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Engineering tobacco for efficient astaxanthin production using a linker-free monocistronic dual-protein expression system and interspecific hybridization method

文献类型： 外文期刊

作者： Fang, Ning 1 ; Du, Zaifeng 1 ; Liu, Xiaofeng 1 ; Tian, Tian 1 ; Chai, Maofeng 2 ; Wang, Wenjing 1 ; Du, Yongmei 1 ; Zhao, Shancen 3 ; Timko, Michael P. 4 ; Xue, Zheyong 5 ; Zhang, Zhongfeng 1 ; Zhang, Hongbo 1 ;

作者机构： 1.Chinese Acad Agr Sci, Key Lab Synthet Biol, Tobacco Res Inst, Minist Agr & Rural Affairs, Qingdao 266101, Peoples R China

2.Qingdao Agr Univ, Coll Grassland Sci, Qingdao 266109, Peoples R China

3.BGI Shenzhen, BGI Inst Appl Agr, Shenzhen 518120, Peoples R China

4.Univ Virginia, Dept Biol, Charlottesville, VA 22904 USA

5.Northeast Forestry Univ, Key Lab Saline Alkali Vegetat Ecol Restorat, Minist Educ, Harbin 150040, Peoples R China

关键词： Metabolic engineering; Astaxanthin; Monocistronic cassette; Protein splicing; Tobacco

期刊名称：PLANT PHYSIOLOGY AND BIOCHEMISTRY （ 影响因子：5.7； 五年影响因子：6.4 ）

ISSN： 0981-9428

年卷期： 2025 年 221 卷

页码：

收录情况： SCI

摘要： The high-value carotenoid astaxanthin is biosynthesized through a dual-enzyme-catalyzed cascade and is getting increased attention for engineered biosynthesis in plants. When developing astaxanthin-producing tobacco by expressing 2A-peptide-linked CBFD (carotenoid (3-ring-4-dehydrogenase) and HBFD (carotenoid 4-hydroxy(3-ring-4-dehydrogenase) from Adonis aestivalis, this work discovered an in-enzyme splicing site at the N-terminus of HBFD that has potentials for multiple protein expression in plant using monocistronic cassette. Based on this finding, we generated astaxanthin-producing tobacco plants expressing a directly fused protein of CBFD and HBFD with a monocistronic cassette. Further integrated IP (immunoprecipitation) and LC-MS/MS assays revealed the presence of an in-enzyme splicing site at the N-terminus of HBFD. Nevertheless, the obtained astaxanthin-producing tobacco plants exhibited a growth retardation as observed by previous researches. Subsequent studies revealed that the astaxanthin-producing caused growth retardation of tobacco was correlated with chloroplast disruption and chlorophyll reduction, and it could be alleviated by expressing a chlorophyll biosynthetic enzyme identified by proteomics. Additionally, crossing the astaxanthin-producing tobacco with a variety having higher chlorophyll content also alleviated the growth retardation caused by astaxanthin production, and improved the total astaxanthin yield per plant by at least threefold along with the biomass increase. This work provides novel approaches for expressing multiple proteins in tobacco and for engineering efficient astaxanthin-producing tobacco.

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