促进米黑根毛霉脂肪酶分泌的毕赤酵母内源信号肽的筛选

Screening Endogenous Signal Peptides in Pichia pastoris for Promoting the Secretion of Lipase from Rhizomucor Miehei

米黑根毛霉脂肪酶(Rhizomucor miehei Lipase,RML)在造纸、食品、化妆品以及医药等行业应用广泛。本研究通过SignalP 4.1在线预测软件预测出9种具有分泌潜力的毕赤酵母内源信号肽序列:FLO10、CPR5、PRY2、DSE4、NUP145、MSB2、SSP120、FRE2、FLO9。以广泛应用的酿酒酵母α-交配因子(α-mating factor,α-MF)信号肽为对照,考察这九种内源信号肽对增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)和RML在蛋白酶缺陷型PichiaPinkTM表达系统的分泌效果。结果发现FLO10、PRY2、DSE4、MSB2、SSP120、FRE2能有效介导EGFP分泌,且PRY2、DSE4、MSB2、FRE2介导EGFP的分泌水平优于α-MF,分别是α-MF的2.15倍、1.15倍、1.33倍、1.30倍;FLO10、PRY2、DSE4、FRE2能有效介导RML的分泌,且FLO10介导RML的分泌水平最高,是α-MF的1.50倍,说明内源信号肽FLO10更能有效分泌RML。本研究为提高米黑根毛霉脂肪酶在蛋白酶缺陷型毕赤酵母的表达量奠定了一定的基础。

Rhizomucor miehei Lipase(RML)is widely used in the paper,food,cosmetics,pharmaceutical industries.In this study,nine potential endogenous signal peptide-like sequences with a secretory potential from Pichia pastoris were predicted by a SignalP 4.1 online software:FLO10,CPR5,PRY2,DSE4,NUP145,MSB2,SSP120,FRE2 and FLO9.Using the widely usedα-mating factor(α-MF)signal peptide of Saccharomyces cerevisiae as the control,the effects of the nine endogenous signal peptides on the secretion of the enhanced green fluorescent protein(EGFP)and RML in a protease-deficient PichiaPinkTM expression system were investigated.The results showed that FLO10,PRY2,DSE4,MSB2,SSP120 and FRE2 could effectively mediate the secretion of EGFP.Moreover,the secretion level of EGFP mediated by PRY2,DSE4,MSB2 and FRE2 were 2.15,1.15,1.33,1.30 folds,respectively,that ofα-MF.FLO10,PRY2,DSE4 and FRE2 could effectively mediate RML secretion,with the highest secretion level of RML with FLO10(which was 1.50 folds that ofα-MF).This study laid a foundation for improving the expression of RML in protease-deficient Pichia pastoris.