Fatty acid biosynthesis in microorganisms being used for Single Cell Oil production

Single cell oils (SCOs) are now produced by various microorganisms as commercial sources of arachidonic acid (ARA) and docosahexaenoic acid (DHA). These oils are now used extensively as dietary supplements in infant formulas. An understanding of the underlying biochemistry and genetics of oil accumulation in such microorganisms is therefore essential if lipid yields are to be improved. Also an understanding of the biosynthetic pathways involved in the production of these polyunsaturated fatty acids (PUFAs) is also highly desirable as a prerequisite to increasing their content in the oils. An account is provided of the biosynthetic machinery that is necessary to achieve oil accumulation in an oleaginous species where it can account for lipid build up in excess of 70% of the cell biomass. Whilst PUFA production in most microorganisms uses a conventional fatty acid synthase (FAS) system followed by a series of desaturases and elongases, in Schizochytrium sp., and probably related thraustochytrid marine protists, PUFA synthesis now appears to be via a polyketide synthase (PKS) route. This route is discussed. It clearly represents a major departure from conventional fatty acid biosynthesis, possibly as a means of decreasing the amount of NADPH that is needed in the overall process.     

Nondestructive analyses of unsaturated fatty acid species in dietary oils by attenuated total reflectance with Fourier transform IR spectroscopy

The aim of this study was to develop a nondestructive method to quantitate relative amounts of n-3 and n-6 polyunsaturated fatty acid (PUFA) species in vegetable oils and oil seeds using Fourier transform IR spectroscopy (FTIR). The alkene Cbond;H stretching vibrations of unsaturated fatty acids in oils showed IR absorption bands with various peak positions and intensities at around 3010 cm(-1), depending on the extent of unsaturation and PUFA species. With the aid of partial least-squares regression analysis, the FTIR measurement could practically predict the content of each PUFA species in the oil to be tested. A calculation method was also presented to directly find PUFA species in oils from the FTIR spectra. This technique was applied to dried soybean seeds to demonstrate a nonhomogenous distribution of saturated fatty acids and PUFAs, as well as glycans, in soybean cross sections.     

Effect of culture media and conditions on polyunsaturated fatty acids production by Mortierella alpina

To improve the polyunsaturated fatty acid (PUFA) production by Mortierella, culture media and conditions were investigated. M. alpina ATCC 32222 had the highest yield of arachidonic acid, gamma-linolenic acid and linoleic acid among 11 test microbes. Soluble starch at 10% and the mixture of KNO3 and yeast extract at 2:1 (w/w) was the best carbon and nitrogen sources for arachidonic acid and total PUFAs production, respectively. The optimal C/N ratio ranged from 5.1 to 9.0. Each gram of carbon produced 17.4 mg of linoleic acid, 17.0 mg of gamma-linolenic acid, 103.0 mg of arachidonic acid and 194.2 mg of total PUFAs at 20 degrees C, while it yielded 21.4 mg of linoleic acid, 25.6 mg of gamma-linolenic acid, 2.6 mg of gamma-linolenic acid, 110.3 mg of arachidonic acid, 4.3 mg of eicosapentaenoic acid and 218.4 mg of total PUFAs at 12 degrees C. A high degree of unsaturation was found at low temperature incubation. Linseed oil supplementation (1%, w/v) increased the PUFAs production and each gram of carbon produced 403.4 mg of alpha-linolenic acid, 123.1 mg of arachidonic acid, 33.6 mg of eicosapentaenoic acid, 1.68 mg of docosahexaenoic acid and 943.2 mg of total PUFAs. From the optimization of culture media and conditions, PUFAs production increased from 30% to 5 times that was optimal for practical use.     

Production and recovery of polyunsaturated fatty acids-added lipids from fermented canola

Canola flake was investigated as a potential substrate for fungal conversion to produce polyunsaturated fatty acids (PUFAs)-added oil in a 7l fermenter. The results showed that yields of total oil were reduced 9-22% compared to initial oil in the canola flake, but as high as 445mg/l arachidonic acid (ARA, C20:4n6) and 67mg/l eicosapentaenoic acid (EPA, C20:5n3) were produced. The percentages of ARA and EPA of total fatty acids in this fermented oil were 15.5% and 2.3%, respectively. Supercritical CO(2) extraction was then investigated for the lipid recovery from fermented canola flake, and extraction kinetics were modeled. The feasibility was demonstrated for production of PUFAs in a laboratory-scale fermentor using canola flake as a single nutrient, and for lipid extraction using supercritical CO(2).     

酵母人工合成细胞生产植物源天然产物北大核心CSCD

植物源天然产物在医疗保健领域有着广泛的应用。目前,生产植物源天然产物的主要方式为从原植物直接提取,但此法面临诸多问题。基于合成生物学的理念,创建酵母人工细胞工厂发酵生产植物源天然产物是一种新的资源获取途径。本文将从植物源天然产物在药物和营养领域的应用前景,发酵法生产青蒿酸的研发历程,部分萜类、生物碱和长链多不饱和脂肪酸的研究进展,以及该领域相关技术前沿4个方面介绍酵母人工合成细胞生产植物源天然产物的近况。     

Thraustochytrid Marine Protists: Production of PUFAs and Other Emerging Technologies

Thraustochytrids, the heterotrophic, marine, straminipilan protists, are now established candidates for commercial production of the omega-3 polyunsaturated fatty acid (ω-3 PUFA), docosahexaenoic acid (DHA), that is important in human health and aquaculture. Extensive screening of cultures from a variety of habitats has yielded strains that produce at least 50% of their biomass as lipids, and DHA comprising at least 25% of the total fatty acids, with a yield of at least 5 g L⁻¹. Most of the lipids occur as triacylglycerols and a lesser amount as phospholipids. Numerous studies have been carried out on salinity, pH, temperature, and media optimization for DHA production. Commercial production is based on a fed batch method, using high C/N ratio that favors lipid accumulation. Schizochytrium DHA is now commercially available as nutritional supplements for adults and as feeds to enhance DHA levels in larvae of aquaculture animals. Thraustochytrids are emerging as a potential source of other PUFAs such as arachidonic acid and oils with a suite of PUFA profiles that can have specific uses. They are potential sources of asataxanthin and carotenoid pigments, as well as other lipids. Genes of the conventional fatty acid synthesis and the polyketide-like PUFA synthesis pathways of thraustochytrids are attracting attention for production of recombinant PUFA-containing plant oils. Future studies on the basic biology of these organisms, including biodiversity, environmental adaptations, and genome research are likely to point out directions for biotechnology explorations. Potential areas include enzymes, polysaccharides, and secondary metabolites.     

Enzyme catalysed production of vegetable oils containing omega-3 polyunsaturated fatty acid

Summary The application of enzymatic interesterification for production of vegetable oils containing omega-3 polyunsaturated fatty acids was investigated. Six veteable oils were used as substrates, together with omega-3 polyunsaturated fatty acid, and reactions were catalysed by immobilized Mucor miehei lipase in organic solvent. The degree of incorporation of eicosapentaenoic acid and docosahexaenoic acid into corn oil, sunflower oil, peanut oil, olive oil and soybean oil were 17.71, 17.59, 16.79, 14.89, 13.91 and 10.48%, respectively, after a 12 h incubation period.     

Beneficial effects of polyunsaturated fatty acids in partially nephrectomized rats

Evening primrose oil, safflower oil, and salmon oil, all with high polyunsaturated fatty acid content, were fed to partially nephrectomized rats; the effects were compared to those of feeding beef tallow. All three oils had favorable effects on progression of renal failure, salmon oil on kidney histology as well. The changes induced in platelet production of thromboxane A2, and in the renal production of various eicosanoids may explain the protective role of these oils.     

Dietary n-3 polyunsaturated fatty acids modify fatty acid composition in hepatic and abdominal adipose tissue of sucrose-induced obese rats

The fatty acid profile of hepatocytes and adipocytes is determined by the composition of the dietary lipids. It remains unclear which fatty acid components contribute to the development or reduction of insulin resistance. The present work examined the fatty acid composition of both tissues in sucrose-induced obese rats receiving fish oil to determine whether the effect of dietary (n-3) polyunsaturated fatty acids (PUFAs) on the reversion of metabolic syndrome in these rats is associated to changes in the fatty acid composition of hepatocyte and adipocyte membrane lipids. Animals with metabolic syndrome were divided into a corn–canola oil diet group and a fish oil diet group, and tissues fatty acids composition were analyzed after 6 weeks of dietary treatment. Fatty acid profiles of the total membrane lipids were modified by the fatty acid composition of the diets fed to rats. N-3 PUFAs levels in animals receiving the fish oil diet plus sucrose in drinking water were significantly higher than in animals under corn–canola oil diets. It is concluded that in sucrose-induced obese rats, consumption of dietary fish oil had beneficial effects on the metabolic syndrome and that such effects would be conditioned by the changes in the n-3 PUFAs composition in hepatic and adipose tissues because they alter membrane properties and modify the type of substrates available for the production of active lipid metabolites acting on insulin resistance and obesity.     

Beneficial effects of polyunsaturated fatty acids in partially nephrectomized rats

Evening primrose oil, safflower oil, and salmon oil, all with high polyunsaturated fatty acid content, were fed to partially nephrectomized rats; the effects were compared to those of feeding beef tallow. All three oils had favorable effects on progression on renal failure, salmon oil on kidney histology as well. The changes induced in platelet production of thromboxane A₂, and in the renal production of various eicosanoids may explain the protective role of these oils.     

Metabolic engineering of plants for polyunsaturated fatty acid production

Very-long-chain polyunsaturated fatty acids (VLCPUFAs) have demonstrated health benefits. Currently, the main sources for these fatty acids are oils from fish and microbes. However, shrinking fish populations and the high cost of microbial oil extraction are making the economic sustainability of these sources questionable. Metabolic engineering of oilseed crops could provide a novel and sustainable source of VLCPUFAs. Recently, genes encoding desaturases and elongases from microbes have been identified and successfully expressed in oilseed plants. However, the levels of VLCPUFAs produced in transgenic plants expressing these genes are still much lower than those found in native microbes. This review assesses the recent progress and future perspectives in the metabolic engineering of PUFAs in plants.     

Polyunsaturated fatty acids production by solid-state fermentation on polyurethane foam by Mortierella alpina

Polyunsaturated fatty acids (PUFAs) are essential in healthy diets and their production is extremely important. Natural sources of PUFAs includes animal and aquatic products such as marine fish oil, however there are several limitations such as the decrease of fish stocks throughout the world. Thus, microbial oils are a preferable source of PUFAs. Herein, it was studied the production of PUFAs by Mortierella alpina under solid-state fermentation (SSF) using polyurethane foam as inert substrate and synthetic medium or lignocellulosic hydrolysate as source of C, N, and other nutrients. Several parameters of fermentation conditions were evaluated as carbon source, inductors addition, ratio C/N and temperature. The highest amount of total PUFAs per mass of solid (535.41 ± 24.12 mg/g), linoleic acid (129.66 ± 5.84 mg/g), and α-linoleic acid (401.93 ± 18.10 mg/g) were produced when the culture medium contained 20 g/L glucose, 10% (w/v) linseed oil, the C/N ratio was adjusted to 25 and the incubation temperature was 25°C for 3 days decreasing to 16°C on the remaining 4 days of fermentation. In addition, a hemicellulosic hydrolysate can be used as low-cost substrate to produce PUFAs, although the production was lower than the achieved with synthetic medium. SSF showed an interesting technology for microbial PUFAs production.     

Digestion and absorption of polyunsaturated fatty acids.

Polyunsaturated fatty acids play an important part in the structure and function of cellular membranes and are precursors of lipid mediators which play a key role in cardiovascular and inflammatory diseases. Dietary sources of essential fatty acids are vegetable oils for either linoleic or alpha-linolenic acids, and sea fish oils for eicosapentaenoic and docosahexaenoic acids. Because of the specificity of the pancreatic lipid hydrolases, triglyceride fatty acid distribution is an essential parameter in the digestibility of fats. The efficiency of the intestinal uptake depends on the hydrolysis and especially on their micellarization. n-3 polyunsaturated fatty acid ethyl ester digestion is recognized to be impaired, but n-3 polyunsaturated fatty acid triglyceride hydrolysis remains a controversial point, and to some authors explains differences observed between vegetable and fish oil absorption. So additional studies are required to investigate this intestinal step. In enterocytes, morphological and biochemical absorption processes involve reesterification of long-chain fatty acids and lipoprotein formation. At this level, specific affinity of I- and L-FABPc (cytosolic fatty acid binding proteins) to polyunsaturated fatty acids requires further investigation. A better understanding of the role of these FABPc might bring to light the esterification step, particularly the integration of polyunsaturated fatty acids into phospholipids. With reference to differences published between fish and vegetable oil absorption, longer-term absorption studies appear essential to some authors. Polyunsaturated fatty acid absorption is thought to be not very dissimilar to that of long-chain mono-unsaturated fatty acid absorption. However, several digestion and absorption specific steps are worth studying with reference to the crucial role of polyunsaturated fatty acids in the organism, and for example adaptation of possible dietary supplements.     

高产PUFAs深黄被孢霉菌株的筛选

以深黄被孢霉(Mortierella isabellina As3.3410)为出发菌株,经微波诱变和紫外诱变,乙酰水杨酸与低温(15℃)相结合的筛选方法,获得1株高产多不饱和脂肪酸菌株A35-4,其生物量为17.9 g/L,油脂含量为67.8%,油脂产量为12.12 g/L,PUFAs含量为20.3%,PUFAs产量为2.46 g/L,上述指标比原始菌株A0分别增加32.6%、49.8%、98.69%、14.0%和125.7%。连续斜面传代培养证实该菌株具有较好的遗传稳定性。本研究为直接利用该菌株生产PUFAs以及克隆高效PUFAs相关基因,创造高含PUFAs转基因植物材料奠定基础。     

Successful high‐level accumulation of fish oil omega‐3 long‐chain polyunsaturated fatty acids in a transgenic oilseed crop

Omega‐3 (also called n‐3) long‐chain polyunsaturated fatty acids (≥C20; LC‐PUFAs) are of considerable interest, based on clear evidence of dietary health benefits and the concurrent decline of global sources (fish oils). Generating alternative transgenic plant sources of omega‐3 LC‐PUFAs, i.e. eicosapentaenoic acid (20:5 n‐3, EPA) and docosahexaenoic acid (22:6 n‐3, DHA) has previously proved problematic. Here we describe a set of heterologous genes capable of efficiently directing synthesis of these fatty acids in the seed oil of the crop Camelina sativa, while simultaneously avoiding accumulation of undesirable intermediate fatty acids. We describe two iterations: RRes_EPA in which seeds contain EPA levels of up to 31% (mean 24%), and RRes_DHA, in which seeds accumulate up to 12% EPA and 14% DHA (mean 11% EPA and 8% DHA). These omega‐3 LC‐PUFA levels are equivalent to those in fish oils, and represent a sustainable, terrestrial source of these fatty acids. We also describe the distribution of these non‐native fatty acids within C. sativa seed lipids, and consider these data in the context of our current understanding of acyl exchange during seed oil synthesis.     

Engineering oilseeds to produce nutritional fatty acids

There is a growing body of evidence suggesting that regular consumption of foods rich in omega-3 long chain polyunsaturated fatty acids has multiple positive health benefits. The fats and oils from marine fish contain high contents of these beneficial fatty acids but increased consumer demand has also increased strain on the ability of the world's fisheries to meet demand from wild capture. Many consumers are choosing fish oil supplements or are eating foods that have been complemented with fish oils instead of consuming fish directly. However, removing undesirable odors, flavors and contaminants is expensive. In contrast, oils derived from land plants such as soybean are inexpensive and contaminant free. Recent strides in plant molecular biology now allow the engineering of oilseeds for the production of novel fats and oils, including those synthesized by complex, multigene biosynthetic pathways such as the omega-3 long chain polyunsaturated fatty acids. Given the potential benefits to the environment with regards to overfishing and the health prospects of increased consumption of these healthy fatty acids, producing these fatty acids in oilseeds is a desirable and worthy goal. In this review, we will describe the recent advances in this field along with some of the technical hurdles encountered thus far.     

Cloning and functional expression of the first plant fatty acid elongase specific for Delta(6)-polyunsaturated fatty acids

In order to elucidate the biosynthesis of long-chain polyunsaturated fatty acids (PUFAs) in plants we searched for a cDNA encoding a Delta(6)-specific PUFA elongase from Physcomitrella patens, which is known to contain high proportions of arachidonic acid (20:4 Delta(5,8,11,14)). An EST clone from P. patens was identified by its low homology to the yeast gene ELO1, which is required for the elongation of medium-chain fatty acids. We functionally characterized this cDNA by heterologous expression in Saccharomyces cerevisiae grown in the presence of several fatty acids. Analysis of the fatty acid profile of the transgenic yeast revealed that the cDNA encodes a protein that leads to the elongation of the C(18) Delta(6)-polyunsaturated fatty acids gamma-linolenic acid (18:3 Delta(6,9,12)) and stearidonic acid (18:4 Delta(6,9,12,15)), which were recovered to 45-51% as their elongation products. In contrast, linoleic and alpha-linolenic acids were hardly elongated and we could not measure any elongation of saturated and mono-unsaturated fatty acids (including 18:1 Delta(6)), indicating that the elongase is highly specific for the polyunsaturated nature of the fatty acid acting as substrate.     

Biotechnological processes for biodiesel production using alternative oils

As biodiesel (fatty acid methyl ester (FAME)) is mainly produced from edible vegetable oils, crop soils are used for its production, increasing deforestation and producing a fuel more expensive than diesel. The use of waste lipids such as waste frying oils, waste fats, and soapstock has been proposed as low-cost alternative feedstocks. Non-edible oils such as jatropha, pongamia, and rubber seed oil are also economically attractive. In addition, microalgae, bacteria, yeast, and fungi with 20% or higher lipid content are oleaginous microorganisms known as single cell oil and have been proposed as feedstocks for FAME production. Alternative feedstocks are characterized by their elevated acid value due to the high level of free fatty acid (FFA) content, causing undesirable saponification reactions when an alkaline catalyst is used in the transesterification reaction. The production of soap consumes the conventional catalyst, diminishing FAME production yield and simultaneously preventing the effective separation of the produced FAME from the glycerin phase. These problems could be solved using biological catalysts, such as lipases or whole-cell catalysts, avoiding soap production as the FFAs are esterified to FAME. In addition, by-product glycerol can be easily recovered, and the purification of FAME is simplified using biological catalysts.