Metabolic engineering of oleaginous yeast in lipogenic phase enhances production of nervonic acid

Insufficient biosynthesis efficiency can be a major obstacle to engineer oleaginous yeasts to overproduce very long-chain fatty acids (VLCFAs) during the lipogenic phase. Taking nervonic acid (NA, C24:1) as an example, we overcame the bottleneck to overproduce NA in engineered Rhodosporidium toruloides by improving the biosynthesis of VLCFAs during the lipogenic phase. First, evaluating the catalytic preferences of three plant-derived ketoacyl-CoA synthases rationally guided reconstructing efficient NA biosynthetic pathway in R. toruloides. More importantly, a genome-wide transcriptional analysis endowed clues to strengthen the fatty acid elongation (FAE) module and identify/use lipogenic phase-activated promoter, collectively addressing the stagnation of NA accumulation during the lipogenic phase. The best-designed strain exhibited a high NA content (major component in total fatty acid [TFA], 46.3%) and produced a titer of 44.2 g/L in a 5 L bioreactor. The strategy developed here provides an engineering framework to establish the microbial process of producing valuable VLCFAs in oleaginous yeasts. Overall design: To understand the underlying mechanism of the weakening productivity of the VLCFA biosynthesis during the lipogenic phase, we first conducted a genome-wide comparative transcriptomic analysis using Rhodosporidium toruloides strains grown at 48h and 120h, respectively.