| Summary: | 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. <i>Mucor circinelloides</i> is 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 strain <i>M. circinelloides</i> MU758, first by integrating heterologous acyl-ACP thioesterase (<i>TE</i>) into fatty acid synthase (FAS) complex and subsequently by modifying the β-oxidation pathway by disrupting the acyl-CoA oxidase (<i>ACOX</i>) and/or acyl-CoA thioesterase (<i>ACOT</i>) 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 of <i>ACOX</i> in MCFAs production. However, MCFAs in M-3 were much decreased compared to M-1,suggesting a positive role of <i>ACOT</i> in MCFAs production. The M-2 strain showed maximum lipid productivity (~1800 milligram per liter per day or mg/L.d) and MCFAs productivity (~1100 mg/L.d). Taken together, this study elaborates on how the combination of two multidimensional approaches, <i>TE</i> gene over-expression and modification of the β-oxidation pathway via substantial knockout of specific <i>ACOX</i> gene, significantly increased the production of MCFAs. This synergistic approach ultimately offers a novel opportunity for synthetic/industrial biologists to increase the content of MCFAs.
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