Genes Linked To Nicotine Metabolism In Aspergillus Oryzae 112822 Revealed Using CRISPR/Cas9

Nicotine is the main alkaloid in tobacco plants.It is well known that nicotine can easily cross the blood-brain barrier and biofilm to interact with the nicotinic acetylcholine receptors of the nervous system,which causing harm to humans,such as nausea,arrhythmia,paralysis,etc.At the same time,in the manufacturing process of cigarettes,tobacco waste is generated,which will bring serious environmental problems.Some microorganisms have the ability to degrade nicotine,use nicotine as a carbon source and nitrogen source,can be used to reduce nicotine content in cigarettes,thereby reducing nicotine dependence of smokers,and can also be applied to nicotine degradation in industry.To alleviate the ecological pollution caused by nicotine-containing waste,the research on microbial degradation of nicotine has significant scientific significance and important application value.Bacterial nicotine metabolism in microorganisms includes two completely different pathways,one is the pyridine pathway of Arthrobacter nicotinovorans,and the other is the pyrrole pathway of the Pseudomonas putida S16.Several fungi that degrade nicotine have been discovered,but the mechanism of nicotine degradation by fungal has not been thoroughly studied so far.Only the first step of Demethylation of nicotine pyrrolidine to demethylated nicotine was studied,but the enzymatic mechanism associated with this step has not been studied.Based on the isolation and identification of nicotine metabolites,our laboratory proposed a new nicotine degradation pathway for Aspergillus oryzae 112822,which is the first detailed description of the nicotine degradation pathway in fungi,it provides a new way for the microbial pathway of nicotine degradation.The CRISPR/Cas9 system is evolving into a mature genome editing technology and successfully editing genes in a variety of species.The CRISPR/Cas9 system constructed in filamentous fungi can edit gene by means of nonhomologous end joining or homologous recombination.In addition,it can simultaneously knock out multiple genes by cotransforming different target sgRNAs and donor DNA(dDNA).Therefore,this thesis aims to study the related candidate genes in the complete nicotine metabolic pathway of Aspergillus oryzae 112822 by constructing the CRISPR/Cas9 system,which was first reported in fungi.Our laboratory constructed the CRISPR/Cas9 system in Aspergillus oryzae 112822 strain and detected the expression of Cas9 protein by Western Blot and green fluorescent protein expression assay,but it is not known whether the established CRISPR/Cas9 system can be really used for gene editing.Therefore,This paper aim to design sgRNA targeting to pyrG gene of Aspergillus oryzae,and to verify whether the established CRISPR/Cas9 system can be used for gene editing.Mutation of pyrG gene can produce uridine uracil auxotrophic mutants,which require growth of exogenous uridine and uracil medium.In this paper,a mutant strain of pyrG gene was successfully obtained by using CRISPR/Cas9 mediated non-homologous end joining,variants need to be exogenously added with uridine and uracil for growth,the positive selection is carried out in a medium containing 5-fluoroorotic acid,so that the uridine uracil auxotroph can be used as a screening marker for transformation of Aspergillus oryzae.The gene editing was first carried out in Aspergillus oryzae 112822 strain using CRISPR/Cas9.It makes great preparation for the the study of genes related to the metabolism of nicotine degradation in Aspergillus oryzae.Based on the results of peptide mapping and transcriptome analysis,three candidate genes that may be involved in the metabolism of nicotine in Aspergillus oryzae were screened:cyp577a4(cypl),cyp620h2 and xdh gene.Among them,the CYP577A4 peptide spectrum matching rate was 10%,and the transcriptional difference level was the highest in the two transcriptome analyses,which was previously estimated to be most related to nicotine demethylation;cyp620h2 gene was up-regulated in both transcriptome analyses.Moreover,To compare the amino acid sequence of the CYP82E4 in plant and CYP2A6 in human with the Aspergillus oryzae genome,we found both have high sequence similarity with the Aspergillus oryzae CYP620 subfamily,which is also likely to catalyze the demethylation of nicotine.The xdh gene is also associated with nicotine metabolism presumed in this experiment,encoding an N-methylnicotinamide ?-hydroxylated molybdenum hydrazide hydroxylase,which may catalyze the nicotine intermediate metabolite N-methylnicotinamide.We design sgRNAs Targeted to three candidate genes and construct corresponding homologous repair donors.The target genes were knocked out by CRISPR/Cas9-mediated homologous recombination technology to investigate whether the three genes are related to the metabolism of nicotine.We successfully knock out the cyp577a4(cyp1)gene by CRISPR/Cas9-mediated homologous recombination and confirm that knockdown of cyp577a4(cyp1)gene do not affect the degradation of nicotine in Aspergillus oryzae by TLC analysis.In addition,We also attempt to knock out the cyp620h2 gene and the xdh gene using CRISPR/Cas9 mediated homologous recombination,Although the pyridine thiamine resistant strain was screened,the off-target effect occurred.This paper provides a new tool for studying the metabolic pathway of nicotine in fungi,in order to use the CRISPR/Cas9 gene editing system constructed in Aspergillus oryzae 112822 to knock out candidate genes,thereby discovering genes related to nicotine degradation in Aspergillus oryzae 112822.Further studies on exogenous expression have laid a solid foundation for elucidating the enzymatic and molecular biological mechanisms of nicotine degradation in fungal.