摘要
毕赤酵母是一种甲基营养型酵母,可以利用甲醇作为唯一的碳源和能源,是应用最为广泛的真核外源蛋白表达系统之一。毕赤酵母的甲醇利用率和同化效率较低,高浓度甲醇对细胞有毒性,其中间代谢产物甲醛、甲酸等毒性物质的积累会严重抑制细胞生长,限制目的蛋白的产量。本研究拟在毕赤酵母过氧化物酶体中,利用多酶组装技术在甲醇代谢关键酶之间构建底物通道,减少甲醛积累,增大同化途径通量,提高甲醇耐受性,提高毕赤酵母表达外源蛋白能力,获得高效利用甲醇的毕赤酵母人工细胞。研究首先采用双分子荧光互补(BiFC)分析技术验证了多酶组装技术—SpyTag/SpyCatcher体系在毕赤酵母中自组装的可行性,随后利用SpyTag和SpyCatcher成功实现了甲醇氧化酶(AOX)和二羟丙酮合酶(DAS)在胞内自组装形成双酶复合体;在此基础上,引入绿色荧光蛋白EGFP作为报告蛋白,考察表达自组装双酶复合体的重组毕赤酵母利用甲醇进行细胞生长和重组蛋白合成的能力。结果显示,在2%(体积分数)甲醇培养基中,相比原始菌株,表达双酶组装体系的重组菌株的生物量提高了2.3倍,单位D(600)细胞表达的EGFP荧光强度提高了4.51倍,是未组装菌株的2.76倍,EGFP产量约为1.52 mg/mL。
As a kind of methanol-trophic yeast,Pichia pastoris can use methanol as the only carbon source and energy source,and it is one of the widely used expression systems for production of eukaryotic exogenous proteins.However,the utilization and assimilation efficiency of methanol in Pichia pastoris is low,and the methanol of high concentration is toxic to the cell.In the process of metabolizing methanol,the accumulate toxic intermediate metabo-lites such as formaldehyde and formic acid will severely repress cell growth,and limit the synthesis of target proteins.This study designed to construct a substrate channel between the key enzymes in methanol metabolism with multi-enzyme-assembly technology,aiming to reduce the accumulation and diffusion of formaldehyde,improve the flux of the assimilation pathway,enhance the tolerability of Pichia pastoris,and improve Pichia pastoris'ability to express foreign protein.Thus Pichia pastoris artificial cell with high methanol utilization can be obtained.In this paper,the feasibility of the multi-enzyme assembly technology—the SpyTag/SpyCatcher system in Pichia pastoris was firstly identified by the bimolecular fluorescence complementation(BiFC)analysis technology.Then alcohol oxidase(AOX)and dihydroxyacetone synthase(DAS)were assembled to form a self-assemble double-enzyme complex with SpyTag and SpyCatcher.On the basis of that,the green fluorescent protein(EGFP)was introduced into the recombinant strain as a reporter protein to analyze the comsumption of methanol for cell growth and the production of recombinant protein.The results show that,when 2%methanol is used as the carbon source,the biomass of the recombinant strain expressing the double-enzyme self-assembly complex is 2.3 times bigger than that of the original strain,and its EGFP fluorescence intensity of per D(600)cell is 4.51 times higher than that of the original strain,and it is 2.76 times of the unassembled strain's EGFP fluorescence intensity;the production of EGFP is about 1.52 mg/mL.
作者
韩双艳
李静文
王媛媛
HAN Shuangyan;LI Jingwen;WANG Yuanyuan(School of Biology and Biological Engineering,South China University of Technology,Guangzhou 510006,Guangdong,China)
出处
《华南理工大学学报（自然科学版）》
EI
CAS
CSCD
北大核心
2022年第1期143-154,共12页
Journal of South China University of Technology(Natural Science Edition)
基金
国家重点研发计划项目(2018YFA0901500)。
