利用GAP启动子在毕赤酵母中表达牛肉风味强化肽

Constitutive Expression of Beefy Meaty Peptide in Pichia pastoris Using the GAP Promoter

牛肉风味强化肽是1种从牛肉木瓜蛋白酶的水解物中分离得到的风味强化八肽。为了提供能够大量且廉价得到这种小肽物质的表达基因,本实验室工作人员构建以甲醇为诱导物并带有16拷贝BMP基因的表达载体pPIC9-16BMP。本文为了提高食品的安全性,避免甲醇作为诱导物,探索用GAP启动子(PGAP)取代AOX1启动子(PAOX1),在毕赤酵母中组成型表达外源蛋白的可能性。应用PCR方法从毕赤酵母染色体中扩增GAP启动子,以其取代诱导型表达载体pPIC9-16BMP上的PAOX1,构建了组成型表达载体pGAP9-16BMP。转化毕赤酵母GS115,对获得的高拷贝转化子毕赤酵母GS115(pGAP9-16BMP)进行组成型表达,并用SDS-PAGE检测到目的产物。

The octapeptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala, also known as ＂beefy meaty peptide＂（BMP）, is considered a savory seasoning in previous literatures. It was first isolated from beef digested by papain. BMP has better flavor cooperating with salt or monosodium glutamate（MSG）. In order to obtain plenty of cheap BMP expression genes,our lab had constructed expression vector pPIC9-16 BMP taking 16 copies BMP genes. The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter（PAOX1） on pPIC9-16 BMP resulting in plasmid pGAP9-16 BMP. pGAP9-16 BMP was then transformed into P. pastoris GS115. The integration transformant P. pastoris GS115（pGAP9-16BMP） was used to investigate the constitutive expression of BMP in P. pastoris.