Study On The Role Of The MAPK Signaling Pathwayof High-yield Phytase Komagataella Phaffii Based On Transcriptome Analysis And Gene Editing Techniques

Komagataella phaffii?syn.Pichia pastoris?has been widely used in industrial production,as one of the present expression host system essential for exogenous protein production.In molecular biology research,the exploration of gene function is the key to revealing the essence of life,how to study the function of genes effectively becomes extremely important.However,at present,little is known about the gene expression during fermentation and the metabolic regulation in this strain,especially the role of the signal transduction pathway in the protein-induced expression process.Due to the lack of effective regulation and control of the fermentation process,the expression of exogenous proteins is also in urgent need of further improvement.Based on this situation,this paper uses the lab to build the high expression of K.phaffii gene engineering of phytase strain K.phaffii GS115/pPIC9K-PhyA for starting strain,study on the fermentation in 10000 L fed-batch culture,then investigated gene expression during the yeast cell culture phase,glycerol feed phase,glycerol-methanol mixture feed?GM?phase,and at different time points following methanol induction using RNA-Seq.The results showed that the significantly up-regulated gene number mainly occurred in the metabolic processes of mitogen-activated protein kinases?MAPK?signaling pathway,peroxisome,methane metabolism,starch and sucrose metabolism,autophagy,mitochondrial phagocytic yeast and meiosis.Among them,the up-regulation of gene number in MAPK signaling pathway was the most significant.It revealed that MAPK signaling pathway may be related to K.phaffii cell growth and may regulate the alcohol oxidase1?AOX1?promoter(P_AOX1)via regulatory factors activated by methanol-mediated stimulation.Next,in order to solve the problem that K.phaffii genetic engineering has been short of useful and straightforward gene knockout tools,we tried to develop CRISPR/Cas9 gene editing system in K.phaffii.First,the mCherry fluorescence system was expression successfully and genetic stability under the P_AOX1 in K.phaffii.Then,the pGAP-spCas9-sgRNA gene editing system was constructed to express spCas9 by GAP promoter(P_GAP)and sgRNA driven by P_ScSNR52.While the editing efficiency of this system was inefficient cause only one converter was obtained.pGAP-spCas9-sgRNA might not the best tool for targeted gene modification in K.phaffii.Subsequently,the CRISPR/Cas9 system pHTX-hsCas9-sgRNA-Z was constructed,which contains an endogenous bidirectional promoter P_HTX,which directs the expression of human codon optimization Cas9?hsCas9?in reverse orientation and the gRNA flanked by the HH and HDV ribozymes in forward orientation.The three targets designed for the mCherry all achieved more than 60%editing efficiency,which proved that this system could carry out effective editing for K.phaffii and was a better choice for precise operation of K.phaffii genome in the future.According to transcriptome analysis,the CRISPR/Cas9 system constructed in this experiment was used to knock out 5 genes that up-regulated significantly in MAPK signaling pathway during the methanol induction stage,including PAS_chr1-1₀273,PAS_chr1-3₀061,PAS_chr1-1₀201,PAS_FragB₀046 and PAS_chr3₀895.The following experimental results were mainly obtained:?1?Besides PAS_chr1-3₀061,the other four target genes were knock out successfully via the CRISPR/Cas9 system.?2?The growth characteristics of PAS_chr1-1₀273,PAS_chr1-1₀201,PAS_FragB₀046,and PAS_chr3₀895 gene deletion strains in the medium with glucose,glycerol and methanol as carbon sources were determined.The results performed that PAS_FragB₀046 promoted strain growth in methanol,the other three knockouts genes showed growth defects in methanol.The deletion of PAS_chr1-1₀201 and PAS_chr3₀895 had significant effects on growth arrest?p<0.05?.?3?All the knockouts showed different levels of phytase expression under the condition of methanol induction,while the activity of phytase was decreased compared with the wild type.After the deletion of PAS_FragB₀046 gene,the activity of P_AOX1was the lowest,the phytase activity was only 4.95 U/mL,which decreased by 98.46%compared with the wild type.Secondly,the phytase activity after the deletion of pas_chr1-1₀273 gene was 27.73 U/mL,which diminished by 91.61%.After the deletion of PAS_chr1-1₀201 and PAS_chr3₀895,the phytase activity was 152.30 U/mL and 66.58 U/mL,which decreased by 51.73%and 78.69%,respectively.In summary,these experimental results and data provide a strong theoretical support for the further transformation of pichia pastoris engineering strains in the future,and also provide a stable foundation for the establishment of a complete cell model.