Increased Accumulation Of Medium-Chain Fatty Acids By Dynamic Degradation Of Long-Chain Fatty Acids In Mucor Circinelloides

Concerns about global warming, fossil-fuel depletion, food security, and human health have promoted metabolic engineers to develop tools/strategies to overproduce microbial functional oils directly from renewable resources. Medium-chain fatty acids (MCFAs, C8-C12) have been shown to be important sources due to their diverse biotechnological importance, providing benefits ranging from functional lipids to uses in bio-fuel production. However, oleaginous microbes do not carry native pathways for the production of MCFAs, and therefore, diverse approaches have been adapted to compensate for the requirements of industrial demand.Mucor circinelloidesis a promising organism for lipid production (15-36% cell dry weight; CDW) and the investigation of mechanisms of lipid accumulation; however, it mostly produces long-chain fatty acids (LCFAs). To address this challenge, we genetically modified strainM. circinelloidesMU758, first by integrating heterologous acyl-ACP thioesterase (TE) into fatty acid synthase (FAS) complex and subsequently by modifying the beta-oxidation pathway by disrupting the acyl-CoA oxidase (ACOX) and/or acyl-CoA thioesterase (ACOT) genes with a preference for medium-chain acyl-CoAs, to elevate the yield of MCFAs. The resultant mutant strains (M-1, M-2, and M-3, respectively) showed a significant increase in lipid production in comparison to the wild-type strain (WT). MCFAs in M-1 (47.45%) was sharply increased compared to the wild type strain (2.25%), and it was further increased in M-2 (60.09%) suggesting a negative role ofACOXin MCFAs production. However, MCFAs in M-3 were much decreased compared to M-1,suggesting a positive role ofACOTin MCFAs production. The M-2 strain showed maximum lipid productivity (similar to 1800 milligram per liter per day or mg/L.d) and MCFAs productivity (similar to 1100 mg/L.d). Taken together, this study elaborates on how the combination of two multidimensional approaches,TEgene over-expression and modification of the beta-oxidation pathway via substantial knockout of specificACOXgene, significantly increased the production of MCFAs. This synergistic approach ultimately offers a novel opportunity for synthetic/industrial biologists to increase the content of MCFAs.