Study On Regulatory Factors Of P_AOX1Promoter And Identification Of Novel Promoters In Pichia Pastoris Expression System

The Pichia pastoris system is wildely applied in recombinant protein expression.So far.it has become one of the most commonly used eukaryotic expression systems.However,compared with other expression systems,the number of available promoters for recombinant protein expression in Pichia pastoris is still limited.Currently,the most frequently used promoter is PAOX1,and this highly efficient promoter is specifically induced by methanol and repressed by other carbon sources such as glucose,glycerol and ethanol.However,besides several transcription factors,the regulatory mechanism of PAOX1 is still unclear.Therefore here we used the transposon tagging technique to screen for more regulators of AOXI promoter and genes involved in peroxisome biogenesis.Besides,because of the toxic and inflammable nature of methanol,the lager-scale application of the Pichia pastoris system in food and medical industries are restricted.Here we also tred to identify more constitutive or inducible promoters under specific carbon sources.First of all,in order to study the regulatory mechanism of PAOX1,we took use of the GS115-AFLDI strain.Since lack of FLD1 blocks formaldehyde oxidation to formate and causes excessive formaldehyde accumulation under high concentration of methanol,the strain will be killed.We relied on the transposon mutation plasmid PMSBHis4MazfARS to introduce random mutations into the GS115-?FLD1 strain.If the mutated gene happened to be transcriptional activators of PAOX1 or involved in peroxisomes biogenesis,the strain will not be able to metabolize methanol and therefore,will stay alive under high methanol concentrations.By adding sorbital as an additional and non-inhibitory carbon source,the mutant could grow.However,if the mutated gene was not related,the mutant would still be killed by metabolizing methanol.As the result of screening,we got 7 viable mutants under high methanol concentrations.Enzymatic activity of Aox and Tail-PCR suggests that 2 of them may be interesting:PEX30 involved in peroxisome biogenesis and PAS_chr 1-4₀251 with unknown function.One the other hand,we compared the RNA-seq data of the wild-type Pichia pastoris GS115 strain under glucose,glycerol and methanol culture.By combining expressional levels and RNA folding free energy ?G.we picked potential promoter candidates with disparate strength under different carbon sources.GFP and recombinant amylase were attached after these promoters to examine their strength and efficiency.Finally we identified two potential novel promoters which could possibly be used in recombinant protein expression:methanol induced promoter P0547 and constitutive promoter P0472.