重组胃蛋白酶原A黑曲霉高效表达及其在食物营养中的应用基础研究

针对胃肠健康问题，和老年人消化功能衰退导致的免疫力低下，基于胃蛋白酶在食物消化中的重要性，本项目拟解决哺乳动物胃蛋白酶（原）的安全性，阐释重组胃蛋白酶原A（rPGA）在黑曲霉中高表达调控机制，探明其消化食物的规律。以食品安全级的糖化酶高产菌黑曲霉为宿主，敲除相关酸性蛋白酶基因，优化启动子和信号肽，将pga表达盒定点替换宿主基因组上糖化酶基因，筛选pga不同拷贝数的重组菌株，并考察这些重组菌株的转录组差异，探讨rPGA表达时关键调控因子和表达调节规律，据此构建rPGA高表达菌株。分析rPGA酶学特性，对比动物来源PGA，评价rPGA对不同动、植物蛋白质的消化效率和热加工对其作用的影响；利用体外消化模型，分析rPGA对不同种类含蛋白食物的消化规律及在食物蛋白消化过程中的贡献度。本项目为rPGA及其它外源蛋白在黑曲霉中高表达提供理论指导，为rPGA在食用助消化制剂中应用提供基础支撑。

In view of the increasingly serious gastrointestinal health problems, as well as the low immunity caused by the decline of digestive function in the elderly, based on the important role of pepsin in the food digestion process, this project aims to solve the safety of mammalian source pepsinogen, illuminate the high level expression mechanism of recombinant pepsinogen A (rPGA) in Aspergillus niger, and find out its rules in food digestion. With GRAS Aspergillus niger AnT21 with high production of glucoamylase as the host, on the basis of knocking out its multiple acid proteinase genes, we’ll optimize the promoter and signal peptides, replace the glucoamylase genes on Aspergillus niger genome with pga expression box, screen out the recombinant strains with different copies of pga and investigate the differences in the transcriptome of these recombinant strains, explore the key regulatory factors and regulatory rules of genes expression in rPGA expression, followed by construction of the strain with higher product of rPGA based on the rules. Further, we’ll analyze the enzyme properties of rPGA, and in comparison with PGA from mammal, evaluate rPGA-catalyzed digestive efficiency of different proteins of plants and animals and the influence of thermal processing on their digestion, and analyze the digestive regularity of rPGA on different kinds of protein-containing foods and its contribution for the whole protein digestion process by using in vitro digestion model. This project can provide theoretical guidance for high-level expressing rPGA and other heterologous proteins in Aspergillus niger, and provide basic support for rPGA as digestion aids in foods.