豆血红蛋白在酿酒酵母中的异源表达  

Heterologous Expression of Leghemoglobin in Saccharomyces cerevisiae

作　　者：薛常鲁 张鹏飞 白丽君 肖功年[1,2] 魏培莲[1,2] XUE Changlu;ZHANG Pengfei;BAI Lijun;XIAO Gongnian;WEI Peilian(Zhejiang University of Science and Technology,Hangzhou 310023,China;Zhejiang Provincial Key Lab for Chem&Bio Processing Technology of Farm Product,Hangzhou 310023,China)

机构地区：[1]浙江科技学院生物与化学工程学院,浙江杭州310023 [2]浙江省农产品化学与生物加工技术重点实验室,浙江杭州310023

出　　处：《食品工业科技》2023年第20期101-107,共7页Science and Technology of Food Industry

基　　金：浙江省重点研发计划项目(2020C02041)。

摘　　要：豆血红蛋白是一种植物源血红蛋白,在植物蛋白肉加工中可作为重要的风味催化剂和着色剂,大大增加植物蛋白肉的拟真性。为实现豆血红蛋白在食品级微生物中的异源表达,将携带豆血红蛋白LBC2基因的质粒PESC-TRP转入酿酒酵母CEN.PK2-1C中,通过添加kozak序列促进其翻译,并进一步对启动子序列进行选择,以提高豆血红蛋白表达量。对重组菌株进行发酵,用Western blot分析LegH蛋白表达水平。结果显示,诱导型启动子GAL1,10较三种组成型启动子TEF1、ADH1、GAP在表达LegH能力上具有显著优势,较产量最低的ADH1启动子提升了3.93倍。在组成型启动子中,TEF1启动子能力较优,是ADH1启动子的2.91倍,是GAP启动子的1.2倍。最后,利用Ni柱亲和层析对蛋白进行浓缩纯化,测得LegH发酵浓度为2.79 mg/L。本研究成功实现了豆血红蛋白在酿酒酵母中的异源表达,经后续优化有可能成为豆血红蛋白异源表达的又一途径。Leghemoglobin is a kind of plant-derived hemoglobin,which can be used as an important flavor catalyst and colorant in the processing of plant protein-based meat because of the property of increasing the fidelity of the product greatly.In order to realize the heterologous expression of leghemoglobin in food-grade microorganisms,the plasmid PESC-TRP carrying leghemoglobin LBC2 gene was transformed into S.cerevisiae CEN.PK2-1C.Kozak sequence was added to promote protein translation,and different promoter sequences were selected to improve the expression of leghemoglobin.The recombinant strain was fermented,and the expression level of LegH protein was analyzed by western blot.The results showed that the inducible promoter GAL1,10 had a significant advantage over the three constitutive promoters TEF1,ADH1 and GAP in LegH expression ability,which was 3.93 times higher than the lowest yield ADH1 promoter.Among the constitutive promoters,TEF1 promoter had the best ability,which was 2.91times that of the ADH1 promoter and 1.2 times that of the GAP promoter.Finally,the protein was concentrated and purified by Ni-column affinity chromatography,and the fermentation concentration of LegH was 2.79 mg/L.This study successfully achieved the heterologous expression of leghemoglobin in Saccharomyces cerevisiae,and after subsequent optimization,it would become another way of leghemoglobin heterologous expression.

关 键 词：酿酒酵母 豆血红蛋白 启动子优化 异源表达 蛋白质印迹 

分 类 号：Q786[生物学—分子生物学]