摘要
通过优化毕赤酵母Komagataella phaffii GS115表达系统,显著提升高倍甜味蛋白Brazzein的分泌表达水平。首先成功获得重组菌株K.phaffii Bra-1,经摇瓶发酵优化,在甲醇诱导体积分数2%、生物素体积分数0.08%、温度30℃、初始pH 6.0的条件下,表达量达到90.2 mg/L。进一步表达元件筛选优化实验表明内源醇氧化酶1启动子(PAOX1)具有最优表达性能;αΔ57-70信号肽(SαΔ57-70)通过增强信号肽柔性使Brazzein表达量达到183.2 mg/L;共表达调控元件蛋白二硫键异构酶进一步将产量提高至209.3 mg/L。在5 L生物反应器中,调控诱导起始OD600为100、pH值恒定在5.0,通过分批补料策略使得Brazzein产量达到1.4 g/L。本研究为甜味蛋白Brazzein的规模化生产与应用奠定了基础。
In this study,the expression system of Komagataella phaffii GS115 was optimized for improved secretion of the sweet protein Brazzein.The recombinant strain K.phaffii Bra-1 was successfully obtained,which produced 90.2 mg/L of Brazzein in shake flask fermentation under 2%methanol,0.08%biotin,30℃and initial pH 6.0.Selection and optimization of expression elements showed that the endogenous alcohol oxidase 1(AOX1)promoter had the best expression performance.TheαΔ57-70 signal peptide increased the expression of Brazzein to 183.2 mg/L,which was further increased to 209.3 mg/L by the co-expression regulatory element,disulfide bond isomerase(PDI).In a 5 L bioreactor,fed-batch fermentation at an initial OD600 of 100 and a constant pH of 5.0 yielded 1.4 g/L of Brazzein.This study lays the foundation for the large-scale production and application of Brazzein.
作者
高鹏
张胜梦
孙元伟
黄巍巍
邱益彬
徐虹
张琦
李莎
GAO Peng;ZHANG Shengmeng;SUN Yuanwei;HUANG Weiwei;QIU Yibin;XU Hong;ZHANG Qi;LI Sha(State Key Laboratory of Materials-Oriented Chemical Engineering,Nanjing Tech University,Nanjing 211800,China;College of Food Science and Light Industry,Nanjing Tech University,Nanjing 211800,China)
出处
《食品科学》
北大核心
2025年第17期111-119,共9页
Food Science
基金
“十四五”国家重点研发计划重点专项(2023YFF1103400)。
