,EC 3.2.1.3)is an exo-amylase which yields glucose by hydrolyzing α-1,4 and α-1,6 glucosidic linkages from the nonreducing ends of starch-type molecule.Glucoamylases have received much attention because of their important applications for the food and biofuel industries.With the rapid development of synthetic biology enabling technologies in recent years,it is an important prerequisite for the industrial production and application of to obtain hyper-producing glucoamylase strains through genetic recombination technology to improve the expression and secretion level of glucoamylase.In order to obtain the improved Aspergillus niger platform strains with enhanced glucoamylase production,the early ancestor of currently used enzyme production strains A.niger strain CBS 513.88 and the industrial glucoamylase-producing strain A.niger N1 were the objects of this study to explore and identify the factors predicted to act in protein synthesis and the secretory pathway.This study performed the genetically modification of the protein secretion pathway in A.niger for improving the glucoamylase production.First,combined with our previous data and the comparative genomics analysis,16 new target factors that have potential effects on the synthesis and secretion of glucoamylase were selected to construct the knockout mutant strains in A.niger CBS 513.88 through our efficient transformation approach and CRISPR-Cas9 genome editing technology in this study.Then,the 16 genes deletion mutants were systematically screened in shake flask medium by measuring the extracellular protein level and glucoamylase activity.Of the 16 deletion mutants screened,3 mutants(CBS-ΔFM1,CBS-ΔFM5 and CBS-ΔFM7)exhibited protein production and glucoamylase activity ranging from 21.8 to 38.5% higher than in its parental strain CBS 513.88.Subsequently,we combined with three new targets screened in this study and the FM17 and FM18 genes previously identified to construct and to generate the deletion mutants of these five genes(N1-ΔFM1,N1-FM5,N1-ΔFM7,N1-ΔFM18 and N1-ΔFM17)in the glucoamylase-producing strain N1.We observed that the four deletion mutants except N1-ΔFM7 led to the significant increase in glucoamylase secretion and activity by approximately 16.0-23.5% compared with host strain N1.Finally,in this study,we developed a flow cytometry-based plating-free(FCPF)system by combining genetic engineering,strain screening,deep-well-plate shaking incubation,and phenotypic analysis in the industrial glucoamylase-producing strain A.niger N1.Triple mutant strain OE-Tegla A-gfpΔFM19ΔFM1 was created by this FCPF system and named An LM3.The An LM3 mutants displayed 1.2-and 1.3-fold higher protein secretion and glucoamylase activity compared with the host strain.