PMC:4301621 / 1678-6496 JSONTXT

{"project":"2_test","denotations":[{"id":"25582171-17439233-14905453","span":{"begin":144,"end":145},"obj":"17439233"},{"id":"25582171-22174787-14905454","span":{"begin":270,"end":271},"obj":"22174787"},{"id":"25582171-9103911-14905455","span":{"begin":391,"end":392},"obj":"9103911"},{"id":"25582171-9304876-14905456","span":{"begin":436,"end":437},"obj":"9304876"},{"id":"25582171-18256790-14905457","span":{"begin":522,"end":523},"obj":"18256790"},{"id":"25582171-15300418-14905458","span":{"begin":730,"end":731},"obj":"15300418"},{"id":"25582171-16473770-14905459","span":{"begin":1345,"end":1346},"obj":"16473770"},{"id":"25582171-16233793-14905460","span":{"begin":1567,"end":1569},"obj":"16233793"},{"id":"25582171-10974120-14905461","span":{"begin":1812,"end":1814},"obj":"10974120"},{"id":"25582171-15825827-14905462","span":{"begin":1815,"end":1817},"obj":"15825827"},{"id":"25582171-16232682-14905463","span":{"begin":2130,"end":2132},"obj":"16232682"},{"id":"25582171-22174787-14905464","span":{"begin":2246,"end":2247},"obj":"22174787"},{"id":"25582171-19565237-14905465","span":{"begin":2248,"end":2250},"obj":"19565237"},{"id":"25582171-14642354-14905466","span":{"begin":2688,"end":2690},"obj":"14642354"},{"id":"25582171-7986045-14905467","span":{"begin":2748,"end":2750},"obj":"7986045"},{"id":"25582171-16357206-14905468","span":{"begin":2802,"end":2804},"obj":"16357206"},{"id":"25582171-15520298-14905469","span":{"begin":2834,"end":2836},"obj":"15520298"},{"id":"25582171-22558935-14905470","span":{"begin":3532,"end":3534},"obj":"22558935"},{"id":"25582171-23236514-14905471","span":{"begin":3552,"end":3554},"obj":"23236514"},{"id":"25582171-23541380-14905472","span":{"begin":3651,"end":3653},"obj":"23541380"},{"id":"25582171-23541380-14905473","span":{"begin":3789,"end":3791},"obj":"23541380"},{"id":"25582171-23541380-14905474","span":{"begin":4007,"end":4009},"obj":"23541380"},{"id":"25582171-21886097-14905475","span":{"begin":4161,"end":4163},"obj":"21886097"},{"id":"25582171-22174787-14905476","span":{"begin":4317,"end":4318},"obj":"22174787"},{"id":"25582171-21336613-14905477","span":{"begin":4460,"end":4462},"obj":"21336613"}],"text":"Background\nMortierella alpina is an oleaginous zygomycete, that can accumulate lipids to 50% of its dry weight in the form of triacylglycerols [1]. The important ω-6 polyunsaturated fatty acid (PUFA) arachidonic acid (ARA) can account for over 50% of the lipid content [2]. M. alpina is nonpathogenic and nonallergenic, including the spores produced during the industrial production of ARA [3] which is widely used in food ingredients [4]. ARA has been produced at levels up to 19.8 g/L in 5 L cultures grown over 7 days [5]. Various methods have been attempted in order to improve ARA production including screening potentially higher yielding mutant strains following treatment with N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) [6]. This work led to the generation of M. alpina strain Y11 which possessed lowered ω-3 desaturation activity, and ARA production was 2.1 fold (2.21 g/L) higher than strain 1S-4. Optimizing the culture medium and fixing the ratio of defatted soybean meal to potassium nitrate at 2:1 gave a fourfold increase (6.0 g/L) in ARA production [7]. In another study the fermentation process was optimized, and a two-stage temperature-shift strategy increased ARA production by 26.1% (9.2 g/L) [8]. Additionally, overexpressing GLELO gene using a genetic manipulation approach increased ARA production by 101.2% (5.05 g/L) [9].\nGenetic engineering of M. alpina for enhanced ARA production remains an attractive proposition and for further research. Early work identified desaturases as key enzymes in PUFA synthesis. Specifically, Δ5 desaturase [10], which catalyzes dehydrogenation of dihomo-γ-linolenic acid (DGLA) to form ARA, was isolated and functionally characterized. The rate-limiting step for ARA biosynthesis is catalyzed by elongase which converts γ-linolenic acid (GLA) to DGLA [11,12]. NADH-cytochrome b5 reductase (Cb5R), an electron carrier and a major component of the cytochrome b5-dependent electron transport system, is also crucial. Cb5R catalyzes several different reduction reactions, including the desaturation and elongation of acyl chains built from acetyl-CoA during PUFA synthesis [13]. Despite various studies that have identified the importance of these enzymes in PUFA synthesis and metabolism [2,14], their exact roles are not completely understood, and neither are the pathways through which glucose relates to PUFA biosynthesis and metabolism.\nA genome-scale metabolic model (GSMM) is an indispensable tool for the study of metabolism that adopts a systems biology approach to integrate data from genomics, transcriptomics, proteomics and metabolomics. It has been widely used in the analysis of the network properties of metabolism [15], prediction and analysis of organism growth phenotypes [16], model-based interpretation of experimental data [17], and metabolic engineering [18]. Oleaginous organisms such as M. alpina can accumulate large quantities of lipids, but maximizing lipid production is complicated by the complexity of the regulatory mechanisms associated with lipid metabolism. It is generally difficult to identify key metabolic modules contributing to lipid physiology. Using reconstruction GSMM, we can systematically analyze the function of each gene and metabolic reaction and model the effects using flux balance analysis (FBA). Specific pathways can be understood based on the model of the whole metabolic network, and strain design strategies can also be used to guide metabolic engineering experiments. Two GSMM studies on Yarrowia lipolytica (iNL895 [19] and iYL619_PCP [20]) have been published along with recent modeled networks of Mucor circinelloides and M. alpina [21]. GSMM studies therefore provide a new approach to investigating the complex lipid metabolism in M. alpina. Vongsangnak et al. (2013) [21] previously published a M. alpina network model, however, this was a refined network that could only be used to investigate genome annotation and metabolic routes, and not flux distribution or phenotypic behaviors [21]. To systematically study flux distribution and the mechanism of lipid accumulation, we reconstructed a new M. alpina GSMM and used the COBRA Toolbox [22] for subsequent research.\nIn this study, a genome-scale metabolic model (iCY1106) of M. alpina ATCC 32222 was reconstructed based on sequencing results [2]. Using this model, we first identified essential genes and reactions in fermentation medium containing sodium nitrate as a nitrogen source [23]. The de novo synthesis pathways of PUFAs such as ARA and eicosapentaenoic acid (EPA) were subsequently resolved. The roles of acetyl-CoA and NADPH in the regulation of PUFA biosynthesis were probed, and important genes and reactions were systematically analyzed using FBA, flux variability analysis (FVA), and minimization of metabolic adjustment (MOMA)."}