融合蛋白和密码子优化策略提高真菌漆酶在黑曲霉中的表达  

Expression of fungal laccase in Aspergillus niger through fusion protein and codon optimization strategy

作　　者：张宇鑫 刘雨 于涛 赵佳东 付欣哲 李杰[1] ZHANG Yuxin;LIU Yu;YU Tao;ZHAO Jiadong;FU Xinzhe;LI Jie(School of Life Sciences,Northeast Agricultural University,Harbin 150030,China;Honggang District Government of Daqing City,Daqing Heilongjiang 163000,China;Inner Mongolia Kunming Cigarette Co.,Ltd.,Hohhot 010018,China)

机构地区：[1]东北农业大学生命科学学院,哈尔滨150030 [2]大庆市红岗区人民政府,黑龙江·大庆163000 [3]内蒙古昆明卷烟有限责任公司,呼和浩特010018

出　　处：《东北农业大学学报》2024年第8期151-159,231,共10页Journal of Northeast Agricultural University

基　　金：中央引导地方科技发展基金项目(ZYYDDF-ZH-202206)。

摘　　要：试验通过融合蛋白和密码子优化策略提高2种真菌漆酶,即灵芝(Ganoderma Karst)漆酶和硬毛粗盖孔菌(Funalia trogii)漆酶在黑曲霉中的分泌表达量。通过对重组菌株进行蛋白水平、转录水平、菌体形态学检测,探究融合蛋白和密码子优化策略对漆酶表达的影响,旨在找出提高蛋白产量的有效策略,构建高效表达漆酶的黑曲霉重组菌株,为低表达蛋白在黑曲霉中的高效表达提供新思路。结果表明:通过融合蛋白策略,以黑曲霉内源高分泌蛋白XynB、FaeA作为载体蛋白,将硬毛粗盖孔菌漆酶的分泌表达量分别提高33.41倍、30.43倍,灵芝漆酶分泌表达量分别提高0.45倍、0.74倍。根据黑曲霉密码子使用偏好性对漆酶基因进行密码子优化,灵芝漆酶分泌表达量提高0.35倍,但硬毛粗盖孔菌漆酶分泌表达量显著降低。In this experiment,a combination of the fusion protein and codon optimization strategy was employed to enhance the secretion levels of two fungal laccases,namely Ganoderma laccase and Funalia trogii laccase,in Aspergillus niger.The impacts of the fusion protein and codon optimization strategies on laccase expression were investigated through protein-level detection,transcription-level detection,and morphological analysis of the recombinant strains.The aim was to identify effective strategies for enhancing protein yield,construct highly efficient expression strains of laccase in Aspergillus niger,and provide novel approaches for the efficient expression of low-expression proteins in this fungus.The results showed that the secretion expression levels of Funalia trogii laccase were enhanced by 33.41-fold and 30.43-fold,respectively,by using the black Aspergillus endogenous highsecretion proteins xynB and faeA as carrier proteins through the fusion protein optimization strategy.Similarly,the secretion expression levels of Ganoderma laccase were increased by 0.45-fold and 0.74-fold,respectively.Furthermore,the laccase gene was codon-optimized based on the codon usage preference of black Aspergillus,resulting in a 0.35-fold increase in the secretion expression level of Ganoderma laccase.However,this optimization significantly reduced the secretion expression level of Funalia trogii laccase.

关 键 词：硬毛粗盖孔菌漆酶 灵芝漆酶 黑曲霉 优化表达策略 高效分泌表达 

分 类 号：Q782[生物学—分子生物学]