Construction Of CRISPR System And Screening NADPH Supply Pathways In Mortierella Alpina

Mortierella alpina is an oleaginous filamentous fungus capable of producing high amount of arachidonic acids and has been widely used in industrial production of arachidonic acids.The genome,transcriptome and lipidomes of M.alpina were identified.In order to demonstrate its lipid accumulation process and construct high oil producing recombinant strains,the multigene management tools need to be constructed as previous gene management methods of M.alpina mainly dealt with one or two genes.The clustered regularly interspaced short palindromic repeats(CRISPR)system is a powerful and widely used tool for target gene disruption.Using inducible promoters,the CRISPR system can repetitively disrupt target genes such as selection marker,thus allowing multigene management in host cells.NADPH is indispensable for lipid biosynthesis in oleaginous microorganisms.Clear demonstration of sources of NADPH is valuable for expounding lipid biosynthesis mechanisms and constructing high-yield strains.The dissertation established a multigene expression strategy in M.alpina and achieved the recycle of the selection marker after a gene was knocked in,showing the successful application of the strategy.The main contents and the results of the dissertation are as follows:1.Improvement of the transformation efficiency of M.alpina.In order to make it easy for larger and multiple fragments to enter into the host cells,the effect of A.tumefaciens species and cell forms of M.alpina on transformation efficiency of ATMT method in M.alpina was explored.Among the four tested A.tumefaciens species(AGL-1,C58C1,EHA105 and LBA4404),A.tumefaciens AGL-1 and EHA105 were found having the highest transformation efficiency.Then,A.tumefaciens AGL-1 was used to transform various cell forms of M.alpina(fresh spores,germinating spores,liquid mycelium and single solid colonies).Results showed that the germinating spores were the most easily transformed cells.2.Establishment of CRISPR system under the guidance of a constitutive promoter.In order to compare the gene editing efficiency of normally used nucleases Cas9 and Cpf1 in M.alpina,this dissertation constructed recombinant strains MA-His550-spCas9 and MA-His550-LbCpf1with CRISPR system under the guidance of the constitutive promoter His550.The guide sequences targeting ura5 sequence named sgRNA(for spCas9)and CrRNA(for LbCpf1)were designed online and transformed into the recombinant strains MA-His550-spCas9 and MA-His550-LbCpf1.The results showed that the gene disruption efficiency of LbCpf1 was higher than spCas9 in M.alpina and was used in subsequent experiments.3.Establishment of CRISPR system under the guidance of an inducible promoter.In order to achieve the recycle of the selection marker,the expression of nuclease LbCpf1 needs to be controlled.This dissertation first verified the function of inducible promoter cbh1 in M.alpina and constructed 6 stable transformants MA-Pchb1-LbCpf1 with CRISPR system under the guidance of the constitutive promoter cbh1.One transformant MA-Pchb1-LbCpf1 with higher biomass and lipid content was selected and induced using optimized induction conditions,then13 uracil auxotrophic strains MA-Pchb1-LbCpf1-ura5~-were achieved.The lipid fermentation of the 13 uracil auxotrophic strains were assessed and one recombinant strain with similar biomass and lipid content as wild type was selected.By this step,the multigene management system of M.alpina was successfully established.4.Exploring and screening the NADPH supply pathways in M.alpina.In order to choose candidate genes for the multigene expression system,this dissertation explored and screened multiple pathways for reduction power supply.Three potential NADPH supply pathways including glyceraldehyde-3-phosphate dehydrogenase(GAPDH,EC 1.2.1.12),NAD kinase(NADK,EC 2.7.1.23)and NADH kinase(NADHK,EC 2.7.1.86)were explored by homologous expression and RNA interference technology in M.alpina.Results indicated that the GAPDH,NADK and NAHDK had ability of providing NADPH for lipid biosynthesis.Comprehensively considering the contribution of reduction power supply pathways to lipid accumulation,me1 was chosen as the candidate gene in the multigene expression strategy.5.Primary application of the multigene expression system in M.alpina.Using the selected recombinant MA-Pchb1-LbCpf1-ura5~-as the beginning strain,the me1 gene was transformed through the optimized transformation method.3 recombinant strains MA-Pchb1-LbCpf1-me1were achieved.One recombinant with the best performance was induced to recycle the selection marker,and 3 uracil auxotrophic strain MA-Pchb1-LbCpf1-me1-ura5~-with correct phenotype and genotype were achieved.This dissertation achieved the repetitive recycle of the selection marker after the knock-in of the aim gene,proving the successful application of the constructed multigene expression system in M.alpina.