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 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 ß-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 ß-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.

Mitochondrial Citrate Transport System in the Fungus Mucor circinelloides: Identification, Phylogenetic Analysis, and Expression Profiling During Growth and Lipid Accumulation.

The mitochondrial citrate transport system, composed of citrate and malate transporters (MTs), can regulate the citrate efflux from mitochondria to cytosol, and then citrate is cleaved into OAA and acetyl-CoA which can be used for fatty acid (FA) biosynthesis. However, in the fungus Mucor circinelloides the molecular mechanism of citrate efflux from the mitochondria by this system and its role in FA synthesis is unclear. In the present study, we have analyzed the genome of high lipid-producing strain WJ11 and the low lipid-producing strain CBS 277.49 to find the potential genes involving in this system. Five potential genes are present in the genome of WJ11. These genes encode one citrate transport protein (CT), one tricarboxylate carrier (TCT), one MT, and two 2-oxoglutarate:malate antiporters (SoDIT-a and SoDIT-b). However, the genome of CBS 277.49 contains the same set of genes, except for the presence of just one SoDIT. The proteins from WJ11 had similar properties as their counterparts in CBS 277.49. Moreover, phylogenetic analyses revealed the evolutionary relationship of these proteins and illuminated their typical motifs related to potential functions. Additionally, the expression of these genes was analyzed to predict the possible functions in lipid metabolism in M. circinelloides. This is the first study to report the in silico analysis of structures and functions of the mitochondrial citrate transport system in M. circinelloides. This work showed a new strategy for research for the selection of candidate genes for further detailed functional investigation of the mitochondrial citrate transport system in lipid accumulation.

Construction of DGLA producing cell factory by genetic modification of Mucor circinelloides.

BACKGROUND: Dihomo-gamma linolenic acid (DGLA, 20:3, n-6) is the elongated product of Gamma linolenic acid (GLA, 18:3, n-6) catalyzed by the enzyme delta-6 elongase (D6E) or gamma linolenic acid elongase (GLELO). Construction of engineered oleaginous microbes have been attracting significant interest to produce DGLA because of its nutritional value and medicinal applications. Mucor circinelloides is a GLA producing filamentous fungus which can be a useful tool to produce DGLA. We have, therefore, overexpressed the D6E (GLELO) gene in this fungus to construct DGLA producing cell factory. RESULT: To produce DGLA in M. circinelloides, homologous overexpression of D6E (GLELO) gene was analyzed. When the gene was overexpressed in M. circinelloides CBS277.49, up to 5.72% DGLA was produced in this strain. CONCLUSION: To our knowledge, this is the first report describing the overexpression of D6E (GLELO) gene in M. circinelloides to construct DGLA producing cell factory. A new scope for further research has been established by this work for improved production of DGLA in this fungus, specifically in its high lipid-producing strain, WJ11.

Genetic Modification of Mucor circinelloides to Construct Stearidonic Acid Producing Cell Factory.

Stearidonic acid (SDA; 18:4, n-3) is the delta 15-desaturase product of gamma linolenic acid (GLA; 18:3, n-6) and delta 6-desaturase product of alpha linolenic acid (ALA; 18:3, n-3). Construction of engineered oleaginous microbes have been attracting significant interest in producing SDA because of its nutritional value and pharmaceutical applications. Mucor circinelloides is a GLA producing filamentous fungus, which can be a useful tool to produce SDA. This study has, therefore, overexpressed the delta-15 desaturase (D15D) gene from Mortierella alpina in this fungus to construct a SDA-producing cell factory. To produce SDA in M. circinelloides, the homologous overexpression of D15D gene was analyzed. When the gene was overexpressed in M. circinelloides CBS 277.49, up to 5.0% SDA was accumulated in this strain. According to current knowledge, this is the first study describing the construction of a SDA-producing cell factory by overexpression of D15D gene in oleaginous fungus M. circinelloides. A new scope for further research has been established by this work to improve SDA production in this fungus, specifically in its high lipid-producing strain, WJ11.

Engineering of Fatty Acid Synthases (FASs) to Boost the Production of Medium-Chain Fatty Acids (MCFAs) in Mucor circinelloides.

Increasing energy demands and health-related concerns worldwide have motivated researchers to adopt diverse strategies to improve medium-chain fatty acid (MCFA) biosynthesis for use in the functional food and aviation industries. The abundance of naturally produced MCFAs from botanical sources (i.e., coconut fruit/seeds and palm tree) has been observed to be insufficient compared with the various microorganisms used to cope with industrial demands. Mucor circinelloides is one of many promising microorganisms; it exhibits diverse biotechnological importance ranging from the production of functional lipids to applications in the manufacture of bio-fuel. Thus, research was conducted to acquire the desired elevated amounts of MCFAs (i.e., C8⁻C12) from metabolically engineered strains of M. circinelloides M65. To achieve this goal, four different acyl-acyl carrier protein (ACP) thioesterase (TE)-encoding genes exhibiting a substrate preference for medium-chain acyl-ACP molecules were expressed in M. circinelloides M65, resulting in the generation of C8⁻C12 fatty acids. Among all the engineered strains, M65-TE-03 and M65-TE-04 demonstrated the highest production of non-native C8⁻C10 and C12 fatty acids, respectively, in comparison to the control. These recombinant strains biosynthesized MCFAs de novo within the range from 28 to 46% (i.e., 1.14 to 2.77 g/L) of total cell lipids. Moreover, the reduction in chain length eventually resulted in a 1.5⁻1.75-fold increase in total lipid productivity in the engineered strains. The MCFAs were also found to be integrated into all lipid classes. This work illustrates how the integration of heterologous enzymes in M. circinelloides can offer a novel opportunity to edit the fatty acid synthases (FAS) complex, resulting in increased production of microbial MFCAs.