| The octapeptide Lys-Gly-Asp-Glu-Glu-Ser-Leu-Ala, known as Beefy Meaty Peptide (BMP), is reported a savory seasoning in previous literatures. The synergisms of BMP with salt and MSG, and its thermal stability suggested that BMP might be an effective alternative to flavor enhancers such as MSG. In this study, we reported the constitutive expression of BMP in P. pas tor is using gene engineering method.The inducible expression vector pPIC9-16BMP with inducible methanol oxidase (AOX1) promoter had already been constructed and studied previously to obtain plenty of BMP by microorganisms. In order to eliminate the hazard associated with methanol and avoid the fed-batch fermentation, the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter was cloned to replace PAOX1reaching the constitutive expression of BMP in P. pastoris.The PGAP was amplified by PCR from P. pastoris chromosome DNA. The amplified PGAP was cloned in pPIC9-16BMP to replace the inducible promoter PAOX1resulting in the constitutive expression vector, pGAP9-16BMP. The pGAP9-16BMP was then linearized and transformed into P. pastoris GS115. the resulting BMP-producing transformant was designated as GS115(pGAP9-16BMP).BMP expression was detected by SDS-PAGE. The fermentation conditions for production of BMP by engineered P. pastoris GS115(pGAP9-16BMP) was optimized in shake-flask cultivation as follows:3%(m/v) glycerol,2%(m/v) yeast powder.4%(m/v) peptone,1.5%(m/v) Yeast Nitrogen Base, medium quantity30%(v/v). temperature32C pH6. cultured for96h. The constitutive expression of BMP by high-density cell culture in a5liter bioreactor using glycerol as the sole carbon source was done, and the production of BMP reached its peak expression at64h of fermentation.After centrifugation and boiled,2.8L supernatant was concentrated to140mL by polypropylene (PP) hollow fiber porous membrane with molecular cut off of60kDa and6000Da, respectively. The BMP fusion peptides with expected sizes were successfully purified by His-tag bind affinity resin. The peptides were testified of having a meaty or savory taste by Maillard reaction. |
