Engineering Promoter And Secretion Pathway Improves ?-Ffase Secretion In Pichia Pastoris

?-Fructofuranosidase(?-Ffase)with fructosyl-transfer activity typically produces neokestose,which is a novel fructooligosaccharide(FOS)exhibiting greater prebiotic effects and chemical stability than commercial FOSs.Based on our previous results,the secretion level of ?-Ffase from Penicillium oxalicum by Pichia pastoris recombinant G/PGK1-BF,in which?-Ffase gene(PoFF32A)was fused to the aF(alpha mating factor signal peptide)driven by the PPGk1(3-phosphoglycerate kinase gene promoter),was only around 9 U/mL in shake flask cultures.In this study,different strategies to improve ?-Ffase level in P.pastoris have been used.Firstly,the truncated PPGK1 varicants(PPE,PPG and PPD)and engineered PPD promoters with 1-5 AFTlp binding sites(PPD1,PPD2 and PPD5)were constructed and characterized,to strong overexpression of ?-Ffase gene(PoFF32A)in P.pastoris.The promoter activity of the strongest variant PPD5 is approximately 1.7-fold greater than that of wild-type PPGK1.Additionally,the PoFF32A gene expression driven by the engineered promoters PPE(G/PE-BF),PPD(G/PD-BF)and PPDS(G/PD5-BF)was 29.9%,32%and 57.7%higher than that of wild-type PPGK1.Since the P.pastoris Afr1p binding sites were found enriched in the promoter regions of secretion enhancing genes and overexpression of AFT1 can significantly enhance the production of heterous protein,the AFT1 was overexpression in ?-Ffase producing strains G/PE-BF(G/AFT1-PE-BF),G/PD-BF(G/AFT1-PD-BF)and G/PD5-BF(G/AFT1-PD5-BF),among which G/AFT1-PD5-BF exhibits the highest extracellular activity of ?-Ffase,with peak value of 26.4 U/mL secretory level of ?-Ffase.Furthermore,we selected four potential secretion helper factors,in which HAC1 and PDI1 were involved in protein folding,SEC4 and SED5 were involved in vesicle trafficking,to investigate the effect of their overexpression on ?-Ffase secretion in the high-producing strain G/PE-BF.The single overexpression of HAC1,PDI1,SEC4 and SED5 enhanced the extracellular activity of ?-Ffase by 113.4%,121.1%,16.7%and 15.7%%,respectively.The results obtained in this study revealed genetic targets of protein secretory pathways that could potentially improve the yield of P-Ffase in P.pastoris.