Refactoring the architecture of a polyketide gene cluster enhances docosahexaenoic acid production in Yarrowia lipolytica through improved expression and genetic stability

Background Long-chain polyunsaturated fatty acids (LC-PUFAs), such as docosahexaenoic acid (DHA), are essential for human health and have been widely used in the food and pharmaceutical industries. However, the limited availa‑ bility of natural sources, such as oily fsh, has led to the pursuit of microbial production as a promising alternative. Yarrowia lipolytica can produce various PUFAs via genetic modifcation. A recent study upgraded Y. lipolytica for DHA pro‑ duction by expressing a four-gene cluster encoding a myxobacterial PKS-like PUFA synthase, reducing the demand for redox power. However, the genetic architecture of gene expression in Y. lipolytica is complex and involves various control elements, ofering space for additional improvement of DHA production. This study was designed to optimize the expression of the PUFA cluster using a modular cloning approach. Results Expression of the monocistronic cluster with each gene under the control of the constitutive TEF promoter led to low-level DHA production. By using the minLEU2 promoter instead and incorporating additional upstream activating UAS1B4 sequences, 5’ promoter introns, and intergenic spacers, DHA production was increased by 16-fold. The producers remained stable over 185 h of cultivation. Benefcially, the diferent genetic control elements acted synergistically: UAS1B elements generally increased expression, while the intron caused gene-specifc efects. Mutants with UAS1B16 sequences within 2–8 kb distance, however, were found to be genetically unstable, which limited pro‑ duction performance over time, suggesting the avoidance of long repetitive sequence blocks in synthetic multigene clusters and careful monitoring of genetic stability in producing strains. Conclusions Overall, the results demonstrate the efectiveness of synthetic heterologous gene clusters to drive DHA production in Y. lipolytica. The combinatorial exploration of diferent genetic control elements allowed the optimiza‑ tion of DHA production. These fndings have important implications for developing Y. lipolytica strains for the indus‑ trial-scale production of valuable polyunsaturated fatty acids