Abstract
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 circinelloides 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 M. circinelloides MU758, first by integrating heterologous acyl-ACP thioesterase (TE) into fatty acid synthase (FAS) complex and subsequently by modifying the β-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 of ACOX in MCFAs production. However, MCFAs in M-3 were much decreased compared to M-1,suggesting a positive role of ACOT 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, TE gene over-expression and modification of the β-oxidation pathway via substantial knockout of specific ACOX 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.
Highlights
Over the last two decades, microbially derived oils have offered a platform to face the challenges related to global warming, scarcity of non-renewable recourses, and food security [1,2,3]
Biokerosene is fundamentally composed of medium-chain-length alkanes that could be derived from saturated fatty acids with medium-chain length (MCFAs) [2]
Our results demonstrated that all engineered strains contained significant quantities of medium-chain fatty acids (MCFAs) in the free fatty acids (FFA) and TAG fractions (Figure 8B)
Summary
Over the last two decades, microbially derived oils have offered a platform to face the challenges related to global warming, scarcity of non-renewable recourses, and food security [1,2,3]. In the current scenario of lowering environmental impact of fossil-fuel, aviation industry is paying a great attention to the development of green-fuels These fuels have a number of necessities: they must exist as liquid at low temperature and have high energy content by volume. MCFAs have been gaining attention in the context of a balanced diet and human health concerns because of their ease of absorption, robust translocation to the liver through the portal veins, and swift metabolism via the β-oxidation pathway, enhancing diet-triggered thermogenesis. This mechanism has prompted interest in their use for the prevention and management of different metabolic disorders, such as hypertension, atherosclerosis, hyperlipidemia, type-II diabetes, obesity, and cardiovascular diseases (CVDs). These microorganisms have been genetically manipulated to produce multi-purpose products of biotechnological interest [5,6]
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