SRA

De novo functional annotation of the genome of C. oleaginosus and compared its transcriptional landscape in conditions with high and low amounts of lipid production. Expression data was analyzed and Gene Ontology enrichment analysis was performed to identify overrepresented pathways and metabolic activities among the upregulated genes; moreover, a metabolic map was reconstructed and integrated with the expression data to gain insight into the transcriptional adaptations. Finally, the genome-scale metabolic model of C. oleaginosus was used to explore metabolism under maximal growth and maximal production conditions. The combination of these four analyses led to the selection of four overexpression targets (ATP-citrate lyase (ACL1), acetyl-CoA carboxylase (ACC), threonine synthase (TS), and hydroxymethylglutaryl-CoA synthase (HMGS)) and five media supplements (biotin, thiamine, threonine, serine, and aspartate). We established an electroporation-based co-transformation method to implement selected genetic interventions. These findings were experimentally validated in the build and test steps of the DBTL approach by adding supplements into the medium and overexpressing the identified genes. Characterization of ACL, ACC, and TS at various C/N ratios, and the addition of medium supplements provided up to 56% (w/w) lipid content, and a 2.5-fold increase in total lipid in the glycerol and urea-based defined medium. In the learn step, quadratic models identified the optimum C/N ratios shifted towards around C/N240. This result firmly confirms C. oleaginous as a sustainable alternative to replace palm as an oil source.