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

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 β-ring-4-dehydrogenase) and HBFD (carotenoid 4-hydroxy-β-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.