[Engineering Rhodotorula toruloides based on exogenous targeting signal peptides]

Rhodotorula toruloides is an excellent oleaginous yeast species characterized by high lipid productivity, a broad substrate spectrum, and strong stress resistance. In addition, it has the suitability for high-density cultivation and the ability to synthesize carotenoids, which make it a potential chassis strain in the field of biotechnology. Signal peptides, as short peptide sequences guiding protein trafficking, have been widely employed in metabolic engineering and synthetic biology research. However, studies on signal peptides in R. toruloides remain limited. In this study, we investigated the organelle-targeting performance of three targeting sequences in R. toruloides, which direct proteins to the endoplasmic reticulum, mitochondria, and peroxisomes. After fusion of the targeting signal peptides with enhanced green fluorescent protein (eGFP), green fluorescence signals were observed in the corresponding organelles, confirming the utility of these signal peptides for protein localization. Furthermore, when hemoglobin VHb as the target protein was linked to the targeting sequences and eGFP, eGFP was still localized in the respective subcellular organelles, demonstrating that fusion with targeting signal peptides enabled the specific compartmentalization of target proteins. Finally, fatty acid photodecarboxylase (CvFAP) was expressed in R. toruloides with targeted localization, and the production of alkanes was detected in the engineered strains after fermentation under blue light. These results provide novel strain resources for the synthetic biology research on R. toruloides and expand its application in cell compartmentalization engineering.